Title: Combination therapy including antibodies that bind EGFR and cMET.

The disclosure relates to the field of antibodies. In particular it relates to the field of therapeutic antibodies, including human antibodies, for the treatment of diseases involving aberrant cells. Further, it relates to antibodies that bind EGFR and cMET, including multispecific antibodies, and their use in the binding of EGFR and cMET positive cells, particularly tumor cells.

The epidermal growth factor (EGF) receptor (EGFR) is a cell-surface receptor for members of the epidermal growth factor family (EGF-family) of extracellular protein ligands. EGFR is also known as the ErbB-1 receptor. The receptor has been given various names in the past (EGFR; ERBB; ERBB1; HER1; PIG61; mENA). In the present disclosure the names ErbB-1, EGFR or HERl in humans will be used interchangeably. EGFR is a member of the ErbB family of receptors, a subfamily of four closely related receptor tyrosine kinases: ErbB-1 (EGFR), ErbB-2 (HER2/c-neu; Her2), ErbB-3 (Her 3) and ErbB-4 (Her 4).

EGFR exists on a cell surface and may be activated by binding of its specific ligands, including epidermal growth factor and transforming growth factor a (TGFa). Upon activation by its growth factor ligands, the receptor may undergo a transition from an inactive mostly monomeric form to an active homodimer. In addition to forming homodimers after ligand binding, EGFR may pair with another member of the ErbB receptor family, such as ErbB2, to create an activated heterodimer. Dimers may also form in the absence of ligand-binding and clusters of activated EGFRs may form after ligand binding.

EGFR dimerization stimulates intrinsic intracellular protein-tyrosine kinase (PTK) activity. This activity induces several signal transduction cascades that lead to cell proliferation and differentiation. The kinase domain of EGFR can cross- phosphorylate tyrosine residues of other receptors it is complexed with, and can itself be activated in that manner.

Mutations involving EGFR have been identified in several types of cancer. It is the target of an expanding class of anticancer therapies. Such therapies include EGFR tyrosine kinase inhibitors (EGFR-TKIs) such as gefitinib and erlotinib for lung cancer, and antibodies as cetuximab and panitumumab for colon cancer and head and neck cancer.

Cetuximab and panitumumab are monoclonal antibodies that inhibit the receptor. Other monoclonals in clinical development are zalutumumab, nimotuzumab, and matuzumab. The monoclonal antibodies aim to block the extracellular ligand-induced receptor activation, mostly by blocking ligand binding to the receptor. With the binding site blocked, signal-inducing molecules may not attach effectively and thereby also not activate downstream signaling. Ligand-induced receptor activation may also be inhibited by stabilization of the inactive receptor conformation (matuzumab).

To date, EGFR targeted therapies have been associated with the development of treatment resistance over time. Various mechanisms for the resistance to EGFR-TKIs have been described. In patients with advanced non-small cell lung cancer (NSCLC) the mechanisms of resistance include the occurrence of secondary or tertiary mutations (e.g., T790M, C797S, L718Q, exon 20 insertion mutations), the activation of alternative signaling (e.g., Met, HGF, AXL, Hh, IGF-1R), aberrant downstream pathways (e.g., AKT mutations, loss of PTEN), the impairment of the EGFR-TKIs-mediated apoptosis pathway (e.g., BCL2-like 11/BIM deletion polymorphism) and histological transformation. Although some mechanisms of resistance have been identified others remain to be identified. Furthermore, in the case of third- generation TKIresistance mechanisms, the molecular heterogeneity of NSCLC influences the contribution to the wide spectrum of resistance aberrations discovered so far. Similarly, patients with colorectal cancer that are treated with EGFR antibodies also develop resistance over time. This may occur through emergence of KRAS mutations. Of those without KRAS mutations; amplification of the MET proto-oncogene maybe associated with acquired resistance during anti-EGFR therapy (Bardelli et al., 2013; Cancer Discov. Jun;3(6):658- 73. doi: 10.1158/2159-8290. CD- 12-0558). The tumor can be resistant ab initio or develop resistance during treatment. Resistance to EGFR-targeted therapy is seen in many EGFR positive cancers and has demonstrated a need in the art for more efficacious EGFR cancer treatments that improve the standard of care, and are superior in terms of the capacity to address EGFR-targeted therapy resistance.

Dysregulation of MET Proto-Oncogene, Receptor Tyrosine Kinase (cMET) and hepatocyte growth factor (HGF) have been reported in a variety of tumors. Ligand- driven cMET activation has been observed in several cancers. Elevated serum and intra- tumoral HGF is observed in lung, breast cancer, and multiple myeloma (J. M. Siegfried et al., Ann Thorac Surg 66, 1915 (1998); P. C. Ma et al., Anticancer Res 23, 49 (2003); B. E. Elliott et al. Can J Physiol Pharmacol 80, 91 (2002); C. Seidel, et al, Med Oncol 15, 145 (1998)). Overexpression of cMET, cMET amplification or mutation has been reported in various cancers such as colorectal, lung, gastric, and kidney cancer and may drive ligand-independent receptor activation (C. Birchmeier et al, Nat Rev Mol Cell Biol 4, 915 (2003); G. Maulik et al., Cytokine Growth Factor Rev 13, 41 (2002)). Expression of HGF is also associated with the activation of the HGF/cMET signaling pathway and is also one of the escape mechanisms of tumors under selection by EGFR targeted therapy. Furthermore, treatment with cMET tyrosine kinase inhibitors, such as capmatinib or tepotinib, has been associated with the emergence of an escape mechanism to cMET aberrations.

The cMET receptor is formed by proteolytic processing of a common precursor into a single-pass, disulphide-linked u/B heterodimer. The extracellular portion of cMET is composed of three domain types. The N-terminal region fold forms a large semaphorin (Serna) domain, which encompasses the whole u-subunit and part of the B-subunit. The plexin-semaphorin-integrin (PSI) domain follows the Serna domain, and includes four disulphide bonds. This domain is connected to the transmembrane helix via four immunoglobulin-plexin-transcription (IPT) domains, which are related to immunoglobulin dike domains. Intracellularly, the cMET receptor contains a tyrosine kinase catalytic domain flanked by distinctive juxtamembrane and carboxy-terminal sequences (Organ and Tsao. Therapeutic advances in medical oncology 3.1_suppl (2011): S7-S19 which is incorporated herein by reference in its entirety).

The ligand of cMET, hepatocyte growth factor (HGF; also known as scatter factor) and its splicing isoforms (NK1, NK2) are known ligands of the cMET receptor. HGF was identified in 1991 as a potent mitogen/morphogen. The HGF/cMET signaling pathway plays important roles in the development and progression of various cancers. Dysregulation and/or hyperactivation of HGF or cMET in human cancers are linked to poor prognosis. cMET can be activated via overexpression, amplification, or mutation. Activation may promote development, progression, invasive growth, and metastasis of cancers. cMET can be activated in an HGF associated and HGF independent fashion. HGF independent activation occurs in cases of cMET over-expression. Abundance of cMET also may trigger (hetero) dimerization and intra-cellular signaling in the absence of ligand. Additional ligand does not appear to affect the function of such cMET over- expression cells. cMET amplification is associated with cMET over-expression and has emerged as a biomarker of tumor subtypes.

HGF is expressed ubiquitously throughout the body, showing this growth factor to be a systemically available cytokine as well as coming from the tumor stroma. A positive paracrine and/or autocrine loop of cMET activation can lead to further cMET expression. The HGF specific antibody Rilotumumab (AMG102) was developed for gastric cancer. Phase I and Phase II trials appeared promising but a phase III study with cisplatin and capecitabine as a first-line therapy in gastric cancer (RILOMET-2) was terminated following a pre-planned data monitoring committee safety review of study 20070622.

The relevance of cMET/HGF signaling in resistance to EGFR-targeted therapies has stimulated the development of ways to deal with the resistance. To date, antibody based approaches include anti-HGF antibodies; anti cMET or cMET antibodies and cMET/EGFR (reviewed in Lee et al., 2015; Immunotargets and Therapy 4: 35-44) have not been clinically effective. The cMET antibodies Onartuzumab (MetMab™) and Emibetuzumab (LY-2875358) have been evaluated in phase II clinical trials. Of these Onartuzumab appeared to be effective against colorectal cancer in a combination treatment together with the EGFR-inhibitor erlotinib. These results could, however, not be repeated in a randomized phase III clinical trial. MetMAb is a monovalent monoclonal antibody (mAh) against cMET, which blocks HGF binding to cMET and subsequent pathway activation (Jin et al., 2008 Cancer Research Vol. 68: pp 4360-68).

Overcoming a problem with anti-EGFR, cMET and HGF immunotherapies, the present disclosure provides novel bispecific antibodies that comprise a first variable domain that can bind an extracellular part of epidermal growth factor receptor (EGFR) and a second variable domain that can bind an extracellular part of cMET Proto- Oncogene, Receptor Tyrosine Kinase (cMET). To date, certain bispecific EGFR x cMET antibodies have been described in the art. Castoldi R. et al. (2013) describe a bispecific EGFR x cMET antibody designated MetHerl with the cMET binding site of the antibody 5D5 (or MetMab) and the EGFR binding site of cetuximab. The bispecific antibody has a fixed EGFR and cMET binding stoichiometry of 2:1 (see Supplemental Figures).

US20140378664 describes a cMET x EGFR bispecific antibody among various other bispecific antibodies. The complete bispecific antibody is produced as a single protein which is later proteolytically cleaved. The two VH/VL domains are produced as single chain Fv fragments. Binding of the antibody induces cMET degradation and Akt phosphorylation in a gastric cancer cell line. Moores et al (2016) describe a bispecific cMET x EGFR antibody designated JNJ-61186372 produced by controlled Fab-arm exchange (cFAE) having mutations at position 405 and 409 according to EU numbering, which may have potential for immunogenicity. JNJ-61186372 was shown to be active in vivo using a xenograft model with tumor cell line H1975 that expresses the cMET ligand HGF. This tumor model is known to be dependent on the ADCC activity of the antibody (Ahmed et al., 2015). JNJ-61186372 has a reported affinity imbalance of approximately 40x greater affinity for cMET than EGFR (Moores et al. (2016)), and the anti-EGFR arm derived from zalutumumab is known to cause infusion related reaction, skin disorders, among other issues.

LY3164530 is a bispecific cMET x EGFR antibody, which contains the EGFR binding domain of cetuximab as a single chain Fv fragment fused to the heavy chain variable domain of the cMET binding antibody LY2875358 (Emibetuzumab; Kim and Kim 2017). It is a so-called dual variable domain antibody that comprises two binding sites for each of the antigens. No data are provided on HGF inhibition for the antibody. The antibody reportedly binds and internalizes cMET and EGFR without agonistic activity. The authors review various cMET, EGFR and cMET x EGFR targeted therapies and draw the conclusion that to date none of these inhibitors have shown significant efficacy in clinical trials.

There is thus a need for novel bispecific cMET x EGFR antibodies, including those which may have superior characteristics as described herein.

SUMMARY OF THE INVENTION

In certain aspects, the present disclosure provides a combination of the bispecific antibody according to the present disclosure that comprises a first variable domain that can bind an extracellular part of human epidermal growth factor receptor (EGFR) and a second variable domain that can bind an extracellular part of human MET Proto- Oncogene, Receptor Tyrosine Kinase (cMET) and a third- generation EGFR tyrosine kinase inhibitor for use in a method of treatment of a cancer.

In certain aspects, the present disclosure provides a method of treatment of a subject having a cancer, comprising administering to the subject an effective amount of a combination of a third- generation EGFR tyrosine kinase inhibitor and the bispecific antibody according to the present disclosure that comprises a first variable domain that can bind an extracellular part of human epidermal growth factor receptor (EGFR) and a second variable domain that can bind an extracellular part of human MET Proto- Oncogene, Receptor Tyrosine Kinase (cMET).

In certain aspects, the present disclosure provides the use of a combination the bispecific antibody according to the present disclosure that comprises a first variable domain that can bind an extracellular part of human epidermal growth factor receptor (EGFR) and a second variable domain that can bind (or binds) an extracellular part of human MET Proto-Oncogene, Receptor Tyrosine Kinase (cMET) and a third- generation EGFR tyrosine kinase inhibitor in the manufacture of a medicament for the treatment of a cancer.

In certain aspects, the present disclosure provides a pharmaceutical combination comprising a third- generation EGFR tyrosine kinase inhibitor and the bispecific antibody according to the present disclosure that comprises a first variable domain that can bind an extracellular part of human epidermal growth factor receptor (EGFR) and a second variable domain that can bind an extracellular part of human MET Proto- Oncogene, Receptor Tyrosine Kinase (cMET).

In certain aspects, the cancer is an EGFR positive cancer, a cMET positive cancer or an EGFR and cMET positive cancer. In certain aspects, the cancer comprises an EGFR aberration, a cMET aberration or an EGFR and cMET aberration

In certain aspects, said cancer or subject has received prior treatment with an EGFR tyrosine kinase inhibitor and/or said cancer or subject is resistant to treatment with a tyrosine kinase inhibitor, in certain aspects to an EGFR and/or cMET tyrosine kinase inhibitor. In certain aspects, said subject has received prior treatment with a third generation EGFR tyrosine kinase inhibitor, such as Osimertinib. Said EGFR tyrosine kinase inhibitor resistance comprises a first, second and/or third generation tyrosine kinase inhibitor. In certain aspects, administration of or treatment with the combination of the bispecific antibody and EGFR tyrosine kinase inhibitor comprises a second line treatment to an EGFR and/or cMET tyrosine kinase inhibitor resistant subject. In other aspects, administration of or treatment with the combination of the bispecific antibody and EGFR tyrosine kinase inhibitor comprises a second line treatment of a subject having received prior treatment with a third-generation EGFR tyrosine kinase inhibitor. In certain aspects, said subject comprises an EGFR and/ or cMET aberration conferring resistance to said third-generation EGFR tyrosine kinase inhibitor. In certain aspects, said subject or cancer is resistant or refractory to a third- generation EGFR tyrosine kinase inhibitor, in certain aspects resistant or refractory to Osimertinib.

In certain aspects, the use or treatment comprises providing the subject with a dose of 1000, 1500 or 2000 mg of the bispecific antibody. In certain aspects, said bispecific antibody is provided once every week or once every two weeks. In certain aspects, said third-generation EGFR tyrosine kinase inhibitor is provided in a daily amount of between about 50 mg and about 400 mg, such as 70 mg, 75 mg, 80 mg, 100 mg, 110 mg or 240 mg.

In certain aspects, the use or treatment comprises providing the subject with a dose of 1500 mg of the bispecific antibody once every two weeks.

In certain aspects, the EGFR tyrosine kinase inhibitor which his administered in said combination treatment is or comprises Osimertinib, Lazertinib, Alflutinib, Rezivertinib, Rociletinib, Olmutinib, Almonertinib, Abivertinib, ASK120067, Befotertinib (also referred to as BPI-D0316 or D-0316), SH-1028, nazartinib (EGF816), naquotinib (ASP8273), mavelertinib (PF-0647775), Olafertinib (CK-101), Keynatinib, ES-072. In certain aspects, said EGFR tyrosine kinase inhibitor is Osimertinib, BPI- D0316/Befotertinib, Lazertinib or Almonertinib.

In certain aspects, the third generation EGFR tyrosine kinase inhibitor is Osimertinib (AZD9291). Osimertinib (AZD9291) is a covalent, orally active, irreversible, and mutant- selective EGFR inhibitor with an apparent IC50 of 12 nM against L858R and 1 nM against L858R/T790M, respectively. Recommended phase 2 dosing was established at a daily dose of 80 mg.

In certain aspects, the third generation EGFR tyrosine kinase inhibitor is Almonertinib (HS- 10296). Almonertinib is an orally available, irreversible, third- generation EGFR tyrosine kinase inhibitor with selectivity for EGFR-sensitizing and T790M resistance mutations. Almonertinib is used for the research of non-small cell lung cancer. Recommended phase 2 dosing was established at a daily dose of 110 mg.

In certain aspects, the third generation EGFR tyrosine kinase inhibitor is Lazertinib. Lazertinib (YH25448) is a potent, mutant- selective, blood-brain barrier permeable, orally available and irreversible third- generation EGFR tyrosine kinase inhibitor, and can be used in the research of non-small cell lung cancer. Recommended phase 2 dosing was established at a daily dose of 240 mg.

In certain aspects, the third generation EGFR tyrosine kinase inhibitor is Befotertinib (or BPLD0316 or sometimes D-0316). Befotertinib (Beta Pharmaceuticals, Co., China) is a third-generation EGFR tyrosine kinase inhibitor. Befotertinib can be used for the research of EGFR positive non-small cell lung cancer (NSCLC). In the phase II, single-arm study NCT05007938, the safety and efficacy of befotertinib was assessed at 25mg three times daily, orally, in combination with icotinib (125mg three times daily, orally) in patients with locally advanced or metastatic NSCLC. In the phase I study NCT04464551, subjects received a single oral dose of 75 mg D-0316 as an oral suspension. In the phase II, single-arm study NCT03861156, patients with locally advance d/metastatic non-small cell lung cancer received an oral dose of 75mg for a cycle of 21 days, and if tolerated, dose was increased to 100mg. Otherwise, the dose was maintained at 75mg. In the phase II/III study NCT04206072, the efficacy and safety of D-0316 at 70 mg once daily for 21 days, then increased to 100 mg once daily was assessed.

In certain aspects, the third generation EGFR tyrosine kinase inhibitor is Alflutinib (AST2818 or Furmonertinib). AST2818 is the subject of clinical trial NCT03787992 into the clinical efficacy thereof in NSCLC.

In certain aspects, the third generation EGFR tyrosine kinase inhibitor is Rezivertinib (BPI-7711) which is an orally active, selective and irreversible third- generation EGFR tyrosine kinase inhibitor (TKI). Rezivertinib is the subject of clinical trial NCT03866499 into the clinical efficacy thereof in NSCLC.

In certain aspects, the third generation EGFR tyrosine kinase inhibitor is Avitinib (Abivertinib/ACOOlO)), which is a pyrrolopyrimi dine -based irreversible epidermal growth factor receptor (EGFR) inhibitor with an IC50 of 7.68 nM. Avitinib is the subject of clinical trial NCT03856697 into the clinical efficacy thereof in NSCLC.

In certain aspects, the third generation EGFR tyrosine kinase inhibitor is ASK120067 which is a potent and orally active inhibitor of EGFR. ASK120067 is a third-generation EGFR-TKI for the research of non-small cell lung cancer (NSCLC). ASK120067 is the subject of a clinical trial (NCT04143607) into the clinical efficacy thereof in NSCLC.

In certain aspects, the third generation EGFR tyrosine kinase inhibitor is Oritinib (SH-1028, Nanjing Sanhome Pharmaceutical Co., Ltd., Nanjing, China). SH-1028 is the subject of clinical trial NCT04239833 into the clinical efficacy thereof in NSCLC.

In certain aspects, the third generation EGFR tyrosine kinase inhibitor is Rociletinib (CO- 1686), which is an orally delivered kinase inhibitor that specifically targets the mutant EGFR forms. Rociletinib was the subject of clinical trial NCT02186301 into the clinical efficacy thereof in NSCLC.

In certain aspects, the third generation EGFR tyrosine kinase inhibitor is Olmutinib (HM61713; BL 1482694) which is an orally active and irreversible third generation EGFR tyrosine kinase inhibitor that binds to a cysteine residue near the kinase domain. Olmutinib can be used in the research into NSCLC. Olmutinib was the subject of clinical trial NCT02485652 into the clinical efficacy thereof in NSCLC.

In certain aspects, the third generation EGFR tyrosine kinase inhibitor is Nazartinib (EGF816), which is a third- generation EGFR TKI that selectively inhibits EGFR activating mutations, in patients with advanced EGFR-mutant NSCLC. Nazartinib was the subject of clinical trial NCT03529084 into the clinical efficacy thereof in NSCLC.

In certain aspects, the third generation EGFR tyrosine kinase inhibitor is Naquotinib, which is an orally available, irreversible, third- generation, mutant- selective, epidermal growth factor receptor (EGFR) inhibitor. Naquotinib was the subject of clinical trial NCT02588261 into the clinical efficacy thereof in NSCLC.

In certain aspects, the third generation EGFR tyrosine kinase inhibitor is Mavelertinib (PF-0647775), which is a selective, orally available and irreversible EGFR tyrosine kinase inhibitor (EGFR TKI) .Mavelertinib was the subject of clinical trial NCT02349633 into the clinical efficacy thereof in NSCLC.

In certain aspects, said subject or cancer is resistant to treatment with a first, second, third- generation EGFR tyrosine kinase inhibitor or a cMET inhibitor.

In certain aspects, said first- generation tyrosine kinase resistance is or comprises resistance to gefitinib, erlotinib or icotinib.

In certain aspects, said second-generation tyrosine kinase resistance is or comprises resistance to afatinib, dacomitinib, XL647, AP26113, CO- 1686 or neratinib.

In certain aspects, said cMET tyrosine kinase resistance is or comprises resistance to capmatinib, tepotinib, crizotenib, cabozantinib, savolitinib, Glesatinib, Sitravatinib, BMS-777607, Merestinib, Tivantinib, Golvatinib, Foretinib, AMG-337 or BMS-794833. In certain aspects, said cMET tyrosine kinase resistance is or comprises resistance to tepotinib. In certain aspects, said cMET tyrosine kinase resistance is or comprises resistance to capmatinib.

In certain aspects, said third-generation tyrosine kinase resistance is or comprises resistance to Osimertinib, Lazertinib, Alflutinib, Rezivertinib, Olmutinib, Almonertinib, Abivertinib, ASK120067, Befotertinib, Rociletinib, Oritinib, Nazartinib, Naquotinib, Mavelertinib. In certain aspects, said third-generation tyrosine kinase resistance is or comprises resistance to Osimertinib. In certain aspects, said third- generation tyrosine kinase resistance is or comprises resistance to Lazertinib. In certain aspects, said third- generation tyrosine kinase resistance is or comprises resistance to Befotertinib. In certain aspects, said third- generation tyrosine kinase resistance is or comprises resistance to Almonertinib.

In certain aspects, the subject has not received prior anti-cancer treatment for an EGFR and/or cMET positive cancer or for a cancer comprising an EGFR and/or cMET aberration. In certain aspects, the subject has not received prior chemotherapy or a treatment comprising an anti-EGFR antibody or anti-cMET antibody. In certain aspects, said subject is EGFR tyrosine kinase inhibitor treatment naive, or cetuximab treatment naive. In certain aspects, administration of or treatment with the combination of the bispecific antibody and EGFR tyrosine kinase inhibitor is a first line treatment. In certain aspects, said first line treatment is to prevent resistance to an EGFR and/or cMET tyrosine kinase mechanism to develop, such as in a lung cancer patient or in a lung cancer, in particular in non-small cell lung cancer. Resistance mechanisms to EGFR and/or cMET tyrosine kinase inhibitors are well known to develop, especially in subjects with non-small cell lung cancer, such as in a metastatic or advanced cancer. Hence, the present disclosure also provides a combination of said third- generation tyrosine kinase inhibitor and said bispecific antibody for use in a method of preventing of a cancer having an EGFR and/or cMET tyrosine kinase inhibitor resistance from developing or occurring in a subject.

In certain aspects, the subject is a human subject.

In certain aspects, the cancer is lung cancer.

In certain aspects, said cancer is non-small cell lung cancer (NSCLC).

In certain aspects, said cancer or subject comprises an activating EGFR mutation, an approved tyrosine kinase inhibitor resistance mutation, a tertiary tyrosine kinase inhibitor resistance mutation, a mutation that reduces binding of a third generation tyrosine kinase inhibitor to EGFR, an acquired tyrosine kinase inhibitor resistance mutation, an EGFR gene amplification, a cMET mutation or cMET aberration.

In certain aspects, said cancer or subject comprises a cMET aberration, such as a cMET amplification, cMET overexpression, increased signaling of the cMET pathway, a cMET gene amplification and/or increased cMET protein activity. In certain aspects, said cancer or subject comprises increased HGF expression. In certain aspects, said cancer comprises a cMET exonl4 skipping mutation.

In certain aspects, the cMET dysregulation comprises a cMET amplification, cMET overexpression, increased signaling of the cMET pathway, a cMET gene amplification and/or increased cMET protein activity, or a cMET exonl4 skipping mutation. In one aspect, said cMET dysregulation is caused by increased HGF expression.

In certain aspects, the present disclosure provides a pharmaceutical combination comprising a third- generation EGFR tyrosine kinase inhibitor and a bispecific antibody that comprises a first variable domain that can bind an extracellular part of human epidermal growth factor receptor (EGFR) and a second variable domain that can bind an extracellular part of human MET Proto-Oncogene, Receptor Tyrosine Kinase (cMET).

In certain aspects, the pharmaceutical combination comprises instructions for use.

In certain aspects, a bispecific antibody of the disclosure exhibits ADCC activity, in certain aspects the antibody has improved ADCC activity. In such aspect the antibody can have altered ADCC activity by means of one or more CH2 variations relative to a fully human CH2 domain. Further provided is therefore a bispecific antibody according to the disclosure, which is afucosylated. In certain aspects, the antibody of the present disclosure comprises two afucosylated CH2 domains. In certain aspects, the antibody of the present disclosure comprises a total of two CH2 domains, both of which are afucosylated. In certain aspects, the antibody of the present disclosure comprises two CH2 domains, both of which are afucosylated. In certain aspects, a bispecific antibody of the disclosure exhibits ADCP activity, in certain aspects the antibody has improved ADCP activity. In certain aspects, both the EGFR and cMET binding arms, or both heavy chains that comprise the EGFR and cMET binding arms, contribute to ADCP. In certain aspects, the bispecific antibody of the present disclosure has or exhibits ADCP activity towards NSCLC cells. In certain aspects, the bispecific antibody of the present disclosure induces ADCP of NSCLC cells.

The bispecific antibody may comprise a common light chain. The first and second variable domains comprise the same or substantially the same (common) light chain variable region in certain aspects. Said common light chain variable region may be one that is known to pair well with a diversity of human variable region gene segments that have undergone recombination. In certain aspects, said common light chain is a variable region encoded by a germline Vk gene segment, such as the 012 I IgVKl-39*01 variable region gene segment. The preferred light chain variable region comprises the rearranged IgVKl-39*01/IGJKl*01 or IgVKl-39*01/IGJK5*01. The light chain of the cMET binding arm and the light chain of the EGFR binding arm is the same (common) light chain in certain aspects. In certain aspects, the common light chain is the rearranged kappa light chain IgVKl-39*01/IGJKl*01 or IgVKl-39*01/IGJK5*01 joined to a human light chain constant region. The bispecific antibody can be a human antibody. The bispecific antibody can be a full length antibody. It may have one variable domain that can bind EGFR and one variable domain that can bind cMET. In certain aspects, the variable domain that can bind human EGFR can also beneficially bind mouse EGFR and/or cynomolgus EGFR. In certain aspects, the variable domain that binds or can bind human EGFR binds to domain III of human EGFR. The variable domain that can bind cMET may block the binding of antibody 5D5 to cMET. The variable domain that can bind cMET may block the binding of HGF to cMET. The Kd of the antibody for cMET can be at least 10 times less than the Kd of the antibody for EGFR. The amino acids at positions 405 and 409 in one CH3 domain may be the same as the amino acids at the corresponding positions in the other CH3 domain (EU-numbering).

In certain aspects, the antibody of the present disclosure comprises a first variable domain comprises a heavy chain variable region with a CDR1 sequence SYGIS; a CDR2 sequence WISAYX1X2NTNYAQKLQG and a CDR3 comprising the sequence X3X4X5X6HWWLX7A wherein X ₁ = N or S; X ₂ = A or G; X ₃ = D or G; X ₄ = R, S or Y; X ₅ = H, L or Y; X ₆ = D or W and X ₇ = D or G; with 0-5 amino acid insertions, deletions, substitutions, additions or a combination thereof at a position other than X ₁ -X ₇ .

In certain aspects, the antibody of the present disclosure comprises a second variable domain which comprises a heavy chain variable region with the amino acid sequence of one of the sequences of SEQ ID NO: 1-23 (Figure 3) with 0-10, in certain aspects 0-5 amino acid insertions, deletions, substitutions, additions or a combination thereof.

Bispecific antibodies are described wherein

X ₁ = N; X ₂ = G; X3 = D; X ₄ = S; X ₅ = Y; X ₆ = W and X ₇ = G; Xi = N; X ₂ = A; X ₃ = D; X ₄ = S; X ₅ = Y; X ₆ = W and X ₇ = G;

Xi = S; X ₂ = G; X ₃ = D; X ₄ = S; X ₅ = Y; X ₆ = W and X ₇ = G;

Xi = N; X ₂ = G; X ₃ = D; X ₄ = R; X ₅ = H; X ₆ = W and X ₇ = D;

Xi = N; X ₂ = A; X ₃ = D; X ₄ = R; X ₅ = H; X ₆ = W and X ₇ = D;

Xi = S; X ₂ = G; X ₃ = D; X ₄ = R; X ₅ = H; X ₆ = W and X ₇ = D;

Xi = N; X ₂ = G; X ₃ = G; X ₄ = Y; X ₅ = L; X ₆ = D and X ₇ = G;

Xi = N; X ₂ = A; X ₃ = G; X ₄ = Y; X ₅ = L; X ₆ = D and X ₇ = G; or Xi = S; X ₂ = G; X ₃ = G; X ₄ = Y; X ₅ = L; X ₆ = D and X ₇ = G.

In certain aspects, Xi = N; X ₂ = G; X ₃ = D; X ₄ = R; Xs = H; Xe = W and X ₇ = D; or

Xi = N; X ₂ = A; X ₃ = D; X ₄ = R; Xs = H; X ₆ = W and X ₇ = D; or Xi = S; X ₂ = G; X ₃ = D; X ₄ =

R; Xs = H; X ₆ = W and X ₇ = D.

In certain aspects, X ₃ -X ₇ = DRHWD and Xi and X ₂ are NG; SG or NA.

Bispecific antibodies are described wherein the heavy chain variable region of the second variable domain comprises the amino acid sequence of one of the sequences of SEQ ID NO: 1-3; 7; 8; 10; 13; 15; 16; 17; 21; 22 or 23 with 0-10, in certain aspects 0-5 amino acid insertions, deletions, substitutions, additions or a combination thereof.

The disclosure also provides a method of treatment of a subject that has a tumor the method comprising administering the bispecific antibody as described herein to the individual in need thereof. Typically, the individual is one suffering from a disease involving aberrant cells, for examples the individual may be suffering from a tumor or a cancer.

The disclosure also provides a bispecific antibody as included in the treatment of the present disclosure that comprises a first variable domain that can bind an extracellular part of epidermal growth factor receptor (EGFR) and a second variable domain that can bind an extracellular part of MET Proto-Oncogene, Receptor Tyrosine Kinase (cMET), wherein the first variable domain comprises a heavy chain variable region with a CDR1 sequence SYGIS; a CDR2 sequence WISAYXIX ₂ NTNYAQKLQG and a CDR3 comprising the sequence X ₃ X ₄ XsX6HWWLX ₇ A wherein

Xi = N or S; X ₂ = A or G; X ₃ = D or G; X ₄ = R, S or Y; X ₅ = H, L or Y; X ₆ = D or W and X ₇ = D or G with 0-5 amino acid insertions, deletions, substitutions, additions or a combination thereof at a position other than Xi-X ₇ and wherein the second variable domain comprises a heavy chain variable region with the amino acid sequence of one of the sequences of SEQ ID NO: 1-23 (Figure 3) with 0-10, in certain aspects 0-5 amino acid insertions, deletions, substitutions, additions or a combination thereof.

The first variable domain in certain aspects comprises a heavy chain variable region with a CDR1 sequence SYGIS; a CDR2 sequence WISAYNGNTNYAQKLQG and a CDR3 comprising the sequence DRHWHWWLDAFDY and the second variable domain in certain aspects comprises a heavy chain variable region with a CDR1 sequence SYSMN; a CDR2 sequence WINTYTGDPTYAQGFTG and a CDR3 sequence ETYYYDRGGYPFDP. The disclosure also provides a bispecific antibody of the present disclosure for use in the treatment of a subject that has a disease involving aberrant cells, such as a tumor.

Also provided is a use of a bispecific antibody of the present disclosure and a third generation tyrosine kinase inhibitor in the manufacture of a medicament for the treatment of a disease involving aberrant cells, such as a tumor or cancer.

Also provided is a method of treatment of a subject that has a tumor, in certain aspects an EGFR positive tumor, a cMET positive tumor or an EGFR and cMET positive tumor, the method comprising administering the bispecific antibody and a third generation tyrosine kinase inhibitor to the individual in need thereof.

An antibody of the present disclosure inhibits HGF and EGF/HGF induced growth of the EGFR TKI resistant tumor cell lines and when used in combination with a third generation TKI. The TKI is in certain aspects Osimertinib. The TKI is in certain aspects Lazertinib. The TKI is in certain aspects Almonertinib. The TKI is in certain aspects Befotertinib.

An antibody of the present disclosure in combination with said third generation tyrosine inhibitor in certain aspects inhibits HGF induced growth of an HGF responsive cell, in certain aspects of an EGFR TKI resistant or refractory tumor of a human subject, tumor model or cell line, such as a cell line or model including an activating EGFR mutation, an approved tyrosine kinase inhibitor resistance mutation, a tertiary tyrosine kinase inhibitor resistance mutation, a mutation that reduces binding of a third generation tyrosine kinase inhibitor to EGFR, an acquired tyrosine kinase inhibitor resistance mutation, an EGFR gene amplification, a cMET mutation or cMET aberration, in certain aspects an in-frame exon 20 insertion mutation. In certain aspects, inhibition is shown in the presence of HGF.

An antibody of the present disclosure combined with a third generation TKI in certain aspects inhibits HGF induced growth of an HGF responsive cell, in certain aspects of an EGFR TKI resistant tumor, tumor model or cell line, such as a cell line or model including an in-frame exon 20 insertion mutation.

Herein, the term “refractory” refers to a disease that does not respond to a treatment. A refractory disease can be resistant to a treatment before or at the beginning of the treatment, or a refractory disease can become resistant during a treatment.

Herein the term “resistant” refers to a cancer or patient which is not responding to treatment when administered to prescribed dose of the therapeutic agent involved.

Herein, the term “1st generation EGFR tyrosine kinase inhibitor” (1st generation TKI) refers to reversible EGFR inhibitors such as gefitinib and erlotinib, which are effective in first-line treatment of NSCLC harboring EGFR activating mutations such as deletions in exon 19 and exon 21 L858R mutation.

Herein, the term “2nd generation EGFR tyrosine kinase inhibitor” (2nd generation TKI) refers to covalent irreversible EGFR inhibitors such as afatinib and dacomitib which are effective in first-line treatment of NSCLC harboring EGFR activating mutations such as deletions in exon 19 and exon 21 L858R mutation.

Herein, the term “3rd generation EGFR tyrosine kinase inhibitor” (3rd generation TKI) refers to covalent irreversible EGFR inhibitors such as osimertinib and lazertinib which are selective to the EGFR activating mutations, such as deletions in exon 19 and exon 21 L858R, alone or in combination with T790M mutation and have lower inhibitory activity against wild-type EGFR.

An antibody of the disclosure inhibits EGF induced growth of an EGF responsive cell, without inducing the toxicities such as rash and diarrhea associated with high affinity bivalent EGFR antibodies. This renders the antibody ideally suited for combination with TKI which have its own toxicity profile.

The disclosure further comprises a pharmaceutical combination, or a kit-of-parts, that comprises a bispecific antibody disclosed herein combined with a third-generation EGFR tyrosine kinase inhibitor. The pharmaceutical combination is in certain aspects not physically linked and comprises a container containing the antibody of the present disclosure and a container containing the third generation EGFR tyrosine kinase inhibitor. The pharmaceutical combination is in certain aspects accompanied by instructions for use. The instructions for use include clinically relevant information, such as instructions for intravenous administration. In certain aspects, the third generation EGFR tyrosine kinase, such as Osimertinib, BPI D-0316/Befotertinib, or Almonertinib, will be administered according to the instructions for use following approval of relevant authorities. In certain aspects, Osimertinib is dosed at a dose of 80mg once per day. In certain aspects, Almonertinib is dosed at a dose of HOmg once per day. In certain aspects, Lazertinib is dosed at a dose of 240 mg once per day. In some aspects, Befotertinib is dosed at 70 mg, 75 mg or 100 mg once per day. The 75 daily dose of Befotertinib may be provided as 3 orally administered dosages of 25 mg each.

In certain aspects, the bispecific antibody of the present disclosure is dosed at 1000 mg, in particular using a flat dose of 1000 mg. In certain aspects, said bispecific antibody is provided in an amount of 1000 mg once every week. In certain aspects, said bispecific antibody is provided in an amount of 1000 mg once every two weeks.

In certain aspects, the bispecific antibody of the present disclosure is dosed at 1500 mg, in particular using a flat dose of 1500 mg. In certain aspects, said bispecific antibody is provided an amount of 1500 mg once every two weeks. In certain aspects, the bispecific antibody of the present disclosure is dosed at 2000 mg, in particular using a flat dose of 2000 mg. In certain aspects, said bispecific antibody is provided in an amount of 2000 mg once every two weeks.

An antibody of the disclosure may be used to treat a tumor which is resistant or has reduced sensitivity to treatment with an EGFR tyrosine kinase inhibitor, for example resistant to Osimertinib, erlotinib, gefitinib, or afatinib, an analogue of Osimertinib, erlotinib, gefitinib or afatinib or a combination of one or more of the respective compounds and/or analogues thereof.

Accordingly, the bispecific antibody of the disclosure may be administered simultaneously, sequentially or separately with the EGFR tyrosine kinase inhibitor of the present disclosure. In certain aspects, said EGFR tyrosine kinase inhibitor comprises or is said third generation EGFR tyrosine kinase inhibitor.

The disclosure further comprises a nucleic acid molecule or a group of nucleic acid molecules that alone or together encode a heavy chain(s) or a heavy chain variable region(s) of a bispecific antibody disclosed herein or a variant thereof. Also provided is a nucleic acid molecule or group of nucleic acid molecules that encode an antibody disclosed herein.

In certain aspects the heavy chain comprises a constant region of an IgGl antibody, in certain aspects a human IgGl antibody. The CH2 region of said IgGl constant region can be engineered to alter ADCC and/or CDC activity of the antibody, or not. In certain aspects, said alteration results in enhanced ADCC and/or CDC activity. In certain aspects the CH3-region of the antibody is engineered to facilitate heterodimerization of heavy chains comprising a first heavy chain that binds EGFR and a second heavy chain binds cMET.

The disclosure further comprises is a cell comprising one or more nucleic acid molecules that alone or together encode a bispecific antibody or a variant thereof as disclosed herein. Also provided are methods of producing a bispecific antibody or a variant thereof disclosed herein using a cell as described, in certain aspects together with the harvesting of the bispecific antibody or variant thereof from a culture of the cells.

The disclosure further comprises a cell system that comprises a bispecific antibody or variant thereof disclosed herein.

The disclosure further provides a cell that expresses the bispecific antibody and/or comprises the nucleic acid molecule(s) that encode said bispecific antibody.

The disclosure further comprises a bispecific antibody as disclosed herein that further comprises a label, in certain aspects a label for in vivo imaging. DETAILED DESCRIPTION OF THE INVENTION

In certain aspects, the present disclosure provides a combination of the bispecific antibody according to the present disclosure that comprises a first variable domain that can bind an extracellular part of human epidermal growth factor receptor (EGFR) and a second variable domain that can bind an extracellular part of human MET Proto- Oncogene, Receptor Tyrosine Kinase (cMET) and a third- generation EGFR tyrosine kinase inhibitor for use in a method of treatment of a cancer.

In certain aspects, the present disclosure provides a method of treatment of a subject having a cancer, comprising administering to the subject an effective amount of a combination of a third- generation EGFR tyrosine kinase inhibitor and the bispecific antibody according to the present disclosure that comprises a first variable domain that can bind an extracellular part of human epidermal growth factor receptor (EGFR) and a second variable domain that can bind an extracellular part of human MET Proto- Oncogene, Receptor Tyrosine Kinase (cMET).

In certain aspects, the present disclosure provides the use of a combination the bispecific antibody according to the present disclosure that comprises a first variable domain that can bind an extracellular part of human epidermal growth factor receptor (EGFR) and a second variable domain that can bind (or binds) an extracellular part of human MET Proto-Oncogene, Receptor Tyrosine Kinase (cMET) and a third- generation EGFR tyrosine kinase inhibitor in the manufacture of a medicament for the treatment of a cancer.

In certain aspects, the bispecific antibody of the disclosure may be administered simultaneously, sequentially or separately with the EGFR tyrosine kinase inhibitor of the present disclosure. Said combination of the bispecific antibody according to the present disclosure and third-generation EGFR tyrosine kinase inhibitor thus encompasses simultaneous, sequential or separate administration. In certain aspects, said EGFR tyrosine kinase inhibitor comprises or is said third generation EGFR tyrosine kinase inhibitor.

Hence, in certain aspects, the present disclosure provides a bispecific antibody according to the present disclosure that comprises a first variable domain that can bind an extracellular part of human epidermal growth factor receptor (EGFR) and a second variable domain that can bind an extracellular part of human MET Proto-Oncogene, Receptor Tyrosine Kinase (cMET) for use in a method of treatment of a cancer, wherein the treatment further comprises administering of a third- generation EGFR tyrosine kinase inhibitor, wherein optionally the bispecific antibody of the present disclosure is administered simultaneously, sequentially or separately with the EGFR tyrosine kinase inhibitor of the present disclosure.

Hence, in certain aspects, the present disclosure provides a method of treatment of a subject having a cancer, comprising administering to the subject an effective amount of a third- generation EGFR tyrosine kinase inhibitor and the bispecific antibody according to the present disclosure that comprises a first variable domain that can bind an extracellular part of human epidermal growth factor receptor (EGFR) and a second variable domain that can bind an extracellular part of human MET Proto-Oncogene, Receptor Tyrosine Kinase (cMET), wherein optionally the bispecific antibody of the present disclosure is administered simultaneously, sequentially or separately with the EGFR tyrosine kinase inhibitor of the present disclosure.

Hence, in certain aspects, the present disclosure provides the use of a bispecific antibody according to the present disclosure that comprises a first variable domain that can bind an extracellular part of human epidermal growth factor receptor (EGFR) and a second variable domain that can bind (or binds) an extracellular part of human MET Proto-Oncogene, Receptor Tyrosine Kinase (cMET) and a third- generation EGFR tyrosine kinase inhibitor in the manufacture of a medicament for the treatment of a cancer, wherein optionally the bispecific antibody of the present disclosure is administered simultaneously, sequentially or separately with the EGFR tyrosine kinase inhibitor of the present disclosure.

EGFR is a member of a family of four receptor tyrosine kinases (RTKs), named Her- or cErbB-1, -2, -3 and -4. The EGFR has an extracellular domain (ECD) that is composed of four sub-domains, two of which are involved in ligand binding and one of which is involved in homo -dimerization and hetero-dimerization Ferguson (2008). The reference numbers used in this section refer to the numbering of the references in the list headed “cited in the specification”, which are each incorporated by reference. EGFR integrates extracellular signals from a variety of ligands to yield diverse intracellular responses (Yarden at al. 2001; and Jorrisen et al. 2003). The EGFR is implicated in several human epithelial malignancies, notably cancers of the breast, bladder, non-small cell lung cancer lung, colon, ovarian head and neck and brain. Activating mutations in the gene have been found, as well as over-expression of the receptor and of its ligands, giving rise to autocrine activation loops (for review, see Robertson et al. 2000). This RTK has therefore been extensively used as target for cancer therapy. Both small-molecule inhibitors targeting the RTK and monoclonal antibodies (mAbs) directed to the extracellular ligand-binding domains have been developed and have shown hitherto several clinical successes, albeit mostly for a select group of patients. Database accession numbers for the human EGFR protein and the gene encoding it are (GenBank NM_005228.3). Other database identifiers for the gene and/or protein are HGNC: 3236; Entrez Gene: 1956; Ensembl: ENSG00000146648; OMIM: 131550 and UniProtKB: P00533. The accession numbers are primarily given to provide a further method of identification of EGFR protein as a target, the actual sequence of the EGFR 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. Where reference herein is made to EGFR, the reference refers to human EGFR unless otherwise stated. The antigen- binding site that binds EGFR, binds EGFR and a variety of variants thereof such as those expressed on some EGFR positive tumors.

The term “EGFR ligand” as used herein refers to polypeptides which bind and activate EGFR. Examples of EGFR ligands include, but are not limited to EGF, TGF-α, HB-EGF, amphiregulin, betacellulin and epiregulin (for review Olayioye MA et al.;

EMBO J (2000) Vol 19: pp 3159-3167). The term includes biologically active fragments and/or variants of a naturally occurring polypeptide

Aberrantly activated forms of EGFR, for instance via mutations in EGFR or EGFR gene amplification, are known to be oncogenic drivers in non-small cell lung cancer (NSCLC) and known to occur in the treatment with EGFR tyrosine kinase inhibitors. The present disclosure provides a combination treatment wherein the antibody of the present disclosure is administered in combination with a third generation EGFR tyrosine kinase inhibitor to treat said oncogenic drivers of EGFR. In certain aspects, said tyrosine kinase inhibitor resistance comprises a resistance to a first, second and/or third generation tyrosine kinase inhibitor.

In certain aspects, treatment comprises treatment of a cancer which results from ligand-independent activation of EGFR and/or ligand-independent activation of cMET. In another aspect, the treatment comprises treatment of a cancer which results from ligand-dependent activation of EGFR and/or ligand- dependent activation of cMET.

In certain aspects, the cancer or subject has received prior treatment with Osimertinib and has an acquired or tertiary Osimertinib resistance. Said prior Osimertinib treatment is in certain aspects first line or second therapy, in certain aspects first line therapy followed by treatment with the combination of the present disclosure as second treatment.

In certain aspects, the cancer or subject comprises an activating EGFR mutation, an approved tyrosine kinase inhibitor resistance mutation, a tertiary tyrosine kinase inhibitor resistance mutation, a mutation that reduces binding of a third generation tyrosine kinase inhibitor to EGFR, an acquired tyrosine kinase inhibitor resistance mutation, an EGFR gene amplification, a cMET mutation, cMET aberration or increased HGF expression.

In certain aspects, the cancer or subject comprises an activating EGFR mutation, such as such as an in-frame exon 19 deletion mutation or exon 21 mutation (in certain aspects L858R). Herein, the term “activating EGFR mutation” means a mutation which develops after progression on a third- generation EGFR tyrosine kinase inhibitor. In NSCLC, the most common activating mutations are an in-frame deletion in exon 19 (de!19) and substitution of leucine for arginine in exon 21 (L858R), which together account for 85-90% of EGFR mutations in NSCLC.

In a clinical study into efficacy of a bispecific antibody of the present disclosure, clinical efficacy was observed in a variety of cancers with different genetic oncogenic backgrounds. In particular, clinical efficacy was observed in NSCLC. For instance, clinical efficacy was observed in patients with an EGFR exon 20 mutation, an EGFR exon 21 mutation, such as L858R, an EGFR exon 19 deletion mutation, a c-MET exonl4 skipping mutation, cMET amplification and EGFR amplification mutations. Thus, in a certain aspect, the cancer is NSCLC and/or the subject suffers from NSCLC which comprises an EGFR exon 21 mutation, such as L858R, an EGFR exon 19 deletion mutation, or a c-MET exonl4 skipping mutation.

In certain aspects, the cancer or subject comprises an approved tyrosine kinase inhibitor resistance mutation. Herein, the term “approved tyrosine kinase inhibitor resistance mutation” means a resistance which develops after progression on an EGFR tyrosine kinase inhibitor which is presently approved for treatment of cancer, such as T790M. Examples of approved tyrosine kinase inhibitors are Osimertinib and Almonertinib.

In certain aspects, the cancer or subject comprises a tertiary tyrosine kinase inhibitor resistance mutation, such as L718X (e.g. L718Q), G719X (e.g. G719A), L792X (e.g. L792H), G796X (e.g. G796R, G796S, G796D), C797X , C797X (e.g. C797S, C797G). Herein, the term “tertiary tyrosine kinase inhibitor resistance mutation” means a resistance which develops after progression on a third-generation EGFR tyrosine kinase inhibitor.

In certain aspects, the cancer or subject comprises a mutation that reduces binding of a third generation tyrosine kinase inhibitor to EGFR, such as L792X, L718X.

In certain aspects, the cancer or subject comprises an acquired tyrosine kinase inhibitor resistance mutation, (such as T790M, L858R, an exon 19 deletion mutation, C797X, L792X, G796X, G724X, S768X, L718X or an exon 20 insertion mutation), in certain aspects a mutation which confers resistance to Osimertinib or which occurred after progression on Osimertinib, including G724X (e.g. G724S), S768X (e.g. S768I), L792X (e.g. L792H), C797X (including C797S and C797G), L798X (e.g. L798I). Herein, the term “ acquired tyrosine kinase inhibitor resistance mutation” means a resistance which is acquired after progression on treatment with a tyrosine kinase inhibitor, such as after progression on a third- generation EGFR tyrosine kinase inhibitor.

In certain aspects, the cancer or subject comprises an EGFR gene amplification, such as an increase in EGFR mRNA or amplification of the wildtype EGFR allele in combination with the presence of an EGFR-exl9del allele after progression on Osimertinib.

In certain aspects, the cancer or subject comprises a cMET mutation, such as a cMET exon 14 skipping mutation.

In certain aspects, the cancer or subject comprises a cMET aberration, such as a cMET amplification, cMET overexpression, increased signaling of the cMET pathway, a cMET gene amplification and/or increased cMET protein activity. In certain aspects, said cancer is NSCLC and said cMET amplification is characterized by MET/CEP7 > 5 or cfDNA > 2 copies or any combination thereof. In certain aspects, the cancer comprises increased HGF expression. In certain aspects, the cMET amplification is characterized by MET/CEP7 > 3, in certain aspects MET/CEP7 > 4, in certain aspects MET/CEP7 > 5 (and up to 15 or 20 or less) or by cfDNA > 1.8 cMET copies, such as (> 1.8, < 2.2), or (> 2.2, <5), or (> 5).

In certain aspects, the cancer or subject comprises an exon 19 deletion mutation, in certain aspects an in-frame exon 19 deletion, an exon 20 missense mutation (e.g. T790M) or an exon 21 mutation, such as L858R.

In certain aspects, the cancer or subject comprises an EGFR exon 20 mutation, in certain aspects an exon 20 insertion mutation, in certain aspects an in-frame exon 20 insertion mutation.

In certain aspects, the cancer or subject comprises an exon 20 mutation selected from a near-loop insertion (positions 767-772), a far-loop insertion (positions 773-775), in certain aspects V769_D770insASV, D770_N771insSVD, H773_V774insNPH, H773_V774insH, D770_N771insG, D770delinsGY, N771_P772insN, V774_C775insHV, D770_N771insGL, H773_V774insPH, A763_Y764insFQEA, D770_N771delinsEGN, D770_N771insGD, D770_N771insH, D770_N771insP, H773_V774insAH, H773_V774insGNPH, H773delinsSNPY, N771_P772insH, N771_P772insVDN, N771delinsGY, N771delinsKH, N771delinsRD, P772_H773delinsHNPY, P772_H773insGT, P772_H773insPNP, P772_H773insT, V769_D770insA, V769_D770insGG, V769_D770insGSV, V769_D770insGW and V769_D770insMASV; or mutations T790M, L792X (e.g. L792H, C796X (e.g. G796R, G796S, G796D), C797X (e.g. C797S, C797G), L798I, or an in-frame exon 20 insertion, such as M766_A767insASV or H773-V774insNPH, Ins761(EAFQ), Ins770(ASV), Ins771(G), Ins774(NPH), M766_A7671ns A, S768_V769InsSVA, P772_H773InsNS, D761_E762InsXl-7, A763_Y764InsXl-7, Y764_Y765 InsXl-7, M766_A767InsXl-7, A767_V768 InsXl-7, S768_V769 InsXl-7) V769_D770 InsXl-7) D770_N771 InsXl-7) N771_P772 InsXl-7) P772_H773 InsXl-7, H773_V774 InsXl-7, or V774_C775 InsXl-7. In certain aspects, the cancer or subject comprises two or more of said mutations.

In certain aspects, the cancer or subject comprises an in-frame exon 19 deletion mutation, an exon 21 mutation, (in certain aspects L858R) or an in-frame deletion in exon 19 (dell9), or substitution of leucine for arginine in exon 21 (L858R), mutation L861X (e.g. L861Q) or L844X (e.g. L844V). In certain aspects, the cancer or subject comprises two or more of said mutations.

In certain aspects, the cancer or subject comprises EGFR mutation T790M.

In certain aspects, the cancer or subject comprises an EGFR mutation selected from L718X (e.g. L718Q, L718V), G719X (e.g. G719A), L792X (e.g. L792H, L792F, L792R, L792Y, L792V and L792P), G796X (e.g. G796R, G796S, G796D), C797X (e.g. C797S, C797G, C797N) , M766X (e.g. M766Q), R776X (e.g. R776C). In certain aspects, the cancer or subject comprises two or more of said mutations. In certain aspects, the cancer or subject comprises an EGFR mutation selected from T790M, L858R, an exon 19 deletion mutation, C797X, L792X, G796X, G724X, S768X, L718X, an exon 20 insertion mutation, mutation G724X (e.g. G724S), S768X (e.g. S768I), L792X (e.g. L792H), C797X (including C797S and C797G), L798X (e.g. L798I) , I941X (e.g. I941R), V948X (e.g. V948R). In certain aspects, the cancer or subject comprises two or more of said mutations.

In certain aspects, the cancer or subject comprises double mutation L858X/T790X (e.g. L858R/T790M), T790X/L798X (e.g. T790M/L798I), T790X/C797X (e.g. /T790M/C797S), G719X/R776X (e.g. G719A/R776C ) or delE746_A750/T790M.

In certain aspects, the cancer or subject comprises double mutation D770insSVD/E762X (e.g. E762K), D770insSVD/L792X (e.g. L792I, L792S), D770insSVD/P794X (e.g. P794S), or D770insSVD/G796X (e.g. G796D).

In certain aspects, the cancer or subject comprises double mutation H773insH /E762X (e.g. E762K), H773insH/L792X (e.g. L792I, L792S), H773insH/P794X (e.g. P794S), or H773insH/G796X (e.g. G796D).

In certain aspects, the cancer or subject comprises double mutation H773insNPH/E762X (e.g. E762K), H773insNPH/L792X (e.g. L792I, L792S), H773insNPH/P794X (e.g. P794S), or H773insNPH/G796X (e.g. G796D).

In certain aspects, the cancer or subject comprises double mutation L858X/L718X (e.g. L858R/cis-L718Q), L858X/C797X (e.g. L858R/cis-C797S), exonl9del/C797X (e.g. exonl9del/cis-C797S).

In certain aspects, the cancer or subject comprises triple mutation L858X/T790X/C797X (e.g. L858R/T790M/C797S), L858X/T790X/M766X (e.g. L858R/T790M/M766Q), L858X/T790X/L718X (e.g. L858R/T790M/cis-L718Q, L858R/T790M/L718Q), L858X/T790X/C797X (e.g. L858R/T790M/cis-C797S), exonl9del/T790X/C797X (e.g. exonl9del/T790M/cis-C797S), L858X/T790X/C941X (e.g. L858R/T790M/I941R), delE746_A750/T790X/C797X (e.g. delE746_A750/T790M/C797S)..

In certain aspects, the cancer or subject comprises an EGFR gene amplification, such as an increase in EGFR mRNA or amplification of the wildtype EGFR allele in combination with the presence of an EGFR-exl9del allele after progression on Osimertinib.

In certain aspects, the cancer comprises a cMET mutation, such as a cMET exon 14 skipping mutation. cMET, also called tyrosine -protein kinase MET or hepatocyte growth factor receptor (HGFR), is a protein that in humans is encoded by the MET gene. The protein possesses tyrosine kinase activity. The primary single chain precursor protein is post- translationally cleaved to produce the alpha and beta subunits, which are disulfide linked to form the mature receptor.

Dysregulation of, or aberrantly activated cMET may induce tumor growth, the formation of new blood vessels (angiogenesis) that supply the tumor with nutrients, and cancer spread to other organs (metastasis). cMET is deregulated in many types of human malignancies, including cancers of kidney, liver, stomach, breast, and brain. The cMET gene is known under a number of different names such as MET Proto-Oncogene, Receptor Tyrosine Kinase; Hepatocyte Growth Factor Receptor; Tyrosine-Protein Kinase Met; Scatter Factor Receptor; Proto-Oncogene C-Met; HGF/SF Receptor; HGF Receptor; SF Receptor; EC 2.7.10.1; Met Proto-Oncogene; EC 2.7.10; DFNB97; AUTS9; RCCP2; C- Met; MET; HGFR; External Ids for cMET are HGNC: 7029; Entrez Gene: 4233; Ensembl: ENSG00000105976; OMIM: 164860 and UniProtKB: P08581. The accession numbers are primarily given to provide a further method of identification of cMET protein as a target, the actual sequence of the cMET 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. Where reference herein is made to cMET, the reference refers to human cMET unless otherwise stated. The antigen-binding site that binds cMET, binds cMET and a variety of variants thereof such as those expressed on some cMET positive tumors. Examples of cMET aberrations or dysregulation include cMET mutations (such as an exonl4 skipping mutation), cMET amplification, cMET overexpression, increased signaling of the cMET pathway, cMET gene amplification and/or increased cMET protein activity. Also, cMET dysregulation may be caused by increased HGF expression. Dysregulation of c-MET is an established driver of tumor invasion, angiogenesis, and metastasis (Birchmeier et al., 2003). Three types of biological alterations of c-MET can lead to oncogenesis: amplification, mutation and fusion. These genomic alterations are found principally as either primary or secondary drivers of tumor growth and such aberrations have been reported to occur after treatment of cancer patients with EGFR tyrosine kinase inhibitors (cf. Suzawa et al., DOI: 10.1200/PG.19.00011 JCO Precision Oncology - May 10, Vol 3, 2019).

An antibody typically recognizes only a part of an antigen. The antigen is typically but not necessarily a protein. The recognition or binding site on an antigen, bound by an antibody is referred to as the epitope, where an epitope may be linear or conformational. Binding of an antibody to an antigen is typically specific. The ‘specificity’ of an antibody refers to its selectivity for a particular epitope, whereas ‘affinity’ refers to the strength of the interaction between the antibody’s antigen binding site and the epitope it binds.

Exemplary antibodies of the disclosure binds to EGFR and cMET, in certain aspects human EGFR and human cMET. An EGFR/cMET bispecific antibody of the disclosure binds to EGFR and, under otherwise identical conditions, at least 100-fold less to the homologous receptors ErbB-2 and ErbB-4 of the same species. An EGFR/cMET bispecific antibody of the disclosure binds to cMET and, under otherwise identical conditions, at least 100-fold less to the receptors ErbB-2 and ErbB-4 of the same species. Considering that the receptors are cell surface receptors, the binding may be assessed on cells that express the receptor(s). A bispecific antibody of the present disclosure in certain aspects binds to human, cynomolgus EGFR and/or to mouse EGFR.

An antibody that binds EGFR and cMET may bind other proteins as well if such other proteins 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 binding is typically referred to as cross-reactivity. An EGFR/cMET bispecific antibody typically does not bind to other proteins than EGFR and/or cMET on the membrane of cells in a post-natal, in certain aspects adult human. An antibody according to the present disclosure is typically capable of binding EGFR with a binding affinity (i.e. equilibrium dissociation constant Kd) of at least lxlOe-6 M, as outlined in more detail below.

The term “antibody” as used herein means a proteinaceous molecule in certain aspects belonging to the immunoglobulin class of proteins. An antibody typically contains two variable domains that bind an epitope on an antigen. Such domains are derived from or share sequence homology with the variable domain of an antibody. A bispecific antibody of the present disclosure in certain aspects comprises two variable domains. Antibodies for therapeutic use are in certain aspects 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. Typically, antibodies for therapeutic applications can have affinities of up to lxl0e-10 M or higher. Antibodies such as bispecific antibodies of the present disclosure in certain aspects comprise the constant domains (Fc part) of a natural antibody. An antibody of the present disclosure is typically a bispecific full length antibody, in certain aspects of the human IgG subclass. In certain aspects, the antibodies of the present disclosure are of the human IgGl subclass. Such antibodies of the present disclosure can have good ADCC properties, have a favorable half-life upon in vivo administration to humans and CH3 engineering technology exists that can provide for modified heavy chains that preferentially form hetero-dimers over homo-dimers upon co-expression in clonal cells. ADCC activity of an antibody can also be improved through techniques known to persons of skill in the art.

An antibody of the present disclosure is in certain aspects a “full length” antibody. The term ‘full length’ according to the disclosure 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 CHI, CH2, CH3, VH, and CL, VL. Typically, 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. Full length antibodies according to the disclosure encompasses antibodies wherein mutations may be present that provide desired characteristics. Antibodies wherein one or several amino acid residues are deleted, without essentially altering the specificity and/or affinity 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 substitutions or a combination thereof in the constant region.

In certain aspects, an antibody of the present disclosure is a bispecific IgG antibody, such as a bispecific full length IgGl antibody or a human IgGl. Full length IgG antibodies are preferred because of their typically favorable half-life and the desire to stay as close to fully autologous (human) molecules for reasons of immunogenicity. In certain aspects, an antibody of the disclosure is a full length IgGl, a full length IgG2, a full length IgG3 or a full length IgG4 antibody.

The variable domain that can bind EGFR and that comprises the amino acid sequence of the MF3370 or variant thereof as indicated herein, in certain aspects binds to EGFR domain III (see table 4 of international patent application PCT/NL2015/050124; WG2015/130172 which is incorporated by reference herein). The variable domain in certain aspects blocks the binding of the ligand EGF to EGFR or competes with the EGF ligand for binding to EGFR. The binding of the variable domain to EGFR can be inhibited by cetuximab. The variable domain binds an epitope that is different from the epitope that is recognized by cetuximab and zalutumumab. For example, the variable domain binds to mouse EGFR whereas cetuximab and zalutumumab do not, indicating that one or more of the residues that differ between mouse and human EGFR domain III play a role in cetuximab and zalutumumab binding, but not in an antibody of the present disclosure. An advantage of a bispecific antibody of the present disclosure having human, mouse, cynomolgus EGFR cross- reactivity is that it permits the use of xenograft studies with human cancer models, which may be more predictive with respect to effectivity and toxicity as the antibody also binds to the normal mouse cells that have the receptor, while also being capable of use in cynomolgus toxicology studies. In one aspect the disclosure provides a bispecific antibody that comprises a first variable domain that can bind an extracellular part of human epidermal growth factor receptor (EGFR) and a second variable domain that can bind an extracellular part of human MET Proto-Oncogene, Receptor Tyrosine Kinase (cMET), wherein said first variable domain can also bind mouse EGFR, cynomolgus EGFR or both.

A cMET variable domain in certain aspects comprises an amino acid sequence of the MF4356 or variant thereof as indicated herein, and in certain aspects blocks the binding of the antibody MetMab to cMET. The variable domain in certain aspects blocks the binding of the ligand HGF to cMET or competes with the ligand HGF for binding to cMET. The variable domain blocks the binding of the antibody MetMab to cMET when the binding of MetMab to cMET at half-maximum binding conditions is reduced by at least 40% and in certain aspects at least 60% in the presence of a saturating amount of said variable domain. The variable domain is in certain aspects provided in the context of a bivalent monospecific antibody. The cMET variable domain can in certain aspects bind the sema domain of cMET. The cMET variable domain of the disclosure may compete with 5D5 for binding cMET or not compete with reported anti-cMET reference antibodies, such as 5D5. See Table 2. A variable domain of the present disclosure can bind EGFR (the first variable domain) and in certain aspects comprises a heavy chain variable region with a CDR1 sequence SYGIS; a CDR2 sequence WISAYX1X2NTNYAQKLQG and a CDR3 comprising the sequence X3X4X5X6HWWLX7A wherein Xi = N or S; X2 = A or G; X3 = D or G; X4 = R, S or Y; X ₅ = H, L or Y; X ₆ = D or W and X ₇ = D or G.

X1-7 is in certain aspects:

Xi = N; X ₂ = G; X3 = D; X ₄ = S; X ₅ = Y; X ₆ = W and X ₇ = G;

Xi = N; X ₂ = A; X3 = D; X ₄ = S; X ₅ = Y; X ₆ = W and X ₇ = G;

Xi = S; X ₂ = G; X3 = D; X ₄ = S; X ₅ = Y; X ₆ = W and X ₇ = G;

Xi = N; X ₂ = G; X3 = D; X ₄ = R; X ₅ = H; X ₆ = W and X ₇ = D;

Xi = N; X ₂ = A; X3 = D; X ₄ = R; X ₅ = H; X ₆ = W and X ₇ = D;

Xi = S; X ₂ = G; X3 = D; X ₄ = R; X ₅ = H; X ₆ = W and X ₇ = D;

Xi = N; X ₂ = G; X3 = G; X ₄ = Y; X ₅ = L; X ₆ = D and X ₇ = G;

Xi = N; X ₂ = A; X3 = G; X ₄ = Y; X ₅ = L; X ₆ = D and X ₇ = G; or

Xi = S; X ₂ = G; X3 = G; X ₄ = Y; X ₅ = L; X ₆ = D and X ₇ = G.

In certain aspects

Xi = N; X ₂ = G; X3 = D; X ₄ = R; X ₅ = H; X ₆ = W and X ₇ = D;

Xi = N; X ₂ = A; X3 = D; X ₄ = R; X ₅ = H; X ₆ = W and X ₇ = D; or

Xi = S; X ₂ = G; X3 = D; X ₄ = R; X ₅ = H; X ₆ = W and X ₇ = D.

In certain aspects Xi = N; X2 = G; X3 = D; X4 = R; X5 = H; Xe = W and X ₇ = D.

The amino acids following the amino acid A in the sequence XBX IX.-XI I WWhXyA in the CDR3 sequence of the first variable domain can vary. The amino acid sequence following the sequence X.'XXrXl I WWI X ₇ A can be FDY. The CDR3 of the first variable domain in certain aspects comprises the sequence XBX4X5X6HWWLX ₇ AF, in certain aspects X3X4X ₅ X ₆ HWWLX ₇ AFD, in certain aspects X3X4X ₅ X ₆ HWWLX ₇ AFDY.

The first variable domain in certain aspects comprises a heavy chain variable region with a CDR1 sequence SYGIS; a CDR2 sequence WISAYNGNTNYAQKLQG and a CDR3 sequence X3X4X ₅ X ₆ HWWLX ₇ A.

The first variable domain in certain aspects comprises a heavy chain variable region with a CDR1 sequence SYGIS; a CDR2 sequence WISAYNGNTNYAQKLQG and a CDR3 comprising the sequence DRHWHWWLDA. The amino acids following the sequence LDA in the CDR3 sequence of the first variable domain can vary. The amino acid sequence following the sequence LDA can be FDY. The CDR3 of the first variable domain in certain aspects comprises the sequence DRHWHWWLDAF, in certain aspects DRHWHWWLDAFD, in certain aspects DRHWHWWLDAFDY.

The first variable domain in certain aspects comprises a heavy chain variable region with the amino acid sequence of MF3353; MF8229; MF8228; MF3370; MF8233; MF8232; MF3393; MF8227 or MF8226 as depicted in figure 2 having at most 10, in certain aspects 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 and in certain aspects having 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or a combination thereof with respect to the indicated sequence. In certain aspects the first variable domain comprises a heavy chain variable region with the amino acid sequence of MF3353; MF8229; MF8228; MF3370; MF8233; MF8232; MF3393; MF8227 or MF8226 as depicted in figure 2. In certain aspects, the first variable domain comprises a heavy chain variable region with the CDR1, CDR2, and CDR3 amino acid sequence of MF3353; MF8229; MF8228; MF3370; MF8233; MF8232; MF3393; MF8227 or MF8226 as depicted in figure 2.

The variable domain that can bind cMET (the second variable domain) in certain aspects comprises a heavy chain variable region that comprises the amino acid sequence of one of the sequences of SEQ ID NO: 1-23 (Figure 3) with 0-10, in certain aspects 0-5 amino acid insertions, deletions, substitutions, additions or a combination thereof. The heavy chain variable region of the second variable domain in certain aspects comprises the amino acid sequence of one of the sequences of SEQ ID NO: 1-3; 7; 8; 10; 13; 15; 16; 17; 21; 22 or 23 with 0-10, in certain aspects 0-5 amino acid insertions, deletions, substitutions, additions or a combination thereof. The heavy chain variable region of the second variable domain in certain aspects comprises the amino acid sequence of one of the sequences of SEQ ID NO: 2; 7; 8; 10; 13 or 23 with 0-10, in certain aspects 0-5 amino acid insertions, deletions, substitutions, additions or a combination thereof. The heavy chain variable region of the second variable domain in certain aspects comprises the amino acid sequence of the sequence of SEQ ID NO: 13 or SEQ ID NO: 23 with 0-10, in certain aspects 0-5 amino acid insertions, deletions, substitutions, additions or a combination thereof. In certain aspects, the second variable domain comprises a heavy chain variable region with the CDR1, CDR2, and CDR3 amino acid sequence of MF8225 (SEQ ID NO: 1), MF8243 (SEQ ID NO: 2), MF8224 (SEQ ID NO:3), MF8239 (SEQ ID NO: 4), MF8242 (SEQ ID NO: 5), MF8237 (SEQ ID NO: 6), MF8240 (SEQ ID NO: 7), MF8234 (SEQ ID NO: 8), MF8245 (SEQ ID NO: 9), MF8231 (SEQ ID NO: 10), MF8247 (SEQ ID NO: 11), MF8238 (SEQ ID NO: 12), MF8230 (SEQ ID NO: 13), MF8248 (SEQ ID NO: 14), MF8246 (SEQ ID NO: 15), MF8223 (SEQ ID NO: 16), MF8222 (SEQ ID NO: 17), MF8235 (SEQ ID NO: 18), MF8236 (SEQ ID NO: 19), MF8241 (SEQ ID NO: 20), MF8244 (SEQ ID NO: 21), MF8221 (SEQ ID NO: 22), or MF4356 (SEQ ID NO: 23).

In certain aspects the first variable domain comprises a heavy chain variable region with a CDR1 sequence SYGIS; a CDR2 sequence WISAYNGNTNYAQKLQG and a CDR3 comprising the sequence DRHWHWWLDA, in certain aspects DRHWHWWLDAFDY and wherein the second variable domain comprises a heavy chain variable region with a CDR1 sequence SYSMN; a CDR2 sequence WINTYTGDPTYAQGFTG and a CDR3 sequence ETYYYDRGGYPFDP. The CDR1, CDR2 and CDR3 of a light chain of the first and second variable domain in certain aspects comprises respectively the amino acid sequence CDR1 - QSISSY, CDR2 - AAS, CDR3 - QQSYSTPPT, i.e. the CDRs of IGKV1-39 (according to IMGT).

In certain aspects the first variable domain comprises a heavy chain variable region with a CDR1 sequence SYGIS; a CDR2 sequence WISAYNGNTNYAQKLQG and a CDR3 comprising the sequence DRHWHWWLDA and wherein the second variable domain comprises a heavy chain variable region with a CDR1 sequence TYSMN; a CDR2 sequence WINTYTGDPTYAQGFTG and a CDR3 comprising the sequence ETYFYDRGGYPFDP. The CDR1, CDR2 and CDR3 of a light chain of the first and second variable domain in certain aspects comprises respectively the amino acid sequence CDR1 - QSISSY, CDR2 - AAS, CDR3 - QQSYSTPPT, i.e. the CDRs of IGKV1- 39 (according to IMGT).

A bispecific antibody that comprises a first variable domain that can bind an extracellular part of EGFR and a second variable domain that can bind an extracellular part of cMET wherein the first variable domain comprises a heavy chain variable region with a CDR1 sequence SYGIS; a CDR2 sequence WISAYNANTNYAQKLQG and a CDR3 comprising the sequence DRHWHWWLDA and wherein the second variable domain comprises a heavy chain variable region with a CDR1 sequence SYSMN; a CDR2 sequence WINTYTGDPTYAQGFTG and a CDR3 sequence ETYYYDRGGYPFDP. The CDR1, CDR2 and CDR3 of a light chain of the first and second variable domain in certain aspects comprises respectively the amino acid sequence CDR1 - QSISSY, CDR2 - AAS, CDR3 - QQSYSTPPT, i.e. the CDRs of IGKV1-39 (according to IMGT).

A bispecific antibody that comprises a first variable domain that can bind an extracellular part of EGFR and a second variable domain that can bind an extracellular part of cMET wherein the first variable domain comprises a heavy chain variable region with a CDR1 sequence SYGIS; a CDR2 sequence WISAYNANTNYAQKLQG and a CDR3 comprising the sequence DRHWHWWLDA and wherein the second variable domain comprises a heavy chain variable region with a CDR1 sequence TYSMN; a CDR2 sequence WINTYTGDPTYAQGFTG and a CDR3 comprising the sequence ETYFYDRGGYPFDP. The CDR1, CDR2 and CDR3 of a light chain of the first and second variable domain in certain aspects comprises respectively the amino acid sequence CDR1 - QSISSY, CDR2 - AAS, CDR3 - QQSYSTPPT, i.e. the CDRs of IGKV1- 39 (according to IMGT).

A bispecific antibody that comprises a first variable domain that can bind an extracellular part of EGFR and a second variable domain that can bind an extracellular part of cMET wherein the first variable domain comprises a heavy chain variable region with a CDR1 sequence SYGIS; a CDR2 sequence WISAYSGNTNYAQKLQG and a CDR3 comprising the sequence DRHWHWWLDA and wherein the second variable domain comprises a heavy chain variable region with a CDR1 sequence SYSMN; a CDR2 sequence WINTYTGDPTYAQGFTG and a CDR3 sequence ETYYYDRGGYPFDP. The CDR1, CDR2 and CDR3 of a light chain of the first and second variable domain in certain aspects comprises respectively the amino acid sequence CDR1 - QSISSY, CDR2 - AAS, CDR3 - QQSYSTPPT, i.e. the CDRs of IGKV1-39 (according to IMGT).

A bispecific antibody that comprises a first variable domain that can bind an extracellular part of EGFR and a second variable domain that can bind an extracellular part of cMET wherein the first variable domain comprises a heavy chain variable region with a CDR1 sequence SYGIS; a CDR2 sequence WISAYSGNTNYAQKLQG and a CDR3 comprising the sequence DRHWHWWLDA and wherein the second variable domain comprises a heavy chain variable region with a CDR1 sequence TYSMN; a CDR2 sequence WINTYTGDPTYAQGFTG and a CDR3 comprising the sequence ETYFYDRGGYPFDP. The CDR1, CDR2 and CDR3 of a light chain of the first and second variable domain in certain aspects comprises respectively the amino acid sequence CDR1 - QSISSY, CDR2 - AAS, CDR3 - QQSYSTPPT, i.e. the CDRs of IGKV1- 39 (according to IMGT).

In certain aspects wherein a cMET binding variable domain is described to have a CDR2 sequence “WINTYTGDPTYAQGFTG” the CDR2 sequence can also be “WINTYTGDPTYAQGFT” .

The CDR1, CDR2 and CDR3 of a light chain of the first and second variable domain as described herein in certain aspects comprises respectively the amino acid sequence CDR1 - QSISSY, CDR2 - AAS, CDR3 - QQSYSTPPT, i.e. the CDRs of IGKV1- 39 (according to IMGT). In some of such embodiments, the CDR3 comprises the amino acid sequence QQSYSTP. In some embodiments of a bispecific antibody as described herein the first and second variable domain comprise a common light chain, in certain aspects a light chain variable region of figure 4B.

In another certain aspect an EGFR/cMET bispecific antibody comprises a first variable domain that can bind an extracellular part of human EGFR that comprises the CDR1, CDR2 and CDR3 of the heavy chain variable region of MF3755 depicted in figure 1 and a second variable domain that can bind an extracellular part of human cMET that comprises the CDR1, CDR2 and CDR3 of the heavy chain variable region of MF4297 depicted in figure 1. The light chain variable region in said first and second variable domain is in certain aspects a common light chain variable region as described herein. The CDR1, CDR2 and CDR3 of a light chain of the first and second variable domain in certain aspects comprises respectively the amino acid sequence CDR1 - QSISSY, CDR2 - AAS, CDR3 - QQSYSTPPT, i.e. the CDRs of IGKV1-39 (according to IMGT). In certain aspects the antibody comprises a heavy chain variable region with the amino acid sequence of MF3755 as depicted in figure 1 having at most 10, in certain aspects 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 and in certain aspects having 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or a combination thereof with respect to the indicated sequence. In certain aspects the first variable domain comprises a heavy chain variable region with the amino acid sequence of MF3755 as depicted in figure 1. The variable domain that can bind cMET (the second variable domain) in certain aspects comprises a heavy chain variable region that comprises the amino acid sequence of MF4297 as depicted in figure 1 with 0-10, in certain aspects 0-5 amino acid insertions, deletions, substitutions, additions or a combination thereof. The heavy chain variable region of the second variable domain in certain aspects comprises the amino acid sequence of MF4297 as depicted in figure 1.

The term ‘bispecific’ (bs) in the context of the present disclosure means that an antibody is capable of binding two different targets or two epitopes on the same target, for example, where one variable domain of the antibody (as defined above) binds to an epitope on EGFR and a second variable domain binds to an epitope on cMET. Depending on the expression level, (sub-)cellular localization and stoichiometry of the two antigens recognized by a bispecific antibody, both Fab arms of the antibody may or may not simultaneously bind their epitope. One arm of the bispecific antibody typically contains the variable domain of one antibody and the other arm contains the variable domain of another antibody (i.e. one arm of the bispecific antibody is formed by one heavy chain paired with one light chain whereas the other arm is formed by a different heavy chain paired with a light chain). Thus, the stoichiometry of a preferred bispecific antibody of the disclosure is 1:1, EGFR:cMET binding.

The heavy chain variable regions of the bispecific antibody of the present disclosure are typically different from each other, whereas the light chain variable regions are the same in certain aspects. A bispecific antibody wherein the different heavy chain variable regions are associated with the same light chain variable region is also referred to as a bispecific antibody with a common light chain variable region (cLcv). It is preferred that the light chain constant region is also the same. Such bispecific antibodies are referred to as having a common light chain (cLc). Further provided is therefore a bispecific antibody according to the present disclosure , wherein both arms comprise a common light chain.

The term ‘common light chain’ according to the disclosure refers to two or more light chains in a bispecific antibody which may be identical or have some amino acid sequence differences while the binding specificity of the full length antibody is not affected. It is for instance possible within the scope of the definition of common light chains as used herein, to prepare or find light chains that are not identical but still functionally equivalent, e.g., by introducing and testing conservative amino acid changes, changes of amino acids in regions that do not or only partly contribute to binding specificity when paired with the heavy chain, and the like. The terms ‘common light chain’, ‘common LC’, ‘cLC’, ‘single light chain’ with or without the addition of the term ‘rearranged’ are all used herein interchangeably. The terms ‘common light chain variable region’, ‘common VL’, ‘common LCv’, ‘cLCv’, ‘single VL’ with or without the addition of the term ‘rearranged’ are all used herein interchangeably. In certain aspects of the present disclosure, a bispecific antibody has a common light chain (variable region) that can combine with at least two, and in certain aspects a plurality of heavy chains (variable regions) of different binding specificity to form antibodies with functional antigen binding domains (e.g., WO2009/157771). The common light chain (variable region) is in certain aspects a human light chain (variable region). A common light chain (variable region) in certain aspects has a germline sequence. A preferred germline sequence is a light chain variable region that has good thermodynamic stability, yield and solubility. A preferred germline light chain is 012. A common light chain in certain aspects comprises the light chain encoded by a germline human Vk gene segment, and is in certain aspects the rearranged germline human kappa light chain IgVKl-39*01/IGJKl*01 (Figure 4A). The common light chain variable region is in certain aspects the variable region of the rearranged germline human kappa light chain IgVKl- 39*01/IGJK1*01. A common light chain in certain aspects comprises a light chain variable region as depicted in figure 4B, or 4D with 0-5 amino acid insertions, deletions, substitutions, additions or a combination thereof. The common light in certain aspects further comprises a light chain constant region, in certain aspects a kappa light chain constant region. A nucleic acid that encodes the common light chain can be codon optimized for the cell system used to express the common light chain protein. The encoding nucleic acid can deviate from a germ-line nucleic acid sequence.

In certain aspects the light chain comprises a light chain region comprising the amino acid sequence of an 012 / IgVKl-39*01 gene segment as depicted in figure 4A with 0-10, in certain aspects 0-5 amino acid insertions, deletions, substitutions, additions or a combination thereof. The phrase “012 light chain” will be used throughout the specification as short for “a light chain comprising a light chain variable region comprising the amino acid sequence of an 012 / IgVKl-39*01 gene segment as part of depicted figure 4A with 0-10, in certain aspects 0-5 amino acid insertions, deletions, substitutions, additions or a combination thereof. IgVKl-39 is short for Immunoglobulin Variable Kappa 1-39 Gene. The gene is also known as Immunoglobulin Kappa Variable 1-39; IGKV139; IGKV1-39; 012a or 012. External Ids for the gene are HGNC: 5740; Entrez Gene: 28930; Ensembl: ENSG00000242371. A preferred amino acid sequence for IgVKl-39 is given in figure 4E. This lists the sequence of the V-region. The V-region can be combined with one of five J-regions. Figure 4B and 4D describe two preferred sequences for IgVKl-39 in combination with a J-region. The joined sequences are indicated as IGKVl-39/jkl and IGKVl-39/jk5; alternative names are IgVKl- 39*01/IGJK1*01 or IgVKl-39*01/IGJK5*01 (nomenclature according to the IMGT database worldwide web at imgt.org).

It is preferred that the 012 / IgVKl-39*01 comprising light chain variable region is a germline sequence. It is further preferred that the IGJK1*01 or /IGJK5*01 comprising light chain variable region is a germline sequence. In certain aspects, the IGKVl-39/jkl or IGKVl-39/jk5 light chain variable regions are germline sequences.

In certain aspects the light chain variable region comprises a germline 012 / IgVKl-39*01. In certain aspects the light chain variable region comprises the kappa light chain IgVKl-39*01/IGJKl*01 or IgVKl-39*01/IGJK5*01. In certain aspects a IgVKl-39*01/IGJKl*01. The light chain variable region in certain aspects comprises a germline kappa light chain IgVKl-39*01/IGJKl*01 or germline kappa light chain IgVKl- 39*01/IGJK5*01, in certain aspects a germline IgVKl-39*01/IGJKl*01.

Mature B-cells that produce an antibody with an 012 light chain often produce a light chain that has undergone one or more mutations with respect to the germline sequence, i.e. the normal sequence in non-lymphoid cells of the organism. The process that is responsible for these mutations is often referred to as somatic (hyper)mutation. The resulting light chain is referred to as an affinity matured light chain. Such light chains, when derived from an 012 germline sequence are 012-derived light chains. In this specification, the phrase “common light chain” will include “common light chain derived light chains and the phrase “012 light chains” will include 012-derived light chains. The mutations that are introduced by somatic hypermutation can also be introduced artificially in the lab. In the lab also other mutations can be introduced without affecting the properties of the light chain in kind, not necessarily in amount. A light chain is at least an 012 light chain if it comprises a sequence as depicted in figure 4A, figure 4B; figure 4D or figure 4E with 0-10, in certain aspects 0-5 amino acid insertions, deletions, substitutions, additions or a combination thereof. In certain aspects the 012 light chain is a light chain comprising a sequence as depicted in figure 4A; 4b; 4d or 4e with 0-9, 0-8, 0-7, 0-6, 0-5, 0-4 amino acid insertions, deletions, substitutions, additions or a combination thereof. In certain aspects the 012 light chain is a light chain comprising a sequence as depicted in figure 4A, figure 4B; figure 4D or figure 4E with 0-5, in certain aspects 0-4, in certain aspects 0-3 amino acid insertions, deletions, substitutions, additions or a combination thereof. In certain aspects the 012 light chain is a light chain comprising a sequence as depicted in figure 4A, figure 4B; figure 4D or figure 4E with 0-2, in certain aspects 0-1, in certain aspects 0 amino acid insertions, deletions, substitutions, additions or a combination thereof. In certain aspects the 012 light chain is a light chain comprising a sequence as depicted in figure 4A or figure 4B with the mentioned amino acid insertions, deletions, substitutions, additions or a combination thereof. In certain aspects the light chain comprises the sequence of figure 4A. In certain aspects the light chain variable region comprises the sequence of figure 4B. The mentioned 1, 2, 3, 4 or 5 amino acid substitutions are in certain aspects conservative amino acid substitutions and may be present in the CDR regions of the heavy and/or light chain; the insertions, deletions, substitutions or combination thereof are in certain aspects not in the CDR3 region of the VL chain, in certain aspects not in the CDR1, CDR2 or CDR3 region or FR4 region of the VL chain.

The common light chain can have a lambda light chain and this is therefore also provided in the context of the disclosure, however a kappa light chain is preferred. The constant part of a common light chain of the disclosure can be a constant region of a kappa or a lambda light chain. It is in certain aspects a constant region of a kappa light chain, in certain aspects said common light chain is a germline light chain, in certain aspects a rearranged germline human kappa light chain comprising the IgVKl-39 gene segment, in certain aspects the rearranged germline human kappa light chain IgVKL 39*01/IGJKl*01 (Figure 4). The terms rearranged germline human kappa light chain IgVKl-39*01/IGJKl*01, IGKV1-39/IGKJ1, IIUVKI-39 light chain or in short IIUVK I-39, or simply 1-39 are used interchangeably throughout the application.

A cell that produces a common light chain can produce for instance rearranged germline human kappa light chain IgVKl-39*01/IGJKl*01 and a light chain comprising the variable region of the mentioned light chain fused to a lambda constant region.

In certain aspects the light chain variable region comprises the amino acid sequence DIQMT QSPSS LSASV GDRVT ITCRA SQSIS SYLNW YQQKP GKAPK LLIYA ASSLQ SGVPS RFSGS GSGTD FTLTI SSLQP EDFAT YYCQQ SYSTP PTFGQ GTKVE IK or DIQMT QSPSS LSASV GDRVT ITCRA SQSIS SYLNW YQQKP GKAPK LLIYA ASSLQ SGVPS RFSGS GSGTD FTLTI SSLQP EDFAT YYCQQ SYSTP PITFG QGTRL EIK with 0-10, in certain aspects 0-5 amino acid insertions, deletions, substitutions, additions or a combination thereof. In certain aspects the light chain variable region comprises 0-9, 0-8, 0-7, 0-6, 0-5, 0-4, in certain aspects 0-3, in certain aspects 0-2, in certain aspects 0- 1 and in certain aspects 0 amino acid insertions, deletions, substitutions, additions with respect to the indicated amino acid sequence, or a combination thereof. A combination of an insertion, deletion, addition or substitution is a combination as claimed if aligned sequences do not differ at more than 5 positions. In certain aspects the light chain variable region comprises the amino acid sequence DIQMT QSPSS LSASV GDRVT ITCRA SQSIS SYLNW YQQKP GKAPK LLIYA ASSLQ SGVPS RFSGS GSGTD FTLTI SSLQP EDFAT YYCQQ SYSTP PTFGQ GTKVE IK or DIQMT QSPSS LSASV GDRVT ITCRA SQSIS SYLNW YQQKP GKAPK LLIYA ASSLQ SGVPS RFSGS GSGTD FTLTI SSLQP EDFAT YYCQQ SYSTP PITFG QGTRL EIK. In certain aspects the light chain variable region comprises the amino acid sequence DIQMT QSPSS LSASV GDRVT ITCRA SQSIS SYLNW YQQKP GKAPK LLIYA ASSLQ SGVPS RFSGS GSGTD FTLTI SSLQP EDFAT YYCQQ SYSTP PTFGQ GTKVE IK. In certain aspects, the light chain variable region comprises the amino acid sequence DIQMT QSPSS LSASV GDRVT ITCRA SQSIS SYLNW YQQKP GKAPK LLIYA ASSLQ SGVPS RFSGS GSGTD FTLTI SSLQP EDFAT YYCQQ SYSTP PITFG QGTRL EIK.

The amino acid insertions, deletions, substitutions, additions or combination thereof are in certain aspects not in the CDR3 region of the light chain variable region, in certain aspects not in the CDR1 or CDR2 region of the light chain variable region. In certain aspects the light chain variable region does not comprise a deletion, addition or insertion with respect to the sequence indicated. In this aspect the heavy chain variable region can have 0-5 amino acid substitutions with respect to the indicated amino acid sequence. An amino acid substitution is in certain aspects a conservative amino acid substitution. The CDR1, CDR2 and CDR3 of a light chain of an antibody of the disclosure in certain aspects comprises respectively the amino acid sequence CDR1 - QSISSY, CDR2 - AAS, CDR3 - QQSYSTPPT, i.e. the CDRs of IGKV1-39 (according to IMGT).

In certain aspects, bispecific antibodies as described herein have one heavy chain variable region/light chain variable region (VI 1/VI d combination that binds an extracellular part of EGFR and a second VH/VL combination that binds an extracellular of cMET. In certain aspects the VL in said first VH/VL combination is similar to the VL in said second VH/VL combination. In a particular aspect, the VLs in the first and second VH/VL combinations are identical. In certain aspects, the bispecific antibody is a full length antibody which has one heavy/light (H/L) chain combination that binds an extracellular part of EGFR and one H/L chain combination that binds an extracellular part of cMET. In certain aspects the light chain in said first H/L chain combination is similar to the light chain in said second H/L chain combination. In a particular aspect, the light chains in the first and second H/L chain combinations are identical.

Several methods have been published to produce a host cell whose expression favors the production of the bispecific antibody or vice versa, the monospecific antibodies. In the present disclosure it is preferred that the cellular expression of the antibody molecules is favored toward the production of the bispecific antibody over the production of the respective monospecific antibodies. Such is typically achieved by modifying the constant region of the heavy chains such that they favor heterodimerization (i.e. dimerization with the heavy chain of the other heavy/light chain combination) over homodimerization. In certain aspects the bispecific antibody of the present disclosure comprises two different immunoglobulin heavy chains with compatible heterodimerization domains. Various compatible heterodimerization domains have been described in the art. The compatible heterodimerization domains are in certain aspects compatible immunoglobulin heavy chain CH3 heterodimerization domains. When wildtype CH3 domains are used, co-expression of two different heavy chains (A and B) and a common light chain will result in three different antibody species, AA, AB and BB. AA and BB are designations for the two mono-specific, bivalent antibodies, and AB is a designation for the bispecific antibody. 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 hetero-dimerization domains, as defined hereunder. The art describes various ways in which such hetero-dimerization of heavy chains can be achieved. One way is to generate 'knob into hole' bispecific antibodies.

The term ‘compatible hetero-dimerization 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, homo -dimerization between A’- A and B’-B’ is diminished.

In US 13/866, 747 (now issued as US 9,248,181), US 14/081,848 (now issued as US 9,358,286) and PCT/NL2013/050294 (published as WO2013/157954; incorporated herein by reference) methods and means are disclosed for producing bispecific antibodies using compatible heterodimerization domains. These means and methods can also be favorably employed in the present disclosure. Specifically, a bispecific antibody of the present disclosure in certain aspects comprises mutations to produce substantial expression of bispecific full length IgG molecules in host cells. Preferred mutations are the amino acid substitutions L351K and T366K in the first CH3 domain (the ‘KK- variant’ heavy chain) and the amino acid substitutions L351D and L368E in the second domain (the ‘DE-variant’ heavy chain), or vice versa. US 9,248,181 and US 9,358,286 patents as well as the WO2013/157954 PCT application (which are incorporated by reference herein) demonstrate that the DE-variant and KK-variant preferentially pair to form heterodimers (so-called ‘DEKK’ bispecific molecules). Homodimerization of DE- variant heavy chains (DEDE homodimers) are disfavored due to repulsion between the charged residues in the CH3-CH3 interface between identical heavy chains.

Bispecific antibodies can be generated by (transient) transfection of plasmids encoding a light chain and two different heavy chains that are CH3 engineered to ensure efficient hetero-dimerization and formation of the bispecific antibodies. The production of these chains in a single cell leads to the favored formation of bispecific antibodies over the formation of monospecific antibodies. Preferred mutations to produce essentially only bispecific full length IgGl molecules are amino acid substitutions at positions 351 and 366, e.g. L351K and T366K (numbering according to EU numbering) in the first CH3 domain (the 'KK-variant' heavy chain) and amino acid substitutions at positions 351 and 368, e.g. L351D and L368E in the second CH3 domain (the 'DE- variant' heavy chain), or vice versa (see for instance figures 5E and 5F).

In one aspect the heavy chain/light chain combination that comprises the variable domain that binds EGFR, comprises a DE variant of the heavy chain. In this aspect the heavy chain/light chain combination that comprises the variable domain that can bind to cMET comprises a KK variant of the heavy chain. The KK variant of the heavy chain that binds cMET do not produce homodimers thereby rendering the observed effect of HGF induced cMET activation inhibition by the bispecific antibody very precise. It avoids activation of cMET sometimes observed with bivalent cMET antibodies (agonism).

The Fc region mediates effector functions of an antibody, such as complement- dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cell phagocytosis (ADCP). Depending on the therapeutic antibody or Fc fusion protein application, it may be desired to either reduce or increase the effector function. Reduced effector function can be desired when an immune response is to be activated, enhanced or stimulated as in some of the aspects of the present disclosure. Antibodies with reduced effector functions can be used to target cell-surface molecules of immune cells, among others. In certain aspects, the antibody of the present disclosure promotes antibody-dependent cellular phagocytosis (ADCP). In certain aspects, the antibody of the present disclosure promotes antibody- dependent cellular cytotoxicity (ADCC). One advantage of the present disclosure is that in certain aspects, bispecific antibodies of the present disclosure show more potent ADCC activity than amivantamab, especially for cells or a cancer comprising a cMET aberration.

Antibodies with reduced effector functions are in certain aspects IgG antibodies comprising a modified CH2/lower hinge region, for instance to reduce Fc-receptor interaction or to reduce Clq binding. In some aspects the antibody of the disclosure is an IgG antibody with a mutant CH2 and/or lower hinge domain such that interaction of the bispecific IgG antibody to a Fc-gamma receptor is reduced. An antibody comprising a mutant CH2 region is in certain aspects an IgGl antibody. Such a mutant IgGl CH2 and/or lower hinge domain in certain aspects comprise an amino substitution at position 235 and/or 236 (EU-numbering), in certain aspects an L235G and/or G236R substitution (Figure 5D).

An antibody of the present disclosure in certain aspects has effector function. A bispecific antibody as disclosed herein in certain aspects comprises antibody- dependent cell-mediated cytotoxicity (ADCC). The antibody can be engineered to enhance the ADCC activity (for review, see Cancer Sci. 2009 Sep; 100(9): 1566-72. Engineered therapeutic antibodies with improved effector functions. Kubota T, Niwa R, Satoh M, Akinaga S, Shitara K, Hanai N). 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 co-incubated with the antibody-labeled target cells. Target cell lysis is subsequently measured by release of intracellular label by a scintillation counter or spectrophotometry. In one aspect a bispecific antibody of the present disclosure exhibits ADCC activity. In such aspect the bispecific antibody can have improved ADCC activity. In such aspect the antibody can have altered ADCC activity by means of one or more CH2 mutations as described elsewhere herein and by techniques known to in the art. 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 according to the disclosure, which is afucosylated. In certain aspects, the antibody of the present disclosure comprises two afucosylated CH2 domains. In certain aspects, the antibody of the present disclosure comprises a total of two CH2 domains, both of which are afucosylated. In certain aspects, the antibody of the present disclosure is a full-length antibody, such as of the IgG-type, having two CH2 domains, both of which are 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, all of which seek to improve Fc binding to low-affinity activating FcyRHIa, and/or to reduce binding to the low affinity inhibitory FcyRHb. A bispecific antibody of the present disclosure is in certain aspects afucosylated in order to enhance ADCC activity. A bispecific antibody of the present disclosure herein in certain aspects 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 variant of an antibody or bispecific antibody as described herein comprises a functional part, derivative and/or analogue of the antibody or bispecific antibody. The variant maintains the binding specificity of the (bispecific) antibody. The functional part, derivative and/or analogue maintains the binding specificity of the (bispecific) antibody. Binding specificity is defined by capacity to bind an extracellular part of a first membrane protein and a second membrane protein as described herein.

A bispecific antibody of the present disclosure is in certain aspects used in humans. A preferred antibody of the disclosure is a humanized or in certain aspects human antibody. The constant region of a bispecific antibody of the present disclosure is in certain aspects a human constant region. The constant region may contain one or more, in certain aspects not more than 10, in certain aspects 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 humanized 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 regions. In such aspects, the VH of a variable domain of an antibody that binds EGFR or cMET of the present disclosure may contain one or more, in certain aspects not more than 10, in certain aspects 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. The light chain variable region of an EGFR binding domain and/or a cMET binding domain in an antibody of the disclosure may contain one or more, in certain aspects not more than 10, in certain aspects 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. The light chain in an antibody of the present disclosure may contain one or more, in certain aspects not more than 10, in certain aspects 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 also occur 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, in certain aspects 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.

Deimmunized murine heavy chain variable regions are less immunogenic in humans than the original murine heavy chain variable region. In certain aspects a variable region or domain of the present disclosure 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 used in the present disclosure is in certain aspects a mammal, in certain aspects a primate, in certain aspects a human.

A bispecific antibody according to the present disclosure in certain aspects 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. A certain aspect comprises an antibody wherein said constant region is selected from the group of IgG, IgA, IgM, IgD, and IgE constant regions, in certain aspects said constant region comprises an IgG constant region, i.e. selected from the group consisting of IgGl, IgG2, IgG3 and IgG4. In certain aspects, said constant region is an IgGl or IgG4 constant region, in certain aspects a mutated IgGl constant region. Some variation in the constant region of IgGl occurs in nature and/or is allowed without changing the immunological properties of the resulting antibody. Variation can also be introduced artificially to install certain preferred features on the antibody or parts thereof. Such features are for instance described herein in the context of CH2 and CH3. Typically between about 1-10 amino acid insertions, deletions, substitutions or a combination thereof are allowed in the constant region. A VH chain of Figure 1, 2 or 3 in certain aspects has at most 15, in certain aspects 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid insertions, deletions, substitutions or a combination thereof with respect to the VH chain depicted in Figures 1, 2 or 3, in certain aspects has 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or a combination thereof with respect to the VH chain depicted in Figures 1, 2 or 3, in certain aspects 0, 1, 2, 3 or 4 insertions, deletions, substitutions or a combination thereof, in certain aspects 0, 1, 2 or 3 insertions, deletions, substitutions or a combination thereof, more in certain aspects 0; 1 or 2 insertions, deletions, substitutions or a combination thereof, and in certain aspects 0 or 1 insertion, deletion, substitution or a combination thereof with respect to the VH chain depicted in Figures 1, 2 or 3. The one or more amino acid insertions, deletions, substitutions or a combination thereof are in certain aspects not in the CDR1, CDR2 and/or CDR3 region of the VH chain. They are also in certain aspects not present in the FR4 region. An amino acid substitution is in certain aspects a conservative amino acid substitution.

Rational methods have evolved toward minimizing the content of non-human residues in the human context. Various methods are available to successfully graft the antigen-binding property of an antibody onto another antibody. The binding properties of antibodies may rest predominantly in the exact sequence of the CDR3 region, often supported by the sequence of the CDR1 and CDR2 regions in the variable domain combined with the appropriate structure of the variable domain as a whole.

CDR sequences can be defined using different methods, including, but not limited to, according to the Kabat numbering scheme (Kabat et al., J. Biol. Chem.252:6609-6616 (1977); and/or Kabat et al., U.S. Dept, of Health and Human Services, “Sequences of proteins of immunological interest” (1991)), the Chothia numbering scheme (Chothia et al., J. Mol. Biol.196:901-917 (1987); Chothia et al., Nature 342: 877-883, 1989; and/or Al- Lazikani B. et al., J. Mol. Biol., 273: 927-948 (1997)), the numbering system of Honegger and Plukthun (Honegger and Pluckthun, J. Mol. Biol., 309:657-670 (2001)), the numbering system of MacCallum (MacCallum et al., J. Mol. Biol.262:732-745 (1996); and/or Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008)), the numbering system of Lefranc (Lefranc M.P. et al., Dev. Comp. Immunol., 27: 55-77 (2003); and/or Honegger and Pluckthun, J. Mol. Biol., 309:657-670 (2001)), or according to IMGT (discussed in Giudicelli et al., Nucleic Acids Res. 25: 206-21 1 (1997)).

Each of these numbering schemes base their definition of CDRs on a predicted contribution of amino acid residues in the heavy or light chain variable region to antigen binding. Hence, each method to identify CDRs can be used to identify the CDRs of the binding domains of the present disclosure. In certain aspects, the heavy chain CDRs of a binding domain of the present disclosure is according to Kabat, Chothia, or IMGT. In certain aspects, the heavy chain CDRs of a binding domain of the present disclosure is according to Kabat. In certain aspects, the heavy chain CDRs of a binding domain of the present disclosure is according to Chothia. In certain aspects, the heavy chain CDRs of a binding domain of the present disclosure is according to IMGT. In certain aspects, the light chain CDRs of a binding domain of the present disclosure is according to Kabat. In certain aspects, the light chain CDRs of a binding domain of the present disclosure is according to Chothia. In certain aspects, the light chain CDRs of a binding domain of the present disclosure is according to IMGT. The amino acid sequence of a heavy chain CDR region as depicted herein determined with the Kabat definition.

Various methods are presently available to graft CDR regions onto a suitable variable domain of another antibody. Some of these methods are reviewed in J.C. Almagrol and J. Fransson (2008) Frontiers in Bioscience 13, 1619-1633, which is included by reference herein. The disclosure therefore further provides a humanized or in certain aspects human bispecific antibody as included in the treatment of the present disclosure comprising a first antigen-binding site that binds EGFR and a second antigen-binding site that binds cMET, wherein the variable domain comprising the EGFR binding site comprises a VH CDR3 sequence as depicted for MF3370 in Figure 1, and wherein the variable domain comprising the cMET binding site comprises a VH CDR3 region as depicted for MF4356 in Figure 1. The VH variable region comprising the EGFR binding site in certain aspects comprises the sequence of the CDR1 region, CDR2 region and the CDR3 region of a VH chain as depicted for MF3370 in Figure 1. The VH variable region comprising the cMET binding site in certain aspects comprises the sequence of the CDR1 region, CDR2 region and the CDR3 region of a VH chain as depicted for MF4356 in Figure 1. CDR grafting may also be used to produce a VH chain with the CDR regions of a VH of Figure 1, but having a different framework. The different framework may be of another human VH, or of a different mammal. The disclosure therefore further provides a humanized or in certain aspects human bispecific antibody comprising a first antigen-binding site that binds EGFR and a second antigen- binding site that binds cMET, wherein the variable domain comprising the EGFR binding site comprises a VH CDR3 sequence as depicted for MF8233 in Figure 2, and wherein the variable domain comprising the cMET binding site comprises a VH CDR3 region as depicted for MF8230 in Figure 3. The VH variable region comprising the EGFR binding site in certain aspects comprises the sequence of the CDR1 region, CDR2 region and the CDR3 region of a VH chain as depicted for MF8233 in Figure 2. The VH variable region comprising the cMET binding site in certain aspects comprises the sequence of the CDR1 region, CDR2 region and the CDR3 region of a VH chain as depicted for MF8230 in Figure 3. CDR grafting may also be used to produce a VH chain with the CDR regions of a VH of Figure 2 or Figure 3, but having a different framework. The different framework may be of another human VH, or of a different mammal.

The disclosure therefore further provides a humanized or in certain aspects human bispecific antibody as included in the treatment of the present disclosure comprising a first antigen-binding site that binds EGFR and a second antigen-binding site that binds cMET, wherein the variable domain comprising the EGFR binding site comprises a VH CDR3 sequence as depicted for MF3370 in Figure 1, and wherein the variable domain comprising the cMET binding site comprises a VH CDR3 region as depicted for MF8230 in Figure 3. The VH variable region comprising the EGFR binding site in certain aspects comprises the sequence of the CDR1 region, CDR2 region and the CDR3 region of a VH chain as depicted for MF3370 in Figure 1. The VH variable region comprising the cMET binding site in certain aspects comprises the sequence of the CDR1 region, CDR2 region and the CDR3 region of a VH chain as depicted for MF8230 in Figure 3. CDR grafting may also be used to produce a VH chain with the CDR regions of a VH of Figure 2 or Figure 3, but having a different framework. The different framework may be of another human VH, or of a different mammal.

The disclosure therefore further provides a humanized or in certain aspects human bispecific antibody as included in the treatment of the present disclosure comprising a first antigen-binding site that binds EGFR and a second antigen-binding site that binds cMET, wherein the variable domain comprising the EGFR binding site comprises a VH CDR3 sequence as depicted for MF8233 in Figure 2, and wherein the variable domain comprising the cMET binding site comprises a VH CDR3 region as depicted for MF4356 in Figure 3. The VH variable region comprising the EGFR binding site in certain aspects comprises the sequence of the CDR1 region, CDR2 region and the CDR3 region of a VH chain as depicted for MF8233 in Figure 2. The VH variable region comprising the cMET binding site in certain aspects comprises the sequence of the CDR1 region, CDR2 region and the CDR3 region of a VH chain as depicted for MF4356 in Figure 3. CDR grafting may also be used to produce a VH chain with the CDR regions of a VH of Figure 2 or Figure 3, but having a different framework. The different framework may be of another human VH, or of a different mammal.

The disclosure therefore further provides a humanized or in certain aspects human bispecific antibody as included in the treatment of the present disclosure comprising a first antigen-binding site that binds EGFR and a second antigen-binding site that binds cMET, wherein the variable domain comprising the EGFR binding site comprises a VH CDR3 sequence as depicted for MF8232 in Figure 2, and wherein the variable domain comprising the cMET binding site comprises a VH CDR3 region as depicted for MF8230 in Figure 3. The VH variable region comprising the EGFR binding site in certain aspects comprises the sequence of the CDR1 region, CDR2 region and the CDR3 region of a VH chain as depicted for MF8232 in Figure 2. The VH variable region comprising the cMET binding site in certain aspects comprises the sequence of the CDR1 region, CDR2 region and the CDR3 region of a VH chain as depicted for MF8230 in Figure 3. CDR grafting may also be used to produce a VH chain with the CDR regions of a VH of Figure 2 or Figure 3, but having a different framework. The different framework may be of another human VH, or of a different mammal.

The disclosure therefore further provides a humanized or in certain aspects human bispecific antibody as included in the treatment of the present disclosure comprising a first antigen-binding site that binds EGFR and a second antigen-binding site that binds cMET, wherein the variable domain comprising the EGFR binding site comprises a VH CDR3 sequence as depicted for MF8232 in Figure 2, and wherein the variable domain comprising the cMET binding site comprises a VH CDR3 region as depicted for MF4356 in Figure 3. The VH variable region comprising the EGFR binding site in certain aspects comprises the sequence of the CDR1 region, CDR2 region and the CDR3 region of a VH chain as depicted for MF8232 in Figure 2. The VH variable region comprising the cMET binding site in certain aspects comprises the sequence of the CDR1 region, CDR2 region and the CDR3 region of a VH chain as depicted for MF4356 in Figure 3. CDR grafting may also be used to produce a VH chain with the CDR regions of a VH of Figure 2 or Figure 3, but having a different framework. The different framework may be of another human VH, or of a different mammal.

Methods for generating sequence variants are well known in the art. One can take a random approach in generating sequence variants or a targeted approach, where one can for instance aim at introducing variations that are likely to increase or decrease binding affinity. Routine methods for affinity maturing antibody binding domains are widely known in the art, see for instance Tabasinezhad M. et al. Immunol Lett. 2019;212:106-113. One can also aim at introducing variations that mitigate develop ability risks with a view on producing a binding domain, or moiety comprising such binding domain, at large scale. Variations may be introduced that are likely not to cause a loss in binding specificity and/or affect binding affinity. Whether amino acid residues within the CDRs and/or framework regions can be substituted, for instance with a conservative amino acid residue, and without, or substantially without, loss in binding specificity and/or affinity, can be determined by methods well known in the art. Experimental examples include, but are not limited to, for instance, alanine scanning (Cunningham BC, Wells JA. Science. 1989;244(4908): 1081-5), and deep mutational scanning (Araya CL, Fowler DM. Trends Biotechnol. 2011;29(9):435-42). Computational methods have also been developed that can predict the effect of amino acid variation, such as for instance described in Sruthi CK, Prakash M. PLoS One. 2020;15(l):e0227621, Choi Y. et al. PLoS One. 2012;7(10):e46688, and Munro D, Singh M. Bioinformatics. 2020;36(22-23):5322-9.

Further provided herein are any variant anti-human EGFR and c-MET binding domains produced by the above described method; binding moieties, such as antibodies, comprising any of said variant binding domains; a pharmaceutical composition comprising any of said variant anti-human EGFR and c-MET binding domains or binding moieties; nucleic acids encoding any of said variant binding domains; vectors and cells comprising said nucleic acids; and use of said variant binding domains or pharmaceutical composition for the treatment of cancer.

The disclosure further provides a humanized or in certain aspects human bispecific antibody as included in the treatment of the present disclosure comprising a first variable domain that binds EGFR and a second variable domain that binds cMET wherein the first variable domain comprises a heavy chain variable region with the amino acid sequence of MF3370 as depicted in figure 2 having at most 10, in certain aspects 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 and in certain aspects having 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or a combination thereof and wherein the second variable domain comprises a heavy chain variable region that comprises the amino acid sequence of MF4356 depicted in figure 3 (SEQ ID NO: 23) with 0-10, in certain aspects 0-5 amino acid insertions, deletions, substitutions, additions or a combination thereof. The disclosure further provides a humanized or in certain aspects human bispecific antibody comprising a first variable domain that binds EGFR and a second variable domain that binds cMET wherein the first variable domain comprises a heavy chain variable region with the amino acid sequence of MF8233 as depicted in figure 2 having at most 10, in certain aspects 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 and in certain aspects having 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or a combination thereof and wherein the second variable domain comprises a heavy chain variable region that comprises the amino acid sequence of MF8230 depicted in figure 3 (SEQ ID NO: 13) with 0-10, in certain aspects 0-5 amino acid insertions, deletions, substitutions, additions or a combination thereof.

The disclosure further provides a humanized or in certain aspects human bispecific antibody as included in the treatment of the present disclosure comprising a first variable domain that binds EGFR and a second variable domain that binds cMET wherein the first variable domain comprises a heavy chain variable region with the amino acid sequence of MF3370 as depicted in figure 2 having at most 10, in certain aspects 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 and in certain aspects having 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or a combination thereof and wherein the second variable domain comprises a heavy chain variable region that comprises the amino acid sequence of MF8230 depicted in figure 3 (SEQ ID NO: 13) with 0-10, in certain aspects 0-5 amino acid insertions, deletions, substitutions, additions or a combination thereof.

The disclosure further provides a humanized or in certain aspects human bispecific antibody as included in the treatment of the present disclosure comprising a first variable domain that binds EGFR and a second variable domain that binds cMET wherein the first variable domain comprises a heavy chain variable region with the amino acid sequence of MF8233 as depicted in figure 2 having at most 10, in certain aspects 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 and in certain aspects having 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or a combination thereof and wherein the second variable domain comprises a heavy chain variable region that comprises the amino acid sequence of MF4356 depicted in figure 3 (SEQ ID NO: 23) with 0-10, in certain aspects 0-5 amino acid insertions, deletions, substitutions, additions or a combination thereof.

The disclosure further provides a humanized or in certain aspects human bispecific antibody as included in the treatment of the present disclosure comprising a first variable domain that binds EGFR and a second variable domain that binds cMET wherein the first variable domain comprises a heavy chain variable region with the amino acid sequence of MF8232 as depicted in figure 2 having at most 10, in certain aspects 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 and in certain aspects having 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or a combination thereof and wherein the second variable domain comprises a heavy chain variable region that comprises the amino acid sequence of MF4356 depicted in figure 3 (SEQ ID NO: 23) with 0-10, in certain aspects 0-5 amino acid insertions, deletions, substitutions, additions or a combination thereof.

The disclosure further provides a humanized or in certain aspects human bispecific antibody as included in the treatment of the present disclosure comprising a first variable domain that binds EGFR and a second variable domain that binds cMET wherein the first variable domain comprises a heavy chain variable region with the amino acid sequence of MF8232 as depicted in figure 2 having at most 10, in certain aspects 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 and in certain aspects having 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or a combination thereof and wherein the second variable domain comprises a heavy chain variable region that comprises the amino acid sequence of MF8230 depicted in figure 3 (SEQ ID NO: 13) with 0-10, in certain aspects 0-5 amino acid insertions, deletions, substitutions, additions or a combination thereof.

The mentioned at most 15, in certain aspects 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 and in certain aspects 0, 1, 2, 3, 4 or 5 amino acid substitutions are in certain aspects conservative amino acid substitutions, the insertions, deletions, substitutions or a combination thereof are in certain aspects not in the CDR3 region of the VH chain, in certain aspects not in the CDR1, CDR2 or CDR3 region of the VH chain and in certain aspects not in the FR4 region.

Various methods are available to produce bispecific antibodies. One method involves the expression of two different heavy chains and two different light chains in a cell and collecting antibody that is produced by the cell. Antibody produced in this way will typically contain a collection of antibodies with different combinations of heavy and light chains, some of which are the desired bispecific antibody. The bispecific antibody can subsequently be purified from the collection. The ratio of bispecific to other antibodies that are produced by the cell can be increased in various ways. In certain aspects, the ratio is increased by expressing not two different light chains but a common light chain in the cell. When a common light chain is expressed with the two different heavy chains, the ratio of bispecific antibody to other antibody that is produced by the cell is significantly improved over the expression of two different light chains. The ratio of bispecific antibody that is produced by the cell can be further improved by stimulating the pairing of two different heavy chains with each other over the pairing of two identical heavy chains. Methods and means are disclosed for producing bispecific antibodies (from a single cell), whereby means are provided that favor the formation of bispecific antibodies over the formation of monospecific antibodies. These methods can also be favorably employed in the present disclosure. Thus the disclosure in one aspect provides a method for producing a bispecific antibody from a single cell, wherein said bispecific antibody comprises two CH3 domains that are capable of forming an interface, said method comprising providing in said cell a) a first nucleic acid molecule encoding a 1st CH3 domain comprising heavy chain, b) a second nucleic acid molecule encoding a 2nd CH3 domain comprising heavy chain, wherein said nucleic acid molecules are provided with means for preferential pairing of said 1st and 2nd CH3 domain comprising heavy chains, said method further comprising the step of culturing said host cell and allowing for expression of said two nucleic acid molecules and harvesting said bispecific antibody from the culture. Said first and second nucleic acid molecules may be part of the same nucleic acid molecule, vector or gene delivery vehicle and may be integrated at the same site of the host cell’s genome. Alternatively, said first and second nucleic acid molecules are separately provided to said cell. A certain aspect provides a method for producing a bispecific antibody according to the present disclosure from a single cell, wherein said bispecific antibody comprises two CH3 domains that are capable of forming an interface, said method comprising providing:

- a cell having a) a first nucleic acid molecule encoding a heavy chain comprising an antigen binding site that binds EGFR and that contains a 1st CH3 domain, and b) a second nucleic acid molecule encoding a heavy chain comprising an antigen-binding site that binds ErbB-3 and that contains a 2nd CH3 domain, wherein said nucleic acid molecules are provided with means for preferential pairing of said 1st and 2nd CH3 domains, said method further comprising the step of culturing said cell and allowing for expression of the proteins encoded by said two nucleic acid molecules and harvesting said bispecific IgG antibody from the culture. In a certain aspect, said cell also has a third nucleic acid molecule encoding a common light chain. Said first, second and third nucleic acid molecule may be part of the same nucleic acid molecule, vector or gene delivery vehicle and may be integrated at the same site of the host cell’s genome. Alternatively, said first, second and third nucleic acid molecules are separately provided to said cell. A preferred common light chain is based on 012, in certain aspects it is the rearranged germline human kappa light chain IgVkl 39*01/IGJK1*01, as described above. Means for preferential pairing of said 1st and said 2nd CH3 domain are in certain aspects the corresponding mutations in the CH3 domain of the heavy chain coding regions. The preferred mutations to preferentially produce bispecific antibodies are the amino acid substitutions L351K and T366K (EU-numbering) in the first CH3 domain and the amino acid substitutions L351D and L368E in the second CH3 domain, or vice versa. Further provided is therefore a method according to the disclosure for producing a bispecific antibody, wherein said first CH3 domain comprises the amino acid substitutions L351K and T366K (EU-numbering) and wherein said second CH3 domain comprises the amino acid substitutions L351D and L368E, said method further comprising the step of culturing said cell and allowing for expression of proteins encoded by said nucleic acid molecules and harvesting said bispecific antibody from the culture. Also provided is a method according to the disclosure for producing a bispecific antibody, wherein said first CH3 domain comprises the amino acid substitutions L351D and L368E (EU-numbering) and wherein said second CH3 domain comprises the amino acid substitutions L351K and T366K, said method further comprising the step of culturing said cell and allowing for expression of said nucleic acid molecules and harvesting said bispecific antibody from the culture. Antibodies that can be produced by these methods are also part of the present disclosure. The CH3 hetero- dimerization domains are in certain aspects IgGl hetero-dimerization domains. The heavy chain constant regions comprising the CH3 hetero-dimerization domains are in certain aspects IgGl constant regions.

One aspect of the disclosure includes a nucleic acid molecule encoding an antibody heavy chain variable region. The nucleic acid molecule (typically an in vitro, isolated or recombinant nucleic acid molecule) in certain aspects encodes a heavy chain variable region as depicted in Figure 2 or Figure 3, or a heavy chain variable region as depicted in Figure 2 or Figure 3 having 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or combination thereof. In certain aspects the nucleic acid molecule comprises codon optimized nucleic acid sequence coding for an amino acid sequence as depicted in Figure 2 or Figure 3. The codon optimization is optimized for the species and/or the cell type of the antibody producing cell. For example, for CHO production the nucleic acid sequence of the molecule is codon optimized for Chinese hamster cells. The disclosure further provides a nucleic acid molecule encoding a heavy chain of Figure 2 or Figure 3.

A nucleic acid molecule as used in the disclosure is typically but not exclusively a ribonucleic acid (RNA) or a deoxyribonucleic acid (DNA). Alternative nucleic acids are available for a person skilled in the art. A nucleic acid according to the disclosure is for instance comprised in a cell. When said nucleic acid is expressed in said cell, said cell can produce an antibody according to the disclosure. Therefore, in one aspect of the disclosure includes a cell comprising an antibody according to the disclosure and/or a nucleic acid according to the disclosure. Said cell is in certain aspects an animal cell, more in certain aspects a mammal cell, in certain aspects a primate cell, in certain aspects a human cell. A suitable cell is any cell capable of comprising and in certain aspects producing an antibody according to the disclosure and/or a nucleic acid according to the disclosure.

The disclosure further provides a cell comprising an antibody according to the disclosure. In certain aspects said cell (typically an in vitro, isolated or recombinant cell) produces said antibody. Said cell can also be a stored cell that is able to produce said antibody when taken out of storage and cultured. In certain aspects said cell is a hybridoma cell, a Chinese hamster ovary (CHO) cell, an NSO cell or a PER-C6™ cell. In a particular aspect said cell is a CHO cell. Further provided is a cell culture comprising a cell according to the disclosure. Various institutions and companies have developed cell lines for the large scale production of antibodies, for instance for clinical use. Non- limiting examples of such cell lines are CHO cells, NSO cells or PER.C6™ cells. These cells are also used for other purposes such as the production of proteins. Cell lines developed for industrial scale production of proteins and antibodies are herein further referred to as industrial cell lines. Thus a certain aspect includes use of a cell line developed for the large scale production of antibody for the production of an antibody of the disclosure, including in certain aspects a cell for producing an antibody comprising a nucleic acid molecule that codes for a VH, a VL, and/or a heavy chain as depicted in Figure 2 or Figure 3.

The disclosure further provides a method for producing an antibody comprising culturing a cell of the disclosure and harvesting said antibody from said culture. In certain aspects said cell is cultured in a serum free medium. In certain aspects said cell is adapted for suspension growth. Further provided is an antibody obtainable by a method for producing an antibody according to the disclosure. The antibody is in certain aspects purified from the medium of the culture. In certain aspects said antibody is affinity purified.

A cell of the disclosure is for instance a hybridoma cell line, a CHO cell, a 293F cell, an NSO cell or another cell type known for its suitability for antibody production for clinical purposes. In a certain aspect said cell is a human cell. In certain aspects a cell that is transformed by an adenovirus El region or a functional equivalent thereof. A preferred example of such a cell line is the PER.C6TM cell line or equivalent thereof. In a certain aspect said cell is a CHO cell or a variant thereof. In certain aspects a variant that makes use of a Glutamine synthetase (GS) vector system for expression of an antibody.

Antibodies of the disclosure can be produced at levels > 50 mg/L after transient transfection in suspension 293F cells. The bispecific antibodies can be purified to greater than 98% purity with yields > 70%. Analytical characterization studies show bispecific IgGl antibody profiles that are comparable to bivalent monospecific IgGl. In terms of functional activity a bispecific antibody of the disclosure can demonstrate superior potency compared to cetuximab in vitro and in vivo.

The disclosure further provides a pharmaceutical composition comprising a combination of an antibody according to the present disclosure and said EGFR tyrosine kinase inhibitor. The pharmaceutical composition in certain aspects comprises a in certain aspects pharmaceutically acceptable excipient or carrier.

An antibody can comprise a label, in certain aspects a label for in vivo imaging. Such a label is typically not necessary for therapeutic applications. In for instance a diagnostic setting, a label can be helpful. For instance in visualizing target cells in the body. Various labels are suited and many are well known in the art. In certain aspects the label is a radioactive label for detection. In another certain aspect, the label is an infrared label. In certain aspects the infrared label is suited for in vivo imaging. Various infrared labels are available to the person skilled in the art. Preferred infrared labels are for instance, IRDye 800; IRDye 680RD; IRDye 680ET; IRDye 750; IRDye 700DX; IRDye 800RS IRDye 650; IRDye 700 phosphoramidite; IRDye 800 phosphoramidite (EI- COR USA; 4647 Superior Street; Lincoln, Nebraska).

The disclosure further provides a method for the treatment of a subject that has a tumor or is at risk of having said tumor comprising administering to the subject in need thereof an antibody and an EGFR tyrosine kinase inhibitor or a pharmaceutical composition of the present disclosure. The tumor is in certain aspects an EGFR, cMET or EGFR/cMET positive tumor. Before start of said treatment, the method in certain aspects further comprises determining whether said subject has such an EGFR, cMET or EGFR/cMET positive tumor. The disclosure further provides an antibody or pharmaceutical composition of the disclosure for use in the treatment of a subject that has or is at risk of having an EGFR, cMET or EGFR/cMET positive tumor.

In certain aspects, said treatment comprises administering to said subject an effective amount of said bispecific antibody and said third-generation tyrosine kinase inhibitor.

In certain aspects, the bispecific antibody binding EGFR and cMET is provided to a subject at a dosage of 1000, 1500 or 2000 mg, in particular using a flat dose regimen. A flat dose regimen offers several advantages over body-surface or weight dosing as it reduces preparation time and reduces potential dose calculation mistakes. In certain aspects, the bispecific antibody is administered once every week (Q1W), once every 2 weeks (Q2W) or once every 3 weeks (Q3W). In certain aspects, the bispecific antibody is administered once every two weeks. In the art, such a dosing scheme is noted as Q2W. In certain embodiments, the flat dose regimen disclosed herein is suitable for use in adults and/or in subjects weighing at least 35kg. As is understood by the skilled person, the dosage can be administered over time. As is understood by the skilled person, the term “flat dose” or “flat dose regimen” means that the subject undergoes a dosing regimen wherein on each day the subject is scheduled to receive the bispecific antibody or third generation EGFR TKI with substantially the same predetermined amount thereof which amount is irrespective of the subjects’ body weight. According to certain aspects, the subject is provided a flat, weekly dose of 1000 mg bispecific antibody. Alternatively, the subject is provided a flat, biweekly dose of 1000 mg bispecific antibody. Alternatively, the subject is provided a flat, biweekly dose of 1500 mg bispecific antibody. Alternatively, the subject is provided a flat, biweekly dose of 2000 mg bispecific antibody. Also, the subject is typically also provided a daily dose of a third generation EGFR tyrosine kinase inhibitor. In certain aspects, the subject is provided a daily dose of 80 mg Osimertinib, a daily dose of 110 mg Almonertinib, a daily dose of 240 mg of Lazertinib, a daily dose of 70 mg D-0316 or a daily dose of 75 mg D-0316. In certain aspects, said daily dose is a flat daily dose. In certain aspects, the subject is provided a daily dose of 70 mg of D-0316 for 21 days, followed by 100 mg as a daily dose or a daily dose of 75 mg of D-0316 for 21 days, followed by 100 mg as a daily dose. The daily dose of 75 mg of D-0316 for 21 days can be provided as 3 oral dosages of 25 mg each.

Thus, in certain aspects, the bispecific antibody of the present disclosure is dosed or administered at 1000 mg, in particular using a flat dose of 1000 mg. In certain aspects, said bispecific antibody is administered in an amount of 1000 mg once every week. In certain aspects, said bispecific antibody is administered in an amount of 1000 mg once every two weeks. Also, the subject is provided the approved dose of a third generation EGFR tyrosine kinase inhibitor, including a daily dose of 80 mg Osimertinib.

Thus, in certain aspects, the bispecific antibody of the present disclosure is dosed or administered at 1500 mg, in particular using a flat dose of 1500 mg. In certain aspects, said bispecific antibody is administered an amount of 1500 mg once every two weeks. Also, the subject is provided the approved dose of a third generation EGFR tyrosine kinase inhibitor, including a daily dose of 80 mg Osimertinib.

Thus, in certain aspects, the bispecific antibody of the present disclosure is dosed or administered at 2000 mg, in particular using a flat dose of 2000 mg. In certain aspects, said bispecific antibody is administered in an amount of 2000 mg once every two weeks. Also, the subject is provided the approved dose of a third generation EGFR tyrosine kinase inhibitor, including a daily dose of 80 mg Osimertinib. In certain other aspects, the bispecific antibody of the present disclosure is dosed or administered at 1000 mg, in particular using a flat dose of 1000 mg. In certain aspects, said bispecific antibody is administered in an amount of 1000 mg once every week. In certain aspects, said bispecific antibody is administered in an amount of 1000 mg once every two weeks. Also, the subject is provided the approved dose of a third generation EGFR tyrosine kinase inhibitor, including a daily dose of 110 mg Almonertinib.

Thus, in certain aspects, the bispecific antibody of the present disclosure is dosed or administered at 1500 mg, in particular using a flat dose of 1500 mg. In certain aspects, said bispecific antibody is administered an amount of 1500 mg once every two weeks. Also, the subject is provided the approved dose of a third generation EGFR tyrosine kinase inhibitor, including a daily dose of 110 mg Almonertinib.

Thus, in certain aspects, the bispecific antibody of the present disclosure is dosed or administered at 2000 mg, in particular using a flat dose of 2000 mg. In certain aspects, said bispecific antibody is administered in an amount of 2000 mg once every two weeks. Also, the subject is provided the approved dose of a third generation EGFR tyrosine kinase inhibitor, including a daily dose of 110 mg Almonertinib.

In certain other aspects, the bispecific antibody of the present disclosure is dosed or administered at 1000 mg, in particular using a flat dose of 1000 mg. In certain aspects, said bispecific antibody is administered in an amount of 1000 mg once every week. In certain aspects, said bispecific antibody is administered in an amount of 1000 mg once every two weeks. Also, the subject is provided the approved dose of a third generation EGFR tyrosine kinase inhibitor, including a daily dose of 240 mg Lazertinib.

Thus, in certain aspects, the bispecific antibody of the present disclosure is dosed or administered at 1500 mg, in particular using a flat dose of 1500 mg. In certain aspects, said bispecific antibody is administered an amount of 1500 mg once every two weeks. Also, the subject is provided the approved dose of a third generation EGFR tyrosine kinase inhibitor, including a daily dose of 240 mg Lazertinib.

Thus, in certain aspects, the bispecific antibody of the present disclosure is dosed or administered at 2000 mg, in particular using a flat dose of 2000 mg. In certain aspects, said bispecific antibody is administered in an amount of 2000 mg once every two weeks. Also, the subject is provided the approved dose of a third generation EGFR tyrosine kinase inhibitor, including a daily dose of 240 mg Lazertinib.

In certain other aspects, the bispecific antibody of the present disclosure is dosed or administered at 1000 mg, in particular using a flat dose of 1000 mg. In certain aspects, said bispecific antibody is administered in an amount of 1000 mg once every week. In certain aspects, said bispecific antibody is administered in an amount of 1000 mg once every two weeks. Also, the subject is provided the approved dose of a third generation EGFR tyrosine kinase inhibitor, including a daily dose of 70 mg, 75 or 100 mg Befotertinib. In certain aspects, a daily dose of 70 mg Befotertinib or a daily dose of 75 mg Befotertinib is provided. In certain aspects, said daily dose is a flat daily dose. In certain aspects, the subject is provided a daily dose of 70 mg of Befotertinib for 21 days, followed by 100 mg as a daily dose or a daily dose of 75 mg of Befotertinib for 21 days, followed by 100 mg as a daily dose. The 75 mg daily dose of Befotertinib can be provided in 3 oral administrations of 25 mg each.

Thus, in certain aspects, the bispecific antibody of the present disclosure is dosed or administered at 1500 mg, in particular using a flat dose of 1500 mg. In certain aspects, said bispecific antibody is administered an amount of 1500 mg once every two weeks. Also, the subject is provided the approved dose of a third generation EGFR tyrosine kinase inhibitor, including a daily dose of 70 mg, 75 or 100 mg Befotertinib. In certain aspects, a daily dose of 70 mg Befotertinib or a daily dose of 75 mg Befotertinib is provided. In certain aspects, said daily dose is a flat daily dose. In certain aspects, the subject is provided a daily dose of 70 mg of Befotertinib for 21 days, followed by 100 mg as a daily dose or a daily dose of 75 mg of Befotertinib for 21 days, followed by 100 mg as a daily dose. The 75 mg daily dose of Befotertinib can be provided in 3 oral administrations of 25 mg each.

Thus, in certain aspects, the bispecific antibody of the present disclosure is dosed or administered at 2000 mg, in particular using a flat dose of 2000 mg. In certain aspects, said bispecific antibody is administered in an amount of 2000 mg once every two weeks. Also, the subject is provided the approved dose of a third generation EGFR tyrosine kinase inhibitor, including a daily dose of 70 mg, 75 or 100 mg Befotertinib. In certain aspects, a daily dose of 70 mg Befotertinib or a daily dose of 75 mg Befotertinib is provided. In certain aspects, said daily dose is a flat daily dose. In certain aspects, the subject is provided a daily dose of 70 mg of Befotertinib for 21 days, followed by 100 mg as a daily dose or a daily dose of 75 mg of Befotertinib for 21 days, followed by 100 mg as a daily dose. The 75 mg daily dose of Befotertinib can be provided in 3 oral administrations of 25 mg each.

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

The cancer or tumor may be an EGFR, cMET or EGFR/cMET positive cancer. In one aspect, the disclosure provides treatment of a positive cancer that is an EGFR, cMET or EGFR/cMET positive cancer that is lung cancer, in certain aspects non-small cell lung cancer. The subject is in certain aspects a human subject. The subject is in certain aspects a subject eligible for antibody therapy using an EGFR specific antibody such as cetuximab. In certain aspects the disclosure may in certain aspects treat a subject that comprises a tumor, in certain aspects an EGFR/cMET positive cancer, in certain aspects a tumor/cancer with an EGFR RTK resistant phenotype, an EGFR monoclonal antibody resistant phenotype or a combination thereof.

As used herein, the term ‘cancer’ applies to the same as the term ‘tumor’, such that treatment of a tumor also applies to treatment of cancer.

The amount of antibody to be administered to a patient is typically in the therapeutic window, meaning that a sufficient quantity is used for obtaining a therapeutic effect, while the amount does not exceed a threshold value leading to an unacceptable extent of side-effects. The lower the amount of antibody needed for obtaining a desired therapeutic effect, the larger the therapeutic window will typically be. An antibody according to the present disclosure exerting sufficient therapeutic effects at low dosage is, therefore, preferred. The dosage can be in range of the dosing regimen of cetuximab. In certain aspects, the dose is 1000 mg, 1500 mg, or 2000 mg. Dosing may be once every week or once every two weeks.

Osimertinib is administered according to the dose approved by the regulatory authorities. Typically, dosing is 80 mg once a day.

Almonertinib is administered according to the dose approved by the regulatory authorities. Typically, dosing is 110 mg once a day.

Lazertinib is administered according to the dose approved by the regulatory authorities. Typically, dosing is 240 mg once a day.

Typically, dosing of Befotertinib is 75 mg per day, provided as three oral administrations of 25 mg each for 21 days, and if tolerated, followed by 100 mg as a daily dose. Alternatively, dosing is 70 mg of Befotertinib for 21 days, and if tolerated, followed by 100 mg as a daily dose.

A bispecific antibody according to the disclosure in certain aspects induces less skin toxicity as compared to cetuximab under otherwise similar conditions. A bispecific antibody according to the disclosure in certain aspects produces less proinflammatory chemokines, in certain aspects of CXCL14 as compared to cetuximab under otherwise similar conditions. A bispecific antibody according to the disclosure in certain aspects induces less impairment of antimicrobial RNAses, in certain aspects Rnase 7, as compared to cetuximab under otherwise similar conditions.

The present disclosure describes among others antibodies that target the EGFR and cMET receptors and result in potent proliferation inhibition of cancer cell lines in vitro and tumor growth inhibition in vivo. A bispecific antibody of the disclosure can combine low toxicity profiles with high efficacy. An antibody of the disclosure can be useful in various types and lines of EGFR-targeted therapies. An antibody of the disclosure can have an increased therapeutic window when compared to an antibody that binds the same antigen(s) with both arms. A bispecific antibody of the disclosure can exhibit better growth inhibitory effects in vitro, in vivo or a combination thereof when compared to the cetuximab antibody.

The disclosure provides a bispecific antibody as disclosed herein, for use in the treatment of subject that may have one or more of a variety of different kinds of tumors. The tumor may be an EGFR positive tumor, a cMET positive tumor or an EGFR and cMET positive tumor. The tumor may be a lung cancer including non-small cell lung cancer. The tumor may be resistant to treatment with an EGFR tyrosine kinase inhibitor. In certain aspects, the EGFR tyrosine kinase inhibitor is a third generation EGFR tyrosine kinase inhibitor, in certain aspects Osimertinib or an analogue thereof. The treatment comprises treatment with said tyrosine kinase inhibitor. When co- treating with said tyrosine kinase inhibitor the tumor can be resistant to the treatment with the EGFR tyrosine kinase inhibitor. The co-treatment at least partly restores sensitivity of the tumor to the tyrosine kinase inhibitor. The EGFR tyrosine kinase inhibitor in certain aspects is a third generation EGFR tyrosine kinase inhibitor. Examples of clinically relevant third generation EGFR tyrosine kinase inhibitors are Osimertinib, Lazertinib, Alflutinib, Rezivertinib, Rociletinib, Olmutinib, Almonertinib, Abivertinib, ASK120067, Befotertinib, Olmutinib, Rociletinib or SH-1028 (nazartinib (EGF816), naquotinib (ASP8273), mavelertinib (PF-0647775), Olafertinib (CK-101), Keynatinib, ES-072. In certain aspects the tyrosine kinase inhibitor is Osimertinib. In certain aspects the tyrosine kinase inhibitor is Lazertinib. In certain aspects the tyrosine kinase inhibitor is Almonertinib. In certain aspects the tyrosine kinase inhibitor is Befotertinib. In this and other aspects the tumor may be an H GF -associated tumor.

In certain aspects, the third generation EGFR tyrosine kinase inhibitor is an irreversible inhibitor, such as Osimertinib, Olmutinib or Almonertinib.

An EGFR-positive tumor is typically a tumor that has an EGFR activating mutation. An EGFR activating mutation is a mutation of EGFR that results in activation of the EGF/EGFR signaling pathway. The EGFR activating mutation may be important for a cancerous state of the tumor. One of the ways in which such tumors can become insensitive to EGFR targeted therapy is by activation of the HGF/cMET signaling pathway. The tumor maybe an H GF- associated tumor. Activation of the cMET/HGF signaling pathway is one of the ways in which an EGFR-positive tumor can escape treatment with an EGFR-targeted therapy. The cMET/HGF pathway can be activated in various ways. Various methods of activation are described in the art some of which are detailed herein. An antibody of the disclosure is particularly suited for the treatment of tumors wherein activation of the cMET/HGF signaling pathway is associated with the presence of or excess of HGF. Such cMET positive tumors are referred to as HGF-associated tumors or HGF-dependent tumors. An antibody of the disclosure can also be used to at least in part inhibit this possible escape mechanism of EGFR positive tumors. Such tumors can escape EGFR-targeted therapy through the selected outgrowth of tumor cells wherein, in addition, the cMET/HGF signaling pathway is activated. Such cells may be present at the start of the EGFR-targeted therapy. Such cells have a selective growth advantage over HGF/cMET signaling negative tumor cells. The tumor may be a tumor wherein the HGF/cMET signaling pathway is activated. The tumor may be a tumor that is associated with elevated levels of hepatocyte growth factor (HGF) or overexpression of the HGF receptor c-Met. The tumor may be a tumor wherein growth is driven by the EGF and/or HGF. A tumor is said to be driven by a certain growth factor if the signaling pathway is activated in cells of the tumor in response to the presence of the growth factor and removal of the growth factor results in inhibition of the growth of the cells of the tumor. Reduction can be measured by reduced cell division and/or induced cell kill such as apoptosis. A tumor is an HGF -associated tumor if under conditions that would otherwise be permissive for the growth of the tumor, the tumor growths or growths faster in the presence of HGF.

EGFR-targeted therapies for various tumors have been reviewed by Vecchione et al., EGFR-targeted therapy." Experimental cell research Vol 317 (2011): 2765-2771. In general EGFR-targeted therapy is a therapy with a molecule that interacts with EGFR and inhibits EGFR-me diate d signaling in the cell.

As used herein, the term “treatment” encompasses prophylaxis. The prophylactic aspect of the present disclosure is specifically given as follows.

The present disclosure also provides a combination of a third- generation tyrosine kinase inhibitor and a bispecific antibody of the present disclosure that comprises a first variable domain that can bind an extracellular part of human epidermal growth factor receptor (EGFR) and a second variable domain that can bind an extracellular part of human MET Proto-Oncogene, Receptor Tyrosine Kinase (cMET) for use in a method of preventing of a cancer having a tyrosine kinase inhibitor resistance from occurring in a subject. In certain aspects wherein the first variable domain comprises a heavy chain variable region with a CDR1 sequence SYGIS; a CDR2 sequence WISAYX1X2NTNYAQKLQG and a CDR3 comprising the sequence X3X4X5X6HWWEX7A, wherein XI = N or S; X2 = A or G; X3 = D or G; X4 = R, S or Y; X5 = H, E or Y; X6 = D or W and X7 = D or G; with 0-5 amino acid insertions, deletions, substitutions, additions or a combination thereof at a position other than X1-X7 and wherein the second variable domain comprises a heavy chain variable region with the amino acid sequence of one of the sequences of SEQ ID NO: 1-23 with 0-10, in certain aspects 0-5 amino acid insertions, deletions, substitutions, additions or a combination thereof.

The prophylactic aspect of the present disclosure also relates to the provision of a method of preventing of a cancer having a tyrosine kinase inhibitor resistance from occurring in a subject, comprising administering to the subject an effective amount of a combination of a third- generation tyrosine kinase inhibitor and the bispecific antibody of the present disclosure that comprises a first variable domain that can bind an extracellular part of human epidermal growth factor receptor (EGFR) and a second variable domain that can bind an extracellular part of human MET Proto-Oncogene, Receptor Tyrosine Kinase (cMET). In particular, the present method of preventing of a cancer having a tyrosine kinase inhibitor resistance from occurring in a subject, is relevant for mutations occurring from progression on treatment with a first, second and/or third generation EGFR tyrosine kinase inhibitors. In certain aspects, said cancer is NSCLC. Treatment of such cancer type with first, second or third generation tyrosine inhibitors have been reported to lead to several mutations, such as acquired resistance mutation T790M or exon 20 insertion mutations.

In certain aspects, said prevention of a cancer having a tyrosine kinase inhibitor resistance from occurring in a subject includes prevention of a cancer having acquired resistance mutations in EGFR to develop or occur, such as T790M or exon 20 insertion mutations.

In certain aspects, said prevention includes treatment of a cancer or a patient comprising an activating EGFR mutation, such as an exon 19 deletion, exon 21 L858R point substitution or an EGFR amplification.

In certain aspects, the subject that benefits from preventing a cancer having a tyrosine kinase inhibitor resistance from occurring is diagnosed for the presence of an EGFR activating mutation. In certain aspects, such a subject is eligible for treatment according to clinically-relevant criteria for first line standard of care, including platinum-based chemotherapy or one of said tyrosine kinase inhibitors. Such criteria for NSCLC or other cancer types, are well-known with a qualified practicing physician. In certain aspects, such criteria include histologically or cytologically confirmed solid tumors with evidence of metastatic or locally advanced unresected disease that is incurable, measurable disease as defined by RECIST version 1.1 by radiologic methods (c.f. Eisenhauer et al., European Journal of Cancer 45 (2009) 228-247), Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1, including or not a life expectancy > 12 weeks, as per investigator judgment.

Herein, the ECOG Performance Status Scale with indicated grade and performance status is as follows:

Grade 0: Fully active, able to carry on all pre-disease performance without restriction. Grade 1: Restricted in physically strenuous activity but ambulatory and able to carry out work of a light or sedentary nature, e.g., light house work, office work. Grade 2: Ambulatory and capable of all selfcare but unable to carry out any work activities; up and about more than 50% of waking hours. Grade 3: Capable of only limited selfcare; confined to bed or chair more than 50% of waking hours. Grade 4: Completely disabled; cannot carry on any selfcare; totally confined to bed or chair. Grade 5: Dead.

In certain aspects, the antibody used in said prevention comprises a first variable domain comprises a heavy chain variable region with a CDR1 sequence SYGIS; a CDR2 sequence WISAYX1X2NTNYAQKLQG and a CDR3 comprising the sequence X3X4X5X6HWWLX7A, wherein XI = N or S; X2 = A or G; X3 = D or G; X4 = R, S or Y; X5 = H, L or Y; X6 = D or W and X7 = D or G; with 0-5 amino acid insertions, deletions, substitutions, additions or a combination thereof at a position other than X1-X7 and wherein the second variable domain comprises a heavy chain variable region with the amino acid sequence of one of the sequences of SEQ ID NO: 1-23 with 0-10, in certain aspects 0-5 amino acid insertions, deletions, substitutions, additions or a combination thereof.

The method of treatment or antibody for use in the treatment as indicated herein in certain aspects further comprises the step of determining whether the tumor is an HGF-associated tumor.

An antibody of the disclosure can inhibit growth of an HGF-associated tumor.

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 number 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.

In certain aspects, at the start of treatment, at least one, more than one or all of the following inclusion factors IF1-IF8 are applicable to subjects for treatment. In certain aspects, the subject comprises or complies with all of inclusion factors IF1-IF8:

IF1. Having an age of equal to or higher than 18 years at signature of informed consent,

IF2. Having a histologically or cytologically confirmed solid tumor with evidence of metastatic or locally advanced unresected disease that is incurable.

IF3.1. For subjects who have failed prior standard first-line treatment: Subjects have progressed on or are intolerant to therapies that are known to provide clinical benefit. Subjects have either NSCEC harboring activating EGFR mutations including tyrosine kinase inhibitor (TKI) sensitizing mutations, and/or approved TKI-resistance mutations, or any activating c-MET mutation/amplification.

IF3.2. For subjects with prior first or higher anti-cancer treatment: Having progressed on or be intolerant to therapies that are known to provide clinical benefit. Patients have either NSCEC EGFR sensitizing mutations (such as Dell9, L858R) and have not received anti-cancer treatment (i.e. first line treatment) or have Osimertinib- resistant NSCLC and are chemotherapy-naive.

IF4. Having availability of FFPE embedded after progression at the latest therapy, archival or a fresh tumor tissue sample.

IF5. Having measurable disease as defined by RECIST version 1.1 by radiologic methods (patients with non-measurable but evaluable disease can be included in the dose escalation part).

IF6. Having an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1.

IF7. Having a life expectancy > 12 weeks. IF8. Having adequate organ function, as established by at least one or all of:

IF8.1 Absolute neutrophil count (ANC) >1.5 X 10 ⁹ /L.

IF8.2 Hemoglobin >9 g/dL.

IF8.3 Platelets >100 x 10 ⁹ /L.

IF8.4 Corrected total serum calcium within normal ranges.

IF8.5 Serum magnesium within normal ranges (or corrected with supplements).

IF8.6 Serum potassium within normal ranges.

IF8.7 Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) equal to or less than 3.0 x the upper limit of normal (ULN) and total bilirubin equal to or less than 1.5 x ULN, with the proviso that in cases of a liver involvement or malignancy, ALT/AST is equal to or less than 5 x ULN and total bilirubin is equal to or less than 2 x ULN.

IF8.8 For patients with Gilbert’s syndrome, conjugated bilirubin has a value within normal limits.

IF8.9 Serum creatinine equal to or less than 1.5 x ULN or creatinine clearance equal to or higher than 50 mL/min calculated according to the Cockroft and Gault formula or the MDRD formula for patients aged over 65 years.

IF8.10 Serum albumin >3.3 g/dL

In certain aspects, all values for organ function measurements according to IF8 have an upper limit observed with healthy subjects.

In certain aspects, the subject for treatment comprises one or more factors selected from the group consisting of IF1-IF8. In certain aspects, the subject for treatment comprises factors IF2, IF3 (such as IF3.1, IF3.2), IF5 and IF8. In certain aspects, the subject for treatment comprises all of the factors IF1-IF8.

In certain aspects, at the start of treatment, at least one, more than one or all of the following exclusion factors EF1-EF16 are applicable to subjects for treatment:

EFl. Having central nervous system metastases that:

EF1.1: are untreated and symptomatic, whereas subjects having asymptomatic lesions can be included if considered stable;

EFl.2 require radiation or surgery;

EFl.3 require continued steroid therapy (> 10 mg prednisone or equivalent) to control symptoms within 14 days of administration prior to administration of the first dose. Subjects with other central nervous system metastasis are allowed.

EF2. Having known leptomeningeal involvement.

EF3. Participation in another clinical study or treatment with any investigational drug within 4 weeks prior to administration of the first dose.

EF4. Administration of systemic anticancer therapy or immunotherapy within 4 weeks or 5 half Jives, whichever is shorter, of the first dose of study drug. For cytotoxic agents that have major delayed toxicity (e.g., mitomycin C, nitrosoureas), a washout period of 6 weeks is required.

EF5. Having undergone major surgery or radiotherapy within 3 weeks of administration of the first dose. Subjects who received prior radiotherapy to equal or more than 25% of bone marrow at any time are not eligible. EF6. Having persistent grade of more than 1 clinically significant toxicity, related to prior antineoplastic therapies (except for alopecia); with the proviso that stable sensory neuropathy with a grade equal to 2 or lower of NCI-CTCAE v5.0 and hypothyroidism with a grade equal to 2 or lower which is stable on hormone replacement is not excluded.

EF7. Having a history of hypersensitivity reaction or any toxicity attributed to human proteins or any of the excipients that warranted permanent cessation of these agents.

EF8. Having a history of clinically significant cardiovascular disease including, but not limited to:

EF8.1 Diagnosis of deep vein thrombosis or pulmonary embolism within 1 month prior to first dose of study drug, or any of the following within 6 months prior to the first dose of study drug: myocardial infarction, unstable angina, stroke, transient ischemic attack, coronary/peripheral artery bypass graft, or any acute coronary syndrome.

EF8.2 Prolonged QT interval > 480 msec or clinically significant cardiac arrythmia or electrophysiologic disease (i.e., placement of implantable cardioverter defibrillator or atrial fibrillation with uncontrolled rate). Patients with cardiac pacemakers who are clinically stable are eligible.

EF8.3 Uncontrolled (persistent) arterial hypertension: systolic blood pressure > 180 mm Hg and/or diastolic blood pressure > 100 mm Hg.

EF8.4 Congestive heart failure (CHF) defined as New York Heart Association (NYHA) class III-IV or hospitalization for CHF within 6 months of the first dose of study drug.

EF8.5 Clinically significant pericardial effusion.

EF8.6 Myocarditis.

EF9. Having a history of interstitial lung disease including drug-induced interstitial lung disease, radiation pneumonitis that requires treatment with prolonged steroids or other immune suppressive agents within 1 year.

EF10. Having a previous or concurrent malignancy, excluding non-basal cell carcinomas of skin or carcinoma in situ of the uterine cervix, unless the tumor was treated with curative or palliative intent and the previous or concurrent malignancy condition does not affect the assessment of safety and efficacy of the study drug.

EF11. Having current serious illness or medical conditions including, but not limited to uncontrolled active infection, clinically significant pulmonary, metabolic or psychiatric disorders.

EF12. Having active Hepatitis B infection (HBsAg positive) without receiving antiviral treatment. Subjects with active hepatitis B (HbsAg positive) must receive antiviral treatment with lamivudine, tenofovir, entecavir, or other antiviral agents, starting at least 7 days or more before administration of the first dose. Subjects with antecedents of Hepatitis B (anti-HBc positive, HbsAg and HBV-DNA negative) are eligible.

EF13. Having a positive test for Hepatitis C ribonucleic acid (HCV RNA); Subjects in whom HCV infection resolved spontaneously (positive HCV antibodies without detectable HCV-RNA) or those who achieved a sustained virological response after antiviral treatment and show absence of detectable HCV RNA equal to or more than 6 months (with the use of IFN-free regimens) or equal to or more than 12 months (with the use of IFN-based regimens) after cessation of antiviral treatment are eligible.

EF14. Having a known history of HIV (HIV 1/2 antibodies). Patients with HIV with undetectable viral load are allowed. HIV testing is not required unless mandated by local health authority or regulations.

EF15. In case of sexually active male and female patients of childbearing potential agree to use one of the following methods of birth control during the entire duration of the study and for 6 months after final administration of PB19478:

• combined (estrogen and progestogen containing) hormonal contraception associated with inhibition of ovulation (oral, intravaginal, transdermal)

• progestogen-only hormonal contraception associated with inhibition of ovulation (oral, injectable, implantable)

• intrauterine device (IUD)

• intrauterine hormone-releasing system (IUS)

• bilateral tubal occlusion

• vasectomized partner

• sexual abstinence

EF16. Being pregnant or breast-feeding.

In certain aspects, the subject for treatment complies with one or more factors selected from the group consisting of EF1-EF16. In certain aspects, the subject for treatment complies with all of the factors EF1-EF16.

A reference herein to a patent document or other matter which is cited is not to be taken as an admission that that document or matter was known or that the information it contains was part of the common general knowledge as at the priority date of any of the claims.

The conjunctive term “and/or” between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by “and/or,” a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or.”

For the purpose of clarity and a concise description, features are described herein as part of the same or separate embodiments, however, it will be appreciated that the scope of the disclosure may include embodiments having combinations of all or some of the features described. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Amino acid sequence of the heavy chain variable regions of variable domains referred to in this application.

Figure 2. MF3370 and variants thereof. The CDR1, CDR2 and CDR3 sequences in MF8226 are underlined from left to right. The CDRs in the other sequences are at the corresponding positions (according to Kabat).

Figure 3. MF4356 and variants thereof. The CDR1, CDR2 and CDR3 sequences in MF4356 are underlined from left to right. The CDRs in the other sequences are at the corresponding positions (according to Kabat).

Figure 4. Common light chain used in mono- and bispecific IgG.

Figure 4A: Common light chain amino acid sequence. Figure 4B: Common light chain variable domain DNA sequence and translation (IGKVl-39/jkl). Figure 4C: Common light chain constant region DNA sequence and translation. Figure 4D: IGKVl-39/jk5 common light chain variable domain translation. Figure 4E: V-region IGKV1-39A.

Figure 5. IgG heavy chains for the generation of bispecific molecules. Figure 5A: CHI region. Figure 5B: hinge region. Figure 5C: CH2 region. Figure 5D: CH2 containing L235G and G236R silencing substitutions. Figure 5E: CH3 domain containing substitutions L351K and T366K (KK). Figure 5F; CH3 domain containing substitutions L351D and L368E (DE).

Figure 6. Overview of the treatment schedule. Antibody, osimertinib, and a combination of antibody and osimertinib, or vehicle were administered the indicated period. At end of treatment (day 31), tumor samples were collected and snap frozen for target expression analysis. Mice were monitored for duration of response (DoR) and relapse after the end of treatment.

Figure 7. Survival curve to 750mm3: Log-rank (Mantel-Cox) test of combination vs each monotherapy group is significant.

Figure 8. Individual tumor volumes at day 28. Statistical analysis was performed using a mixed model with Tukey’s post test in Graphpad Prism

Figure 9. Mean Tumor volume ± SEM during the full treatment period. Observation period terminated on day 100.

Figure 10. Overview of the treatment schedule. Groups were administered over a period of 3 weeks.

Figure 11. Effects of treatment with Groups comprising antibody, osimertinib, combination treatment and only vehicle on tumor volume in NSCLC CDX model HCC827/ER1 harboring a mutation in exon 19. Tumor volume growth curve representing vehicle and treatment groups shown at different time points (mean TV ± SEM).

Figure 12. Animal survival curves of mice carrying NSCLC CDX model HCC827/ER1 harboring a mutation in exon 19 in different treatment Groups vs vehicle Group.

Clauses

1. A combination of a third-generation EGFR tyrosine kinase inhibitor and a bispecific antibody that comprises a first variable domain that can bind an extracellular part of human epidermal growth factor receptor (EGFR) and a second variable domain that can bind an extracellular part of human MET Proto-Oncogene, Receptor Tyrosine Kinase (cMET), wherein the first variable domain comprises a heavy chain variable region with a CDR1 sequence SYGIS; a CDR2 sequence WISAYX1X2NTNYAQKLQG and a CDR3 comprising the sequence X3X4X5X6HWWLX7A, wherein Xi = N or S; X2 = A or G; X3 = D or G; X4 = R, S or Y; X5 = H, L or Y; Xe = D or W and X7 = D or G; with 0-5 amino acid insertions, deletions, substitutions, additions or a combination thereof at a position other than X1-X7 and wherein the second variable domain comprises a heavy chain variable region with the amino acid sequence of one of the sequences of SEQ ID NO: 1-23 with 0-10, in certain aspects 0-5 amino acid insertions, deletions, substitutions, additions or a combination thereof, for use in a method of treatment of a cancer in a subject.

2. Method of treating a subject having a cancer, said treatment comprising administering to the subject an effective amount of a combination of a third-generation EGFR tyrosine kinase inhibitor and a bispecific antibody that comprises a first variable domain that can bind an extracellular part of human epidermal growth factor receptor (EGFR) and a second variable domain that can bind an extracellular part of human MET Proto-Oncogene, Receptor Tyrosine Kinase (cMET), wherein the first variable domain comprises a heavy chain variable region with a CDR1 sequence SYGIS; a CDR2 sequence WISAYX1X2NTNYAQKLQG and a CDR3 comprising the sequence X3X4X5X6HWWLX7A, wherein XI = N or S; X2 = A or G; X3 = D or G; X4 = R, S or Y; X5 = H, L or Y; X6 = D or W and X7 = D or G; with 0-5 amino acid insertions, deletions, substitutions, additions or a combination thereof at a position other than X1-X7 and wherein the second variable domain comprises a heavy chain variable region with the amino acid sequence of one of the sequences of SEQ ID NO: 1-23 with 0-10, in certain aspects 0-5 amino acid insertions, deletions, substitutions, additions or a combination thereof.

3. Use of a combination of a bispecific antibody that comprises a first variable domain that can bind an extracellular part of human epidermal growth factor receptor (EGFR) and a second variable domain that can bind (or binds) an extracellular part of human MET Proto-Oncogene, Receptor Tyrosine Kinase (cMET) wherein the first variable domain comprises a heavy chain variable region with a CDR1 sequence SYGIS; a CDR2 sequence WISAYX1X2NTNYAQKLQG and a CDR3 comprising the sequence X3X4X5X6HWWLX7A, wherein XI = N or S; X2 = A or G; X3 = D or G; X4 = R, S or Y; X5 = H, L or Y; X6 = D or W and X7 = D or G; with 0-5 amino acid insertions, deletions, substitutions, additions or a combination thereof at a position other than X1-X7 and wherein the second variable domain comprises a heavy chain variable region with the amino acid sequence of one of the sequences of SEQ ID NO: 1-23 with 0-10, in certain aspects 0-5 amino acid insertions, deletions, substitutions, additions or a combination thereof, and a third- generation EGFR tyrosine kinase inhibitor in the manufacture of a medicament for the treatment of a cancer in a subject.

4. The use or method of any one of the preceding clauses, wherein said cancer is an EGFR positive cancer, a cMET positive cancer or an EGFR and cMET positive cancer.

5. The use or method of any one of the preceding clauses, wherein said cancer comprises an EGFR aberration, a cMET aberration or an EGFR and cMET aberration.

6. The use or method of any one of the preceding clauses, wherein said cancer is resistant to treatment with a tyrosine kinase inhibitor.

7. The use or method of any one of the preceding clauses, wherein said cancer is resistant to an EGFR and/or cMET tyrosine kinase inhibitor.

8. The use or method of clause 7, wherein the EGFR tyrosine kinase inhibitor resistance comprises a resistance to a first, second and/or third generation tyrosine kinase inhibitor, preferably to a third generation EGFR tyrosine kinase inhibitor.

9. The use or method of clause 7, wherein the cMET tyrosine kinase inhibitor resistance comprises a resistance to capmatinib, tepotinib, crizotenib, cabozantinib, savolitinib, Glesatinib, Sitravatinib, BMS-777607, Merestinib, Tivantinib, Golvatinib, Foretinib, AMG-337 or BMS-794833, preferably capmatinib or tepotinib.

10. The use or method of any one of the preceding clauses, wherein the subject has received prior treatment with a tyrosine kinase inhibitor, preferably an EGFR and/or cMET tyrosine kinase inhibitor.

11. The use or method of any one of the preceding clauses, wherein the subject has received prior treatment with a first, second or third generation EGFR tyrosine kinase inhibitor.

12. The use or method of any one of the preceding clauses, wherein the subject has received prior treatment with a cMET tyrosine kinase inhibitor.

13. The use or method of any one of the preceding clauses, wherein said cancer comprises an activating EGFR mutation, an approved tyrosine kinase inhibitor resistance mutation, a tertiary tyrosine kinase inhibitor resistance mutation (such as L718X (e.g. L718Q), G719X (e.g. G719A), L792X (e.g. L792H), G796X (e.g. G796R, G796S, G796D), C797X , C797X (e.g. C797S, C797G), a mutation that reduces binding of a third generation tyrosine kinase inhibitor to EGFR (e.g. L792X, L718X), an acquired tyrosine kinase inhibitor resistance mutation (such as C797X, L792X, G796X, G724X, S768X, L718X or an exon 20 insertion mutation), an EGFR gene amplification, a cMET mutation or cMET aberration.

14. The use or method according to any one of the preceding clauses, wherein said cancer comprises an exon 19 deletion mutation, preferably an in-frame exon 19 deletion, an exon 20 missense mutation (e.g. T790M) or an exon 21 mutation, such as L858R.

15. The use or method according to any one of the preceding clauses, wherein said cancer comprises an EGFR exon 20 mutation, preferably an exon 20 insertion mutation.

16. The use or method according to any one of the preceding clauses, wherein said cancer comprises an acquired tyrosine kinase inhibitor resistance mutation such as a mutation which confers resistance to Osimertinib, including G724X (e.g. G724S), S768X (e.g. S768I), T790X (e.g. T790M), L792X (e.g. L792H), C797X (including C797S and C797G), L798X (e.g. L798I).

17. The use or method according to any one of the preceding clauses, wherein said cancer comprises an exon 20 (762-823) mutation selected from a near-loop insertion (positions 767-772), a far-loop insertion (positions 773-775), preferably V769_D770insASV, D770_N771insSVD, H773_V774insNPH, H773_V774insH, D770_N771insG, D770delinsGY, N771_P772insN, V774_C775insHV, D770_N771insGL, H773_V774insPH, A763_Y764insFQEA, D770_N771delinsEGN, D770_N771insGD, D770_N771insH, D770_N771insP, H773_V774insAH, H773_V774insGNPH, H773delinsSNPY, N771_P772insH, N771_P772insVDN, N771delinsGY, N771delinsKH, N771delinsRD, P772_H773delinsHNPY, P772_H773insGT, P772_H773insPNP, P772_H773insT, V769_D770insA, V769_D770insGG, V769_D770insGSV, V769_D770insGW and V769_D770insMASV; or mutations T790M, L792X (e.g. L792H, C796X (e.g. G796R, G796S, G796D), C797X (e.g. C797S, C797G), L798I, or an in-frame exon 20 insertion, such as M766_A767insASV or H773-V774insNPH, Ins761(EAFQ), Ins770(ASV), Ins771(G), Ins774(NPH), M766_A7671ns A, S768_V769InsSVA, P772_H773InsNS, D761_E762InsXl-7, A763_Y764InsXl-7, Y764_Y765 InsXl-7, M766_A767InsXl-7, A767_V768 InsXl-7, S768_V769 InsXl-7) V769_D770 InsXl-7) D770_N771 InsXl-7) N771_P772 InsXl-7) P772_H773 InsXl-7, H773_V774 InsXl-7, or V774_C775 InsXl-7.

18. The use or method according to any one of the preceding clauses, wherein said cancer comprises a cMET aberration, such as a cMET amplification, cMET overexpression, increased signaling of the cMET pathway, a cMET gene amplification, , increased cMET protein activity and/or increased HGF expression.

19. The use or method according to any one of the preceding clauses, wherein said cancer comprises a cMET exonl4 skipping mutation.

20. The use or method according to any one of the preceding clauses, wherein said first-generation EGFR tyrosine kinase inhibitor comprises or is gefitinib, erlotinib or icotinib. 21. The use or method according to any one of the preceding clauses, wherein said second- generation EGFR tyrosine kinase inhibitor comprises or is afatinib, dacomitinib, XL647, AP26113, CO- 1686 or neratinib.

22. The use or method according to any one of the preceding clauses, wherein said third-generation EGFR tyrosine kinase comprises or is Osimertinib, Lazertinib, Alflutinib, Rezivertinib, Rociletinib, Olmutinib, Almonertinib, Abivertinib, ASK120067, Befotertinib, SH-1028, Nazartinib (EGF816), Naquotinib (ASP8273), Mavelertinib (PF- 0647775), Olafertinib (CK-101), Keynatinib, or ES-072, preferably Osimertinib.

23. The use or method according to any one of the preceding clauses, wherein said cMET tyrosine kinase inhibitor comprises or is capmatinib, tepotinib, crizotenib, cabozantinib, savolitinib, Glesatinib, Sitravatinib, BMS-777607, Merestinib, Tivantinib, Golvatinib, Foretinib, AMG-337 or BMS-794833.

24. The use or method according to any one of the preceding clauses, wherein the treatment comprises administering said combination of said bispecific antibody and said tyrosine kinase inhibitor to a subject in need thereof, and wherein preferably said bispecific antibody is administered simultaneously, sequentially or separately with said third generation tyrosine kinase inhibitor.

25. The use or method according to any one of clauses 1-5, wherein the subject has not received prior anti-cancer treatment, such as a subject which is tyrosine kinase inhibitor treatment or anti- EGFR treatment naive.

26. The use or method according to any one of clauses 1-5, wherein the administration of the bispecific antibody and TKI inhibitor is administered as first line treatment.

27. The use or method according to any one of clauses 1-5, wherein the subject or cancer comprises an EGFR and/or cMET activating mutation, such as an exon 19 deletion mutation or exon 21 mutation (such as L858R).

28. The use or method according to any one of the preceding clauses, wherein the subject is a human subject.

29. The use or method according to any one of the preceding clauses, wherein the cancer is lung cancer, in particular non-small cell lung cancer, preferably metastatic or advanced non-small cell lung cancer.

30. The use or method according to any one of the preceding clauses, wherein said cancer is NSCLC comprising an activating EGFR mutation, EGFR tyrosine kinase inhibitor sensitizing mutation (such as an exonl9deletion and L858X), an acquired EGFR tyrosine kinase inhibitor resistance mutation, (such as T790X, C797X, L792X, L798X) an approved EGFR tyrosine kinase inhibitor resistance mutation, an EGFR exon 20 insertion mutation, an activating c-MET mutation, preferably an exon 14 skipping mutation, or a cMET amplification, preferably comprising MET/CEP7 > 5 or cfDNA > 2 copies or any combination thereof.

31. The use or method according to any one of the preceding clauses, wherein said cancer is advanced or metastatic cancer.

32. A pharmaceutical combination comprising a third-generation EGFR tyrosine kinase inhibitor and a bispecific antibody that comprises a first variable domain that can bind an extracellular part of human epidermal growth factor receptor (EGFR) and a second variable domain that can bind an extracellular part of human MET Proto- Oncogene, Receptor Tyrosine Kinase (cMET), wherein the first variable domain comprises a heavy chain variable region with a CDR1 sequence SYGIS; a CDR2 sequence WISAYX1X2NTNYAQKLQG and a CDR3 comprising the sequence X3X4X5X6HWWLX7A, wherein XI = N or S; X2 = A or G; X3 = D or G; X4 = R, S or Y; X5 = H, L or Y; X6 = D or W and X7 = D or G; with 0-5 amino acid insertions, deletions, substitutions, additions or a combination thereof at a position other than X1-X7 and wherein the second variable domain comprises a heavy chain variable region with the amino acid sequence of one of the sequences of SEQ ID NO: 1-23 with 0-10 preferably 0- 5 amino acid insertions, deletions, substitutions, additions or a combination thereof.

33. The use, method or pharmaceutical combination according to any one of the preceding clauses, wherein the antibody is a human antibody.

34. The use, method or pharmaceutical combination according to any one of the preceding clauses, wherein the antibody is ADCC enhanced.

35. The use, method or pharmaceutical combination according to any one of the preceding clauses, wherein the antibody is an IgGl format antibody having an anti- EGFR, anti-cMET stoichiometry of 1:1.

36. The use, method or pharmaceutical combination according to any one of the preceding clauses, wherein the antibody has one variable domain that can bind EGFR and one variable domain that can bind cMET.

37. The use, method or pharmaceutical combination according to any one of the preceding clauses, wherein the variable domain that can bind human EGFR can also bind cynomolgus and mouse EGFR.

38. The use, method or pharmaceutical combination according to any one of the preceding clauses, wherein the variable domain that can bind human EGFR binds to domain III of human EGFR.

39. The use, method or pharmaceutical combination according to any one of the preceding clauses, wherein the variable domain that can bind cMET blocks the binding of antibody 5D5 to cMET. 40. The use, method or pharmaceutical combination according to any one of the preceding clauses, wherein the variable domain that can bind cMET blocks the binding of HGF to cMET.

41. The use, method or pharmaceutical combination according to any one of the preceding clauses, wherein the amino acids at positions 405 and 409 in one CH3 domain are the same as the amino acids at the corresponding positions in the other CH3 domain (EU-numbering).

42. The use, method or pharmaceutical combination according to any one of the preceding clauses, wherein

Xi = N; X ₂ = G; X ₃ = D; X ₄ = S; X ₅ = Y; X ₆ = W and X ₇ = G;

Xi = N; X ₂ = A; X ₃ = D; X ₄ = S; X ₅ = Y; X ₆ = W and X ₇ = G;

Xi = S; X ₂ = G; X ₃ = D; X ₄ = S; X ₅ = Y; X ₆ = W and X ₇ = G;

Xi = N; X ₂ = G; X ₃ = D; X ₄ = R; X ₅ = H; X ₆ = W and X ₇ = D;

Xi = N; X ₂ = A; X ₃ = D; X ₄ = R; X ₅ = H; X ₆ = W and X ₇ = D;

Xi = S; X ₂ = G; X ₃ = D; X ₄ = R; X ₅ = H; X ₆ = W and X ₇ = D;

Xi = N; X ₂ = G; X ₃ = G; X ₄ = Y; X ₅ = L; X ₆ = D and X ₇ = G;

Xi = N; X ₂ = A; X ₃ = G; X ₄ = Y; X ₅ = L; X ₆ = D and X ₇ = G; or

Xi = S; X ₂ = G; X ₃ = G; X ₄ = Y; X ₅ = L; X ₆ = D and X ₇ = G

43. The use, method or pharmaceutical combination according to any one of the preceding clauses, wherein Xi = N; X ₂ = G; X ₃ = D; X ₄ = R; Xs = H; Xe = W and X ₇ = D; or Xi = N; X ₂ = A; X ₃ = D; X ₄ = R; Xs = H; X ₆ = W and X ₇ = D; or Xi = S; X ₂ = G; X ₃ = D; X ₄ = R; Xs = H; X ₆ = W and X ₇ = D.

44. The use, method or pharmaceutical combination according to any one of the preceding clauses, wherein Xi = N; X ₂ = G; X ₃ = D; X ₄ = R; Xs = H; Xe = W and X ₇ = D; or Xi = N; X ₂ = A; X ₃ = D; X ₄ = R; Xs = H; X ₆ = W and X ₇ = D.

45. The use, method or pharmaceutical combination according to any one of the preceding clauses, wherein the heavy chain variable region of the second variable domain comprises the amino acid sequence of one of the sequences of SEQ ID NO: 1-3; 7; 8; 10; 13; 15; 16; 17; 21; 22 or 23 with 0-10 preferably 0-5 amino acid insertions, deletions, substitutions, additions or a combination thereof.

46. The use, method or pharmaceutical combination according to any one of the preceding clauses, wherein the heavy chain variable region of the second variable domain comprises the amino acid sequence of one of the sequences of SEQ ID NO: 2; 7; 8; 10; 13 or 23 with 0-10 preferably 0-5 amino acid insertions, deletions, substitutions, additions or a combination thereof.

47. The use, method or pharmaceutical combination according to any one of the preceding clauses, wherein the first variable domain comprises a heavy chain variable region with a CDR1 sequence SYGIS; a CDR2 sequence WISAYNGNTNYAQKLQG and a CDR3 comprising the sequence DRHWHWWLDA and wherein the second variable domain comprises a heavy chain variable region with a CDR1 sequence SYSMN; a CDR2 sequence WINTYTGDPTYAQGFTG and a CDR3 sequence ETYYYDRGGYPFDP.

48. The use, method or pharmaceutical combination according to any one of the preceding clauses, wherein the first variable domain comprises a heavy chain variable region with a CDR1 sequence SYGIS; a CDR2 sequence WISAYNANTNYAQKLQG and a CDR3 comprising the sequence DRHWHWWLDA and wherein the second variable domain comprises a heavy chain variable region with a CDR1 sequence TYSMN; a CDR2 sequence WINTYTGDPTYAQGFTG and a CDR3 comprising the sequence ETYFYDRGGYPFDP.

49. The use, method or pharmaceutical combination according to any one of the preceding clauses, wherein the first and second variable domain comprise a common light chain, preferably a light chain of figure 4B.

50. The use, method or pharmaceutical combination according to any one of the preceding clauses, wherein the first and second variable domain comprise a light chain with a CDR1, CDR2 and CDR3 amino acid sequence of QSISSY, AAS, and QQSYSTP, respectively (according to IMGT).

51. The use, method or pharmaceutical combination according to any one of the preceding clauses, which antibody inhibits HGF induced growth of an HGF-growth responsive cell.

52. The use, method or pharmaceutical combination according to any one of the preceding clauses, which antibody inhibits EGF induced growth of an EGF-growth responsive cell.

53. The use, method or pharmaceutical combination according to any one of the preceding clauses, wherein the subject is administered with 1000 mg bispecific antibody, in particular using a flat dose of 1000 mg.

54. The use, method or pharmaceutical combination according to clause 53, wherein the bispecific antibody is administered once every week.

55. The use, method or pharmaceutical combination according to clause 53 or 54, wherein the subject is further administered with Osimertinib, preferably comprising a 80 mg daily dose.

56. The use, method or pharmaceutical combination according to clause 53 or 54, wherein the subject is further administered with Almonertinib, preferably comprising a 110 mg daily dose. 57. The use, method or pharmaceutical combination according to clause 53 or 54, wherein the subject is further administered with Lazertinib, preferably comprising a 240 mg daily dose.

58. The use, method or pharmaceutical combination according to clause 53 or 54, wherein the subject is further administered with Befotertinib, preferably comprising a 75 mg daily dose.

59. The use, method or pharmaceutical combination according to clause 53, wherein the bispecific antibody is administered once every two weeks.

60. The use, method or pharmaceutical combination according to clause 53 or 59, wherein the subject is further administered with Osimertinib, preferably comprising a 80 mg daily dose.

61. The use, method or pharmaceutical combination according to clause 53 or 59, wherein the subject is further administered with Almonertinib, preferably comprising a 110 mg daily dose.

62. The use, method or pharmaceutical combination according to clause 53 or 59, wherein the subject is further administered with Lazertinib, preferably comprising a 240 mg daily dose.

63. The use, method or pharmaceutical combination according to clause 53 or 59, wherein the subject is further administered with Befotertinib, preferably comprising a 75 mg daily dose.

64. The use, method or pharmaceutical combination according to any one of clauses 1-52, wherein the subject is administered with 1500 mg bispecific antibody, in particular using a flat dose of 1500 mg.

65. The use, method or pharmaceutical combination according to clause 64, wherein the bispecific antibody is administered once every two weeks.

66. The use, method or pharmaceutical combination according to clause 64 or 65, wherein the subject is further administered with Osimertinib, preferably comprising a 80 mg daily dose.

67. The use, method or pharmaceutical combination according to clause 64 or 65, wherein the subject is further administered with Almonertinib, preferably comprising a 110 mg daily dose.

68. The use, method or pharmaceutical combination according to clause 64 or 65, wherein the subject is further administered with Lazertinib, preferably comprising a 240 mg daily dose. 69. The use, method or pharmaceutical combination according to clause 64 or 65, wherein the subject is further administered with Befotertinib, preferably comprising a 75 mg daily dose.

70. The use, method or pharmaceutical combination according to any one of clauses 1-51, wherein the subject is administered with 2000 mg bispecific antibody, in particular using a flat dose of 2000 mg.

71. The use, method or pharmaceutical combination according to clause 70, wherein the bispecific antibody is administered once every two weeks.

72. The use, method or pharmaceutical combination according to clause 70 or 71, wherein the subject is further administered with Osimertinib, preferably comprising a 80 mg daily dose.

73. The use, method or pharmaceutical combination according to clause 70 or 71, wherein the subject is further administered with Almonertinib, preferably comprising a 110 mg daily dose.

74. The use, method or pharmaceutical combination according to clause 70 or 71, wherein the subject is further administered with Lazertinib, preferably comprising a 240 mg daily dose.

75. The use, method or pharmaceutical combination according to clause 70 or 71, wherein the subject is further administered with Befotertinib, preferably comprising a 75 mg daily dose.

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 Figure 4A, typically 4B. “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 differ 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 (see for reference PCT/NL2013/050294 (published as WO2013/157954).

Reference is made to PCTNL/2018/050537 (published as WO2019/031965) for details on production of antibodies of the present disclosure. Bispecific antibodies binding EGFR and cMET suitable for use in the appended examples and for use in the methods of the disclosure include those of Tables 3, 4, 5 and 6. In particular, bispecific antibody PB19478 is suitably used in the appended examples. Each bispecific antibody comprises two VH as specified by the MF numbers capable of binding EGFR and cMET respectively, further comprises an Fc tail with a KK/DE CH3 heterodimerization domain as indicated in Figure 5e and Figure 5f, respectively, a CH2 domain as indicated by Figure 5d, a hinge domain as indicated by Figure 5b, a CHI domain as indicated by Figure 5a and a common light chain as indicated by Figures 4a-e. For example, a bispecific antibody indicated by MF8233 x MF8230 has the above general sequences and a variable domain with a VH with the sequence of MF8233 and a variable domain with a VH with the sequence of MF8230 and is preferably used in the appended examples.

Example 1: Materials and Methods

Cell lines:

EBC-1 [JCRB0820], PC-9 [RCB0446], H358 [ATCC® CRL-5807™], HCC827 [ATCC® CRL-2868™],MKN-45 [DSMZ ACC 409] N87 [ATCC®CRE-5822™] and A431 [ATCC® CRE-1555™] cell lines were purchased and routinely maintained in growth media supplemented with 10% heat inactivated fetal bovine serum (FBS). HEK293F Freestyle cells were obtained from Invitrogen and routinely maintained in 293 FreeStyle medium. cDNA constructs:

Generation of cMET and EGFR expression vectors for generation of stable cell lines (cMET and EGFR) and for immunization (cMET)

Full length cDNA of each target including unique restriction sites for cloning and kozak consensus sequence for efficient translation was either synthetized, or obtained via PCR amplification on a commercially available expression construct, containing the target cDNA, with specific primers that introduced unique restriction sites for cloning and kozak consensus sequence for efficient translation. The full length cDNA of each target was cloned into a eukaryotic expression construct such as pcDNA3.1, whereas the extracellular domains were cloned into pVAXl and pDisplay. The insert sequences were verified by comparison with NCBI Reference amino acid sequences.

Amino acid sequence full length human EGFR insert for expression on the cell surface (Identical to GenBank: NP_00533):

MRPSGTAGAALLALLAALCPASRALEEKKVCQGTSNKLTQLGTFEDHFLSLQRMFNN CEVVLGNLEIT YVQRNYDLSFLKTIQEVAGYVLIALNTVERIPLENLQI IRGNMYYENSYALAVLSNYDANKTGLKELP MRNLQEILHGAVRFSNNPALCNVESIQWRDIVSSDFLSNMSMDFQNHLGSCQKCDPSCPN GSCWGAGE ENCQKLTKIICAQQCSGRCRGKSPSDCCHNQCAAGCTGPRESDCLVCRKFRDEATCKDTC PPLMLYNP TTYQMDVNPEGKYSFGATCVKKCPRNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGP CRKVCNGI GIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKE ITGFLLIQ AWPENRTDLHAFENLE 11 RGRTKQHGQFSLAVVSLNIT SLGLRSLKE I SDGDVI I SGNKNLCYANT IN WKKLFGTSGQKTKI ISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEG EPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENN TLVWKYAD AGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFMRRRH IVRKRTLR RLLQERELVEPLTPSGEAPNQALLRILKETEFKKIKVLGSGAFGTVYKGLWIPEGEKVKI PVAIKELR EATSPKANKEILDEAYVMASVDNPHVCRLLGICLTSTVQLITQLMPFGCLLDYVREHKDN IGSQYLLN WCVQIAKGMNYLEDRRLVHRDLAARNVLVKTPQHVKITDFGLAKLLGAEEKEYHAEGGKV PIKWMALE SILHRIYTHQSDVWSYGVTVWELMTFGSKPYDGIPASEISSILEKGERLPQPPICTIDVY MIMVKCWM IDADSRPKFRELIIEFSKMARDPQRYLVIQGDERMHLPSPTDSNFYRALMDEEDMDDVVD ADEYLIPQ QGFFSSPSTSRTPLLSSLSATSNNSTVACIDRNGLQSCPIKEDSFLQRYSSDPTGALTED SIDDTFLP VPEYINQSVPKRPAGSVQNPVYHNQPLNPAPSRDPHYQDPHSTAVGNPEYLNTVQPTCVN STFDSPAH WAQKGSHQISLDNPDYQQDFFPKEAKPNGI FKGSTAENAEYLRVAPQSSEFIGA

Of which:

MRPSGTAGAALLALLAALCPASR : signal peptide.

ALEEKKVCQGTSNKLTQLGTFEDHFLSLQRMFNNCEVVLGNLEITYVQRNYDLSFLK TIQEV AGYVLIALNTVERIPLENLQIIRGNMYYENSYALAVLSNYDANKTGLKELPMRNLQEILH GAVRFSNN PALCNVESIQWRDIVSSDFLSNMSMDFQNHLGSCQKCDPSCPNGSCWGAGEENCQKLTKI ICAQQCSG RCRGKSPSDCCHNQCAAGCTGPRESDCLVCRKFRDEATCKDTCPPLMLYNPTTYQMDVNP EGKYSFGA TCVKKCPRNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIGIGEFKDSL SINATNIK HFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDL HAFENLEI IRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSG QKTKI ISN RGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENS ECIQCHPE CLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCH PNCTYGCT GPGLEGCPTNGPKIPS : ECD of human EGFR .

IATGMVGALLLLLVVALGIGLFM : predicted TM region.

RRRHIVRKRTLRRLLQERELVEPLTPSGEAPNQALLRILKETEFKKIKVLGSGAFGT VYKGL WIPEGEKVKIPVAIKELREATSPKANKEILDEAYVMASVDNPHVCRLLGICLTSTVQLIT QLMPFGCL LDYVREHKDNIGSQYLLNWCVQIAKGMNYLEDRRLVHRDLAARNVLVKTPQHVKITDFGL AKLLGAEE KEYHAEGGKVPIKWMALESILHRIYTHQSDVWSYGVTVWELMTFGSKPYDGIPASEISSI LEKGERLP QPPICTIDVYMIMVKCWMIDADSRPKFRELIIEFSKMARDPQRYLVIQGDERMHLPSPTD SNFYRALM DEEDMDDVVDADEYLIPQQGFFSSPSTSRTPLLSSLSATSNNSTVACIDRNGLQSCPIKE DSFLQRYS SDPTGALTEDSIDDTFLPVPEYINQSVPKRPAGSVQNPVYHNQPLNPAPSRDPHYQDPHS TAVGNPEY LNTVQPTCVNSTFDSPAHWAQKGSHQISLDNPDYQQDFFPKEAKPNGIFKGSTAENAEYL RVAPQSSE FIGA: intracellular tail.

Amino acid sequence of extracellular domain of human EGFRvarlll a natural occurring EGFR variant VAR_066493 [Ji H„ Zhao X; PNAS 103:7817-7822(2006)] caused by an in- frame deletion of exons 2-7. The > below indicates the location lacking amino acids 30 - 297

MRPSGTAGAALLALLAALCPASRALEEKK_GNYVVTDHGSCVRACGADSYEMEEDGV RKCKKCEGPCR KVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDI LKTVKEIT GFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVI ISGNKNLC YANTINWKKLFGTSGQKTKI ISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDK CNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAG VMGENNTL VWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPS

Of which:

MRPSGTAGAALLALLAALCPASR : signal peptide. ALEEKK_GNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIGIGEFKDSL SI NATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWP ENRTDLHA FENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVI ISGNKNLCYANTINWKKLFGTSGQK TKI ISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFV ENSEC IQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGH VCHLCHPN CTYGCTGPGLEGCPTNGPKI PS : ECD of EGFRvarlll

Amino acid sequence chimeric macaque (Macaca mulatta) extra cellular EGFR domain hybrid with human EGFR transmembrane and intracellular domain for expression on the cell surface (Identical to GenBank: XP_014988922.1. Human EGFR sequence underlined in the example below.

MGPSGTAGAALLALLAALCPASRALEEKKVCQGTSNKLTQLGTFEDHFLSLQRMFNN CEVVLGNLEIT YVQRNYDLSFLKTIQEVAGYVLIALNTVERIPLENLQI IRGNMYYENSYALAVLSNYDANKTGLKELP MRNLQEILHGAVRFSNNPALCNVESIQWRDIVSSEFLSNMSMDFQNHLGSCQKCDPSCPN GSCWGAGE ENCQKLTKIICAQQCSGRCRGKSPSDCCHNQCAAGCTGPRESDCLVCRKFRDEATCKDTC PPLMLYNP TTYQMDVNPEGKYSFGATCVKKCPRNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGP CRKVCNGI GIGEFKDTLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKE ITGFLLIQ AWPENRTDLHAFENLE 11 RGRTKQHGQFSLAVVSLNIT SLGLRSLKE I SDGDVI I SGNKNLCYANT IN WKKLFGTSSQKTKI ISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCQNVSRGRECVDKCNILEG EPREFVENSECIQCHPECLPQVMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENN TLVWKYAD AGHVCHLCHPNCTYGCTGPGLEGCARNGPKIPSIATGMLGALLLLLVVALGIGLFMRRRH IVRKRTLR RLLQERELVEPLTPSGEAPNQALLRILKETEFKKIKVLGSGAFGTVYKGLWIPEGEKVKI PVAIKELR EATSPKANKEILDEAYVMASVDNPHVCRLLGICLTSTVQLITQLMPFGCLLDYVREHKDN IGSQYLLN WCVQIAKGMNYLEDRRLVHRDLAARNVLVKTPQHVKITDFGLAKLLGAEEKEYHAEGGKV PIKWMALE SILHRIYTHQSDVWSYGVTVWELMTFGSKPYDGIPASEISSILEKGERLPQPPICTIDVY MIMVKCWM IDADSRPKFRELIIEFSKMARDPQRYLVIQGDERMHLPSPTDSNFYRALMDEEDMDDVVD ADEYLIPQ QGFFSSPSTSRTPLLSSLSATSNNSTVACIDRNGLQSCPIKEDSFLQRYSSDPTGALTED SIDDTFLP VPEYINQSVPKRPAGSVQNPVYHNQPLNPAPSRDPHYQDPHSTAVGNPEYLNTVQPTCVN STFDSPAH WAQKGSHQISLDNPDYQQDFFPKEAKPNGI FKGSTAENAEYLRVAPQSSEFIGA

Of which:

MGPSGTAGAALLALLAALCPASR : signal peptide .

LEEKKVCQGTSNKLTQLGTFEDHFLSLQRMFNNCEVVLGNLEITYVQRNYDLSFLKT IQEVA GYVLIALNTVERIPLENLQI IRGNMYYENSYALAVLSNYDANKTGLKELPMRNLQEILHGAVRFSNNP ALCNVESIQWRDIVSSDFLSNMSMDFQNHLGSCQKCDPSCPNGSCWGAGEENCQKLTKI ICAQQCSGR CRGKSPSDCCHNQCAAGCTGPRESDCLVCRKFRDEATCKDTCPPLMLYNPTTYQMDVNPE GKYSFGAT CVKKCPRNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIGIGEFKDSLS INATNIKH FKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLH AFENLEI I RGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVI ISGNKNLCYANTINWKKLFGTSGQKTKIISNR GENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSE CIQCHPEC LPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHP NCTYGCTG PGLEGCPTNGPKIPS : ECD of cyEGFR

Amino acid sequence full length human cMET insert for expression on the cell surface (Identical to GenBank: P08581-2). The sequence differs from the reference sequence at position with an insertion at 755-755: S STWWKEPLNIVSFLFCFAS MKAPAVLAPGILVLLFTLVQRSNGECKEALAKSEMNVNMKYQLPNFTAETPIQNVILHEH HIFLGATN YIYVLNEEDLQKVAEYKTGPVLEHPDCFPCQDCSSKANLSGGVWKDNINMALVVDTYYDD QLISCGSV NRGTCQRHVFPHNHTADIQSEVHCIFSPQIEEPSQCPDCVVSALGAKVLSSVKDRFINFF VGNTINSS YFPDHPLHSISVRRLKETKDGFMFLTDQSYIDVLPEFRDSYPIKYVHAFESNNFIYFLTV QRETLDAQ TFHTRIIRFCSINSGLHSYMEMPLECILTEKRKKRSTKKEVFNILQAAYVSKPGAQLARQ IGASLNDD ILFGVFAQSKPDSAEPMDRSAMCAFPIKYVNDFFNKIVNKNNVRCLQHFYGPNHEHCFNR TLLRNSSG CEARRDEYRTEFTTALQRVDLFMGQFSEVLLTSISTFIKGDLTIANLGTSEGRFMQVVVS RSGPSTPH VNFLLDSHPVSPEVIVEHTLNQNGYTLVITGKKITKIPLNGLGCRHFQSCSQCLSAPPFV QCGWCHDK CVRSEECLSGTWTQQICLPAIYKVFPNSAPLEGGTRLTICGWDFGFRRNNKFDLKKTRVL LGNESCTL TLSESTMNTLKCTVGPAMNKHFNMSI IISNGHGTTQYSTFSYVDPVITSISPKYGPMAGGTLLTLTGN YLNSGNSRHISIGGKTCTLKSVSNSILECYTPAQTISTEFAVKLKIDLANRETSI FSYREDPIVYEIH PTKSFISTWWKEPLNIVSFLFCFASGGSTITGVGKNLNSVSVPRMVINVHEAGRNFTVAC QHRSNSEI ICCTTPSLQQLNLQLPLKTKAFFMLDGILSKYFDLIYVHNPVFKPFEKPVMISMGNENVL EIKGNDID PEAVKGEVLKVGNKSCENIHLHSEAVLCTVPNDLLKLNSELNIEWKQAISSTVLGKVIVQ PDQNFTGL IAGVVSISTALLLLLGFFLWLKKRKQIKDLGSELVRYDARVHTPHLDRLVSARSVSPTTE MVSNESVD

YRATFPEDQFPNSSQNGSCRQVQYPLTDMSPILTSGDSDISSPLLQNTVHIDLSALN PELVQAVQHVV IGPSSLIVHFNEVIGRGHFGCVYHGTLLDNDGKKIHCAVKSLNRITDIGEVSQFLTEGI IMKDFSHPN VLSLLGICLRSEGSPLVVLPYMKHGDLRNFIRNETHNPTVKDLIGFGLQVAKGMKYLASK KFVHRDLA ARNCMLDEKFTVKVADFGLARDMYDKEYYSVHNKTGAKLPVKWMALESLQTQKFTTKSDV WSFGVLLW ELMTRGAPPYPDVNTFDITVYLLQGRRLLQPEYCPDPLYEVMLKCWHPKAEMRPSFSELV SRI SAIFS TFIGEHYVHVNATYVNVKCVAPYPSLLSSEDNADDEVDTRPASFWETS

Of which:

MKAPAVLAPGILVLLFTLVQRSNG : signal peptide

ECKEALAKSEMNVNMKYQLPNFTAETPIQNVILHEHHI FLGATNYIYVLNEEDLQKVAEYKT

GPVLEHPDCFPCQDCSSKANLSGGVWKDNINMALVVDTYYDDQLISCGSVNRGTCQR HVFPHNHTADI QSEVHCIFSPQIEEPSQCPDCVVSALGAKVLSSVKDRFINFFVGNTINSSYFPDHPLHSI SVRRLKET KDGFMFLTDQSYIDVLPEFRDSYPIKYVHAFESNNFIYFLTVQRETLDAQTFHTRIIRFC SINSGLHS YMEMPLECILTEKRKKRSTKKEVFNILQAAYVSKPGAQLARQIGASLNDDILFGVFAQSK PDSAEPMD RSAMCAFPIKYVNDFFNKIVNKNNVRCLQHFYGPNHEHCFNRTLLRNSSGCEARRDEYRT EFTTALQR VDLFMGQFSEVLLTSISTFIKGDLTIANLGTSEGRFMQVVVSRSGPSTPHVNFLLDSHPV SPEVIVEH TLNQNGYTLVITGKKITKIPLNGLGCRHFQSCSQCLSAPPFVQCGWCHDKCVRSEECLSG TWTQQICL PAIYKVFPNSAPLEGGTRLTICGWDFGFRRNNKFDLKKTRVLLGNESCTLTLSESTMNTL KCTVGPAM

NKHFNMSI IISNGHGTTQYSTFSYVDPVITSISPKYGPMAGGTLLTLTGNYLNSGNSRHISIGGKTCT LKSVSNSILECYTPAQTISTEFAVKLKIDLANRETSIFSYREDPIVYEIHPTKSFISGGS TITGVGKN LNSVSVPRMVINVHEAGRNFTVACQHRSNSEI ICCTTPSLQQLNLQLPLKTKAFFMLDGILSKYFDLI YVHNPVFKPFEKPVMISMGNENVLEIKGNDIDPEAVKGEVLKVGNKSCENIHLHSEAVLC TVPNDLLK LNSELNIEWKQAISSTVLGKVIVQPDQNFT : ECD of human cMET

GLIAGVVSISTALLLLLGFFLWL : transmembrane region

KKRKQIKDLGSELVRYDARVHTPHLDRLVSARSVSPTTEMVSNESVDYRATFPEDQF PNSSQ

NGSCRQVQYPLTDMSPILTSGDSDISSPLLQNTVHIDLSALNPELVQAVQHVVIGPS SLIVHFNEVIG RGHFGCVYHGTLLDNDGKKIHCAVKSLNRITDIGEVSQFLTEGIIMKDFSHPNVLSLLGI CLRSEGSP LVVLPYMKHGDLRNFIRNETHNPTVKDLIGFGLQVAKGMKYASKKFVHRDLAARNCMLDE KFTVKVAD FGLARDMYDKEYYSVHNKTGAKLPVKWMALESLQTQKFTTKSDVWSFGVLLWELMTRGAP PYPDVNTF DITVYLLQGRRLLQPEYCPDPLYEVMLKCWHPKAEMRPSFSELVSRISAIFSTFIGEHYV HVNATYVN VKCVAPYPSLLSSEDNADDEVDTRPASFWETS: intracellular region Reference antibodies

Anti-cMET Antibodies are known in the art (Table 1). Monospecific bivalent cMET antibodies were constructed according to published information and expressed in 293F Freestyle cell. Table 1 shows the related disclosed information. Monospecific bivalent antibodies directed against cMET were constructed according to published information and expressed in 293F Freestyle cells. For HGF ligand blocking assays VH- and VL- encoding gene segments of patent- derived anti-cMET antibodies were re-cloned in a phage display vector for display on filamentous bacteriophage.

Reference antibody cetuximab (Erbitux) was used as reference antibody for the EGFR Fab panel.

2994 Fab protein was generated from purified PG2994 IgG by papain digestion. Therefore PG2994 was incubated with papain coupled on beads (Pierce #44985), and allowed to digest for 5.5 hour at 37°C under rotation. Fab fragments were purified from the digestion mixture by filtration over MabSelectSure LX. Flow through fractions containing Fab protein, concentrated to 3 ml using vivaspin20 10 kDa and further purified by gel filtration using a superdex75 16/600 column in PBS.

Example 2

Generation of bivalent monoclonal antibodies and antibody characterization

VH genes of unique antibodies, as judged by VH gene sequence and some sequence variants thereof, were cloned in the backbone IgGl vector. Suspension adapted 293F Freestyle cells were cultivated in T125 flasks at a shaker plateau until a density of 3.0 x 10 ⁶ cells/ml. Cells were seeded at a density of 0.3-0.5 x 10 ⁶ viable cells/ml in each well of a 24-deep well plate. The cells were transiently transfected with the individual sterile DNA: PEI mixture and further cultivated. Seven days after transfection, supernatant was harvested and filtrated through 0.22 uM (Sartorius) and purified on protein A beads using batch purification followed by a buffer exchange to PBS.

Cross block assay cMET antibodies cMET specific phages were tested for competition with cMET reference antibodies in ELISA. Therefore 2.5pg/ml of cMET-Fc fusion protein was coated overnight to MAXISORPTM ELISA plates at 4°C. Wells of the ELISA plates were blocked with PBS (pH 7.2) containing 2% ELK for 1 H at RT while shaking (700rpm). Next reference or negative control IgG was added at a concentration of 5 ug/ml and allowed to bind for 15 min at RT at700rpm. Next, 5id of PEG precipitated phage was added and allowed to bind for 1H at RT at 700rpm. Bound phages were detected with HRP labelled anti-M13 antibody for 1H at RT at 700rpm. As a control the procedure was performed simultaneously with an antibody specific for the coated antigens and a negative control phage. Bound secondary antibody was visualized by TMB/H2O2 staining and staining was quantified by means of OD450nm measurement. Table 2 demonstrates that MF4040 and MF4356 show competition with the 5D5 reference antibody. MF4297 competes with 13.3.2 and C8H241 to a lesser extent. The positive control phages all show complete competition with the corresponding IgG, whereas the no antibody control, does not influence the competition assay. Generation of bispecific antibodies

Bispecific antibodies were generated by transient co-transfection of two plasmids encoding IgG with different VH domains, using a proprietary CH3 engineering technology to ensure efficient heterodimerisation and formation of bispecific antibodies. The common light chain is also co-transfected in the same cell, either on the same plasmid or on another plasmid. In our co-pending applications (e.g. WO2013/157954 and WO2013/157953; incorporated herein by reference) we have disclosed methods and means for producing bispecific antibodies from a single cell, whereby means are provided that favor the formation of bispecific antibodies over the formation of monospecific antibodies. These methods can also be favorably employed in the present disclosure. Specifically, preferred mutations to produce essentially only bispecific full length IgG molecules are amino acid substitutions at positions 351 and 366, e.g. L351K and T366K (numbering according to EU numbering) in the first CH3 domain (the 'KK- variant' heavy chain) and amino acid substitutions at positions 351 and 368, e.g. L351D and 10 L368E in the second CH3 domain (the 'DE-variant' heavy chain), or vice versa. It was previously demonstrated in our co-pending applications that the negatively charged DE-variant heavy chain and positively charged KK- variant heavy chain preferentially pair to form heterodimers (so-called 'DEKK' bispecific molecules). Homodimerization of DE-variant heavy chains (DE-DE homodimers) or KK-variant heavy chains (KK-KK homodimers) are disfavored due to strong repulsion between the charged residues in the CH3-CH3 interface between identical heavy chains.

Table 3 shows which cMET and EGFR Fab arms were cloned in the appropriate KK and DE vectors. After production, bispecific IgG were purified by protein-A batch purification and the buffer was exchanged to PBS. Successful productions resulted in an IgGl full length antibody, with a minimal concentration of 0.1 mg/ml, which were assigned a unique code (PBnnnnn; where nnnnn represents a randomly generated number) to identify the specific combination of 2 different target binding Fab fragments. Successfully produced bispecific IgG were tested for binding to their respective targets in ELISA. Reference is made herein to PCTNL/2018/050537 (published as WO2019/031965) for more details on production of bispecific antibodies.

Example 3

Screening of c-MET x EGFR bispecific antibodies in an EGF/HGF and HGF and EGF proliferation assay

The potency of a panel of cMET x EGFR bispecific antibodies was tested in N87 cells using an HGF/EGF, HGF and EGF assays. The N87 cell line, official name NCI- N87, is a gastric carcinoma cell line derived from a metastatic site and has high EGFR expression levels and intermediate cMET expression levels (Zhang et al, 2010). Antibodies were tested in an 8 steps semi-log titration ranging from 10 pg/ ml to 3.16 ng/ml. Each antibody was tested in duplicate. The anti-RSV-G antibody PG2708 was used as negative control. The reference antibody 2994 Fab was used as positive control for the HGF assay and the reference antibody cetuximab was used as positive control for the EGF assay.

An equimolar 1:1 cetuximab/5D5 Fab was used as positive control for the EGF, HGF and EGF/HGF assays. Wells with either one, or a combination of ligand, as well as medium control were included to determine the assay window. Antibodies were diluted in chemically defined starvation medium (CDS: RPMI1640 medium, containing 80U penicillin and 80pg of streptomycin per ml, 0.05% (w/v) BSA and lOpg/ml holo-transferrin) and 50pl of diluted antibody was added to the wells of a 96 wells black well clear bottom plate (Costar). Ligand was added (50pl per well of a stock solution containing 400ng/ml HGF and 4ng/ml of EGF, and a EGF/HGF concentration of 4 ng/ml EGF/400 ng/ml HGF diluted in CDS: R&D systems, cat. nr. 396-HB and 236-EG). N87 cells were trypsinised, harvested and counted and 8000 cells in lOOpl of CDS were added to each well of the plate. To avoid edge effects, plates were left for an hour at RT before being put in a container inside a 37°C cell culture incubator for three days. On the fourth day, Alamar blue (Invitrogen, # DALI 100) was added (20pl per well) and the fluorescence was measured after 6 hours of incubation (at 37°C) with Alamar blue using 560nm excitation and 590nm readout on a Biotek Synergy 2 Multi-mode microplate reader. Fluorescence values were normalised to uninhibited growth (no antibody, but both ligands added).

Table 4 lists the results of the various experiments. In the N87 HGF/EGF assay, fourteen different cMETxEGFR bispecifics with potency comparable to the reference monospecific antibodies (equimolar mix of cetuximab and 5D5 Fab) were identified: PB7679, PB7686, PB8218, PB8244, PB8292, PB8316, PB8340, PB8364, PB8388, PB8511, PB8535, PB8583, PB8607 and PB8640.

In the N87 EGF assay, eleven different cMETxEGFR bispecifics with potency comparable to monospecific cetuximab were identified: PB7679, PB8244, PB8292, PB8340, PB8364, PB8388, PB8511, PB8535, PB8583, PB8607 and PB8640. They all contain the EGFR Fab arm MF3755. In the HGF N87 assay nine bispecifics were identified that showed a higher potency compared to the monospecific 5D5 Fab reference antibody: PB8218, PB8388, PB8511, PB8532, PB8535, PB8545, PB8583, PB8639 and PB8640. They contain six different cMET Fab arms MF4040, MF4297, MF4301, MF4356, MF4491 and MF4506.

ADCC activity

The ADCC activity of the 24 cMetxEGFR bispecifics was tested to the tumor cell lines N87 (EGFR-high, cMET-low) and MKN-45 (EGFR-low, cMET- amplified). The ADCC assay was performed using the Promega ADCC Bioassay kit in 384-well plate format. Antibodies were tested in duplicate at 9 different concentrations in semi-log serial dilutions ranging from 10 pg/ml to 1 ng/ml.

The reference cetuximab antibody was included as a positive control for the assay and PG2708 was used as negative control antibody. Antibodies or assay medium control (no IgG) were incubated for 6 hours of induction at 37°C with ADCC effector cells, and target cells (N87 or MKN-45). Luciferase activity was quantified using Bio-Gio luciferase reagent.

An example of the ADCC assay is shown in Figure 15 of WO2019/031965. None of the cMETxEGFR bispecifics showed a significant ADCC activity in both cell lines. The positive control reference cetuximab antibody showed a dose-dependent ADCC activity to both cell lines. Five bispecifics composed of EGFR and cMet arms which did show high efficacy in the N87 HGF/EGF assay and showed high sequence diversity (Table 5) were selected for further analysis. Two from the five bispecifics contain MF4356, which competes with 5D5 for binding to cMET (Table 2). Table 5 summarizes the characteristics of the selected candidates.

Example 4

Figure 2 depicts various sequences for alternative variable regions of the heavy chain of an EGFR binding variable domain as disclosed herein. Figure 3 depicts various sequences for alternative variable regions of the heavy chain of a cMET binding variable domain as disclosed herein. The heavy chain variable regions were used to create a number of different cMET x EGFR bispecific antibodies. The light chain in these antibodies has the sequence as depicted in figure 4B. Bispecific antibodies were produced as described in example 1. The antibodies were also produced as an ADCC enhanced version. ADCC enhanced versions were produced by including in the co- transfection of the antibody constructs, a DNA encoding a reductase enzyme that removes a fucose residue from the Fc region of IgGl. See table 6 for a list of bispecific antibodies used and their PB coding.

Example 5

The heavy chain variable region (VH) of the cMET variable domain of PB8532 comprises the amino acid of MF4356 as depicted for instance in figure 3. The VH of the cMET variable domain of PB 19748 comprises the amino acid sequence of MF8230 (see figure 3). The VH of the EGFR variable domain of PB8532 comprises the amino acid of MF3370 as depicted for instance in figure 2. The VH of the EGFR variable domain of PB19748 comprises the amino acid sequence of MF8233 of figure 2. The light chain in PB8532 and PB19748 is the same and is depicted in figure 4B. The cMET antibody LY2875358 antibody is among other described in Kim and Kim 2017.

Example 6

Efficiency of bispecific antibody PB19478 and Osimertinib in EGFR exon 20 insertion NSCLC models

The aim of this study was to evaluate the anti-tumor efficacy of antibody PB 19478 alone and in combination with osimertinib in patient-derived tumor xenograft (PDX) non- small cells lung cancer (NSCLC) model with EGFR exon 20 insertions. The EGFR exon 20 insertions (“EGFRex20ins”) represent a class that encode mutants with amino acid insertions clustered between positions 762 and 774 that result in constitutive activation of EGFR. EGFR exon 20 insertions confer resistance to approved EGFR TKIs in human subjects with cancer haboring such mutations and are associated with poor prognosis.

Material:

PB19748 was generated by Merus and control material was the PB19748 vehicle and consisted of 12% of the PB19748 formulation buffer without antibody, although other negative controls such as saline or PBS may also be used. PDX Model characteristics:

The model LXFE2478 was generated at Charles River in nude mice which have functional Fc-effector cells.

This model carries mutation EGFRex20ins (M766_A767insASV). It also carries a point mutation (E168D) in the SEMA domain of c-MET, which is located at the ligand binding site of c-MET.

Insertion of 9 nucleotides in exon 20 in this model affects the EGFR tyrosine kinase domain (M766X) and confers resistance to small molecule EGFR inhibitors. This model carries no mutations in B-RAF, H-/N- and KRAS and PTEN genes (whole exome sequencing was done for both patient tumor and tumor xenograft with matching results).

Expression of EGFR, c-MET and HGF in LXFE2478 PDX model

The model LXFE2478 was analysed for expression of EGFR and c-MET receptors and ligand huHGF, in order to investigate its suitability for in vivo studies.

Two tumor pieces were evaluated for expression of the EGFR and c-MET receptors, and that of the ligand huHGF, via western blot. huHGF was included in the analysis. Tumor samples obtained from the PDX model were assessed by Simple Western Size technology (SWS) to confirm expression of EGFR, HGF, and c-MET.

Western blotting revealed that LXFE2478 model expressed EGFR, HGF and MET on protein level (Table 7).

Statistical methods:

Antitumor efficacy of all groups was assessed using the control vehicle/placebo buffer group as a reference. Tumor growth inhibition was determined by the comparison of RTVs of the test groups with the control group and is expressed as minimum T/C value in percent. For the evaluation of the statistical significance of antitumor efficacy, the non-parametric Kruskal-Wallis test followed by Dunn’s method for multiple comparisons was performed. Individual RTVs of test and control groups were compared on days on which the minimum T/C values were achieved in the test groups. Statistical analysis was only carried out if at least 50% of the initially randomized animals still remained in the relevant group. Comparisons between test groups were carried out for the same days. All p-values < 0.05 were considered statistically significant. Statistical calculations were performed using GraphPad Prism bioanalytic software (version 9.10 for Microsoft Windows, GraphPad Software, San Diego, California, USA. Treatment schedules and methods:

The anti-tumor efficacy of the combination of antibody and osimertinib was assessed in LXFE2478 NSCLC PDX tumor model. Figure 6 shows a schematic overview of the treatment schedules.

Tumors were harvested from the donor PDX mice, cut into fragments (LXFE2478: 3-4 mm edge length) and inoculated into recipient nude mice subcutaneously (SC) in the flank. When the tumor implants reached approximately 80- 200 mm3 in a sufficient number of animals, mice were assigned to groups. Randomization was performed based on “stratified distribution” method. Treatment started on the same day as randomization (day 0).

Mice were treated with antibody, osimertinib, or with a combination of the two compounds for 4 weeks, followed by a dose-free observation period of up to 100 days. Treatment started the same day as enrollment (day 0). Mice were terminated during the observation period if the tumor volume was >1000 mm3.

Antibody was administered once a week intra peritoneally (IP) at 5 mg/kg and 25 mg/kg dose over a period of 5 weeks. Osimertinib was administered every day (QD) per oral gauge (PO) at 5 mg/kg, 25 mg/kg over a period of 30 days. Mice in group 1 were treated with vehicle 1 (antibody buffer) once a week for 5 weeks, or with vehicle 2 (osimertinib buffer) every day for 30 days. The treatment plan is shown in Table 8.

After randomization, animals were routinely monitored for morbidity and mortality, they were weighted twice a week, and tumor volumes (TV) were determined twice a week with a caliper. Relative body weight (RBW) was calculated by dividing the absolute weight or volume on a certain day by the absolute weight at day 0 multiplied by 100. Relative tumor volumes (RTV) were calculated by dividing the absolute individual tumor volume on a certain day by the absolute tumor volume at day 0 multiplied by 100.

For mice that lost >10% body weight, weight was measured daily, and animals had facilitated access to feed and water, and access to DietGel. For mice that lost >15% body weight, therapy was suspended until they regained a RBW of >90%.

A dose-free observation period was included to compare duration of response and relapse (tumor re-growth) after treatment end. In addition, a low dose of antibody was included to create a window for tumor growth inhibition comparison.

Due to significant body weight loss (>10%) observed with Osimertinib 25 mg/kg treatment (Groups 2 and 6), these 2 groups were not administered the therapeutic compound between Day 11-14. Body weight recovered during this dosing-free period. Body weight loss (>10%) was again observed on Day 28 in these groups, after which it was decided to stop the Osimertinib 25 mg/kg dosing on Day 28. Combination of Osimertinib at 25 mg/kg with bispecific antibody PB19478 did not increase any adverse effects observed with 25 mg/kg osimertinib treatment.

Animal survival curves of this experiment are shown in Figure 7. Log-rank (Mantel-Cox) test of combination vs. each corresponding monotherapy group is significant as follows:

• Osimertinib 25 mg/kg vs. antibody + Osimertinib 25 mg/kg P=0.0102

• Antibody 25 mg/kg vs. antibody + Osimertinib 25 mg/kg P=0.0344

• Osimertinib 5 mg/kg vs. antibody + Osimertinib 5 mg/kg P=0.0006

• Antibody 5 mg/kg vs. antibody + Osimertinib 5 mg/kg P=0.0162

Individual tumor volumes at day 28 are shown in Figure 8. Statistical analysis was done using mixed model with Tukey’s post-test (Graphpad Prism). All 25 mg/kg groups induced tumor stasis or regression, so comparisons of combination groups with the corresponding monotherapy group was significant for the 5 mg/kg groups only. All groups were significantly different to the Vehicle group on Day 28.

Figure 9 shows the effects of bispecific antibody PB 19478 and osimertinib monotherapies and combination therapy on tumor volume in NSCLC PDX model during the complete observation period with treatment stop indicated on Day 28.

For monotherapy treatment, a dose-dependent anti-tumor efficacy of bispecific antibody PB19478 and osimertinib was observed. At 5mg/kg, the model shows reduced sensitivity to Osimertinib treatment. In addition, combination of antibody and osimertinib enhanced tumor growth inhibition compared to both corresponding monotherapy groups.

After treatment stop (Day 28), antibody and osimertinib combination therapy significantly prolonged progression-free survival (defined as a tumor size < 750 mm3) compared with antibody or osimertinib alone. Log-rank (Mantel-Cox) test showed P<0.05 for each combination group vs. the corresponding monotherapy groups. At 11 weeks post-treatment, only 1 of 9 mice (11%) in the 25 mg/kg combination group of showed xenograft progression, whereas 5/9 and 6/9 xenografts in the 25 mg/kg antibody and 25 mg/kg osimertinib arms progressed to 750 mm3 (Figure 9).

In summary, these results show preclinical antitumor activity of antibody against an NSCLC PDX model harbouring an EGFR exon20 insertion mutation (H773- V774insNPH). The combination of antibody with osimertinib enhances tumor growth inhibition and extends progression-free survival in this model.

Example 7

Efficiency of bispecific antibody PB19478 and osimertinib in EGFR exon 19 deletion NSCLC model. The aim of this study was to evaluate the anti-tumor efficacy of antibody PB19478 alone and in combination with osimertinib in a cell line-derived tumor xenograft (CDX) non-small cell lung cancer (NSCLC) model with an EGFR exon 19 deletion. This adenocarcinoma cell line, HCC827-ER1, harbors an activating EGFR mutation in exon 19 (deletion E746-A750), and is resistant to approved EGFR TKIs such as erlotinib.

Material:

PB19748 was generated by Merus as mentioned in Example 5.

CDX Model characteristics:

The model HCC827-ER1 was generated in BALB/c nude mice. It carries an EGFR mutation in exon 19 (deletion E746-A750), and has amplified c-MET copy number and Axl expression compared to the wild type HCC827 cell line. The HCC827-ER1 cell line is resistant to EGFR TKI erlotinib and was generated via repeated in vitro exposure of the wild type cell line to escalating concentrations of erlotinib.

Experimental procedures:

Each mouse was inoculated in the right front flank region with HCC827-ER1 tumor cells mixed with Matrigel for tumor development. The randomization started when the mean tumor size reached approximately 125 (75-175) mm ³ . Totally 32 tumor-bearing mice were enrolled in the tumor efficacy study and randomly allocated to 4 different groups, as shown in table 9, with 8 mice per group. The date of randomization was denoted as day 0 and dosing was initiated from day 0. Mice were treated with antibody, osimertinib, or with a combination of the two compounds for 3 weeks, followed by a dose- free observation period of up to 74 days. The dose-free observation period was included to compare duration of response and relapse (tumor re-growth) after treatment end. Mice with tumor volume exceeding 1500 mm ³ , or with body weight loss over 20% relative to the weight at the first day of treatment were terminated during the treatment period. Antibody was administered twice a week intra peritoneally (i.p.) at 25 mg/kg dose over a period of 21 days, with a total of 7 doses. Osimertinib was administered every day (QD) per oral gauge (p.o.) at 25 mg/kg over a period of 21 days with a total of 22 doses. Eight mice in group 1 were treated with vehicle control (placebo buffer). A schematic overview of the treatment schedules is shown in figure 10. Tumor volume (mm ³ ) was measured twice per week in 2 dimensions using a caliper. The animals were checked for any effects of tumor growth and treatments on behavior such as mobility, food and water consumption, body weight gain/loss, eye/hair matting and any other abnormalities. Body weights were measured twice per week after randomization. None of the mice lost more than 15% body weight, and there was no suspension of administration of a therapeutic agent in any of the treatment Groups.

Figure 11 shows the effects of bispecific antibody PB19478 and osimertinib monotherapies and combination therapy on tumor volume in NSCLC CDX model during the complete observation period with treatment stop indicated on day 21. The monotherapies with antibody or osimertinib showed tumor regression compared to the vehicle control group. However, the combination therapy of antibody with osimertinib showed a strong synergistic effect and complete tumor regression compared to any other treatment groups. All treatments were well tolerated. No mice in the study showed body weight loss >10% from starting body weight. Apart from tumor scabbing or tumor ulceration in some mice in Groups 1, 2 and 3, no adverse events were observed. No adverse events were reported for Group 4. Mild body weight loss (between 5-10%) was observed in only one mouse in Group 4.

Animal survival curves are shown in figure 12. Log-rank (Mantel-Cox) test of combination vs. each corresponding monotherapy group is significant as follows:

• Antibody 25 mg/kg+ Osimertinib 25 mg/kg vs. Antibody 25 mg/kg P=0.0009

• Antibody 25 mg/kg+ Osimertinib 25 mg/kg vs. Osimertinib 25 mg/kg P<0.0001

In summary, these results show preclinical antitumor activity of the antibody under investigation against an NSCLC CDX model harboring a mutation in exon 19 (deletion E746-A750). The combination of antibody with osimertinib enhances tumor growth inhibition and extends progression-free survival in this model.

Example 8

In the dose escalation phase, bispecific antibody PB 19478 will be administered with increasing doses to patients with NSCLC harboring an activating EGFR mutation (TKI sensitizing mutations and/or approved TKLresistance mutations) or an activating c- MET mutation (exon 14 skipping)/amplification (MET/CEP7 > 5 or cfDNA > 2 copies), or c-MET amplification (MET/CEP7 > 5 or cfDNA > 2 copies), who in all cases, have progressed after receiving prior therapy for advance d/metastatic disease.

Allometric scaling of a preclinical PK model was used to predict antibody exposure in humans. The antibody starting dose is 100 mg (flat dose, intravenously) once every 2 weeks (q2w), with 4- week cycles (28 days). Five dose levels are planned to be investigated between 100-3000 mg. Cohorts of patients will be treated with antibody until the MTD is reached or a lower recommended dose(s) is established.

The RP2D (recommended phase 2 dosing) is defined as the dose at or below the MTD, taking into account available data for PK, pharmacodynamic activity, and preliminary antitumor activity.

Dose expansion

Using an RP2D of 1500 mg in combination with Osimertinib given at 80 mg oral daily dose, the planned expansion cohorts in the phase 2 part can be initiated. The safety of the RP2D will be confirmed during dose expansion in the first 12 patients treated with antibody alone, for at least 2 cycles (recruitment will continue in the meanwhile). Antitumor activity of antibody (alone or in combination with osimertinib) will be evaluated in terms of ORR, and an evaluation of other efficacy parameters, safety, tolerability, PK, immunogenicity, and biomarkers will be performed.

The following cohorts of locally advanced unresectable/metastatic solid tumors may be opened:

Expansion cohort 1: PB19478 + Osimertinib as first line treatment of NSCLC Expansion cohort 2: PB19478 + Osimertinib as second line treatment of NSCLC in an Osimertinib resistant population

Osimertinib will be administered at 80mg dose once per day. Administration may be with or without food. It’s preferable to take the dose in the morning (i.e. after waking up). On days of antibody infusion, the dose has to be taken before the infusion. If a dose is missed it should not be substituted, rather the patient should wait until the next planned dose.

STUDY POPULATION

Inclusion criteria

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

1. Signed informed consent before initiation of any study procedures.

2. Age > 18 years at signature of informed consent.

3. Histologically or cytologically confirmed solid tumors with evidence of metastatic or locally advanced unresected disease that is incurable.

1. Dose Escalation Part - Patients who have failed prior standard firsLline treatment. Patients must have progressed on or be intolerant to therapies that are known to provide clinical benefit. There is no limit to the number of prior treatment regimens. Patients must have:

• Non-small cell lung cancer (NSCLC) harboring activating EGFR mutations including tyrosine kinase inhibitor (TKI) sensitizing mutations (e.g., 19del and L858R), and/or approved TKLresistance mutations (e.g., acquired TKLresistance mutations, i.e., T790M, C797S, L792, L798I, exon 20 insertion), or any activating c-MET mutation/amplification (e.g., high-level c-MET amplification [MET/CEP7 > 5 or cfDNA > 2 copies], or c-MET exon 14 skipping mutation).

*NOTE: Patient identification will be based on previous treatment history with EGFR tyrosine kinase inhibitors and on local tests performed in CLIA-certified laboratories. 2. Cohort Expansion Part - For > 2L patients must have progressed on or be intolerant to therapies that are known to provide clinical benefit. There is no limit to the number of prior treatment regimens.

Patients must have:

Cohort 1: NSCLC first line treatment, harboring EGFR sensitizing mutations (such as Del 19, L858R) or NSCLC treatment naive for advanced disease with EGFR sensitizing mutation.

Cohort 2: NSCLC osimertinib resistance I chemo naive patients, or NSCLC osimertinib resistance and progressed after 3 months of treatment with osimertinib with at least a stable disease reported.

4. Availability of archival or a fresh tumor tissue sample (preferred in escalation, mandatory in expansion).

5. Measurable disease as defined by RECIST version 1.1 by radiologic methods (patients with non-measurable but evaluable disease can be included in the dose escalation part).

6. Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1.

7. Life expectancy > 12 weeks, as per Investigator judgment

8. Adequate organ function, as per Investigator judgment

• Absolute neutrophil count (ANC) >1.5 X 109/L

• Hemoglobin >9 g/dL

• Platelets >100 x 109/L

• Corrected total serum calcium within normal ranges

• Serum potassium within normal ranges

• Serum magnesium within normal ranges (or corrected with supplements)

• Alanine aminotransferase (ALT), aspartate aminotransferase (AST) <3 x upper limit of normal (ULN) and total bilirubin <1.5 x ULN (patients with Gilbert’s syndrome are eligible if conjugated bilirubin value is within normal limits); in cases of liver involvement, ALT/AST <5 x ULN and total bilirubin <2 x ULN will be allowed

• Serum creatinine <1.5 x ULN or creatinine clearance >50 mL/min calculated according to the Cockroft and Gault formula or MDRD formula for patients aged >65 years

• Serum albumin >3.3 g/dL

Exclusion Criteria

The presence of any of the following criteria excludes a patient from participating in the study:

1. Central nervous system metastases that (not exclusionary in escalation, mandatory in expansion):

• Are untreated or symptomatic (patients with untreated, asymptomatic lesions can be included if in the investigator judgment considered stable)

• require radiation or surgery

• require continued steroid therapy (> 10 mg prednisone or equivalent) to control symptoms within 14 days of study entry.

2. Known leptomeningeal involvement.

3. Participation in another clinical study or treatment with any investigational drug within 4 weeks prior to study entry. 4. Systemic anticancer therapy or immunotherapy within 4 weeks or 5 half-lives, whichever is shorter, of the first dose of study drug. For cytotoxic agents that have major delayed toxicity (e.g., mitomycin C, nitrosoureas), a washout period of 6 weeks is required. Note: For agents with long half-lives, enrollment before the fifth half-life requires Sponsor approval.

5. Major surgery or radiotherapy within 3 weeks of the first dose of study drug. Patients who received prior radiotherapy to >25% of bone marrow at any time are not eligible.

6. Persistent grade >1 clinically significant toxicities, in the Investigator judgment, related to prior antineoplastic therapies (except for alopecia); stable sensory neuropathy < grade 2 NCI-CTCAE v5.0 and hypothyroidism < grade 2 which is stable on hormone replacement are allowed.

7. History of hypersensitivity reaction or any toxicity attributed to human proteins or any of the excipients that warranted permanent cessation of these agents.

8. History of clinically significant cardiovascular disease including, but not limited to:

• Prolonged QT interval > 480 msec, obtained from 3 electrocardiograms (ECGs), or clinically significant cardiac arrythmia or electrophysiologic disease (i.e., placement of implantable cardioverter defibrillator or atrial fibrillation with uncontrolled rate), or any factors that increase the risk of QTc prolongation or risk of arrhythmic events such as electrolyte abnormalities. Patients with cardiac pacemakers who are clinically stable are eligible.

• Heart failure, congenital long QT syndrome, family history of long QT syndrome, or unexplained sudden death under 40 years of age in first-degree relatives or any concomitant medication known to prolong the QT interval and cause Torsades de Pointes.

• Uncontrolled (persistent) arterial hypertension: systolic blood pressure > 180 mm Hg and/or diastolic blood pressure > 100 mm Hg.

• Congestive heart failure (CHF) defined as New York Heart Association (NYHA) class III-IV or hospitalization for CHF within 6 months of the first dose of study drug.

9. History of interstitial lung disease including drug-induced interstitial lung disease, radiation pneumonitis that requires treatment with prolonged steroids or other immune suppressive agents within 1 year.

10. Previous or concurrent malignancy, excluding non-basal cell carcinomas of skin or carcinoma in situ of the uterine cervix, unless the tumor was treated with curative or palliative intent and in the opinion of the Investigator, with Sponsor agreement, the previous or concurrent malignancy condition does not affect the assessment of safety and efficacy of the study drug.

11. Current serious illness or medical conditions including, but not limited to uncontrolled active infection, clinically significant pulmonary, metabolic or psychiatric disorders.

12. Active Hepatitis B infection (HBsAg positive) without receiving antiviral treatment. Note: Patients with active hepatitis B (HbsAg positive) must receive antiviral treatment with lamivudine, tenofovir, entecavir, or other antiviral agents, starting at least > 7 days before the initiation of the study treatment. Patients with antecedents of Hepatitis B (anti-HBc positive, HbsAg and HBV-DNA negative) are eligible.

13. Positive test for Hepatitis C ribonucleic acid (HCV RNA); Note: Patients in whom HCV infection resolved spontaneously (positive HCV antibodies without detectable HCV-RNA) or those who achieved a sustained virological response after antiviral treatment and show absence of detectable HCV RNA > 6 months (with the use of IFN- free regimens) or > 12 months (with the use of IFN-based regimens) after cessation of antiviral treatment are eligible.

14. Known history of HIV (HIV 1/2 antibodies). Patients with HIV with undetectable viral load are allowed. HIV testing is not required unless mandated by local health authority or regulations.

15. Sexually active male and female patients of childbearing potential must agree to use one of the following highly effective methods of birth control during the entire duration of the study and for 6 months after final administration of PB19478:

• combined (estrogen and progestogen containing) hormonal contraception associated with inhibition of ovulation (oral, intravaginal, transdermal)

• progestogen-only hormonal contraception associated with inhibition of ovulation (oral, injectable, implantable)

• intrauterine device (IUD)

• intrauterine hormone-releasing system (IUS)

• bilateral tubal occlusion

• vasectomized partner

• sexual abstinence

16. Pregnant or breast-feeding women are excluded from this study.

INVESTIGATIONAL THERAPY AND REGIMEN

Antibody will be administered as an IV infusion at a dose level of 1500 mg (flat dose) as the RP2D level. The administered dose, dose increments, and frequency of dosing for each patient (including the expansion cohorts) is subject to change based on patient safety, PK and pharmacodynamic data, and upon recommendation of the Sponsor. The Sponsor may recommend the use of an alternate weekly dosing schedule for Cycle 1.

Treatment duration

Study treatment will be administered until confirmed progressive disease (as per RECIST vl.l), unacceptable toxicity, withdrawal of consent, patient non-compliance, Investigator decision (e.g., clinical deterioration), or Antibody interruption >6 consecutive weeks.

Patients will be followed up for safety for at least 30 days following the last Antibody infusion and until recovery or stabilization of all related toxicities, and for disease progression and survival status every 3 months for up to 1 year.

Example 9

A 54 year-old female patient with non-small cell lung cancer that carried an EGFR 19 deletion and an EGFR mutation was dosed a combination of osimertinib and 1500mg q2w of the bispecific antibody PB19478 of the clinical trial protocol of Example 8. The patient had received prior treatment with osimertinib and an investigational antineoplastic drug. The patient exhibited a clinically relevant effect in the form of a confirmed Partial Response (PR) after 6 cycles of said antibody and 6 cycles of osimertinib. No adverse effects were observed with a grade other than Grade 1 or 2.

Example 10

A 61 year-old female patient with non-small cell lung cancer that carried an EGFR 19 deletion, an EGFR amplification and cMET amplification was dosed a combination of osimertinib (80mg q2w) and the bispecific antibody (1500mg q2w) PB19478 of the clinical trial protocol of Example 8. The patient had received prior treatment with Osimertinib. The patient exhibited a clinically relevant effect in the form of a confirmed PR after 3 cycles of said antibody and 4 cycles of osimertinib. The most severe adverse effect observed was hypokalaemia. No further adverse effects were observed with a grade other than Grade 1 or 2.

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Table 3. List of the 24 cMETxEGFR bispecifics antibodies selected after dose dependent titration experiments in a N87 HGF/EGF proliferation assay. The MF number of the EGFR and cMET arms in each individual PB as well as the their HCDR3 sequence are indicated.

Table 4. Summary of the antibody titration experiments done using N87 HGF/EGF, HGF and EGF proliferation assays with the 24 cMETxEGFR bispecific antibodies. Bispecifics are indicated as PBXXXX and the different Fab arms with MGXXXX. The activity of the bispecifics in the individual assays is indicated as: - no effect; + inhibition of proliferation lower than positive control; ++ = inhibition of proliferation comparable to positive control antibody 5D5 Fab; +++ = Inhibition of proliferation higher than positive control antibody 5D5 Fab.

Table 5. Composition of the most potent EGFRxcMET bispecific antibodies and their competition with reference antibodies. Table 6. Composition of bispecific antibodies. The pXX number indicates the number of the production run and can be used to identify whether the antibody was produced in an ADCC version or not.

Table 7: Relevant characteristics of model LXFE2478. AUC = area under the curve.

Table 8 I Treatment plan for PDX model LXFE2478. Table 9 I Treatment plan for CDX model HCC827-ER1. QD = once a day, BIW = twice a week, i.p. = intraperitoneal, p.o. = oral.