WO2025036910A1

WO2025036910A1 - Novel c-met binders and uses thereof

Info

Publication number: WO2025036910A1
Application number: PCT/EP2024/072831
Authority: WO
Inventors: Achim Doerner; Andreas Evers; Simon Krah
Original assignee: Merck Patent GmbH
Current assignee: Merck Patent GmbH
Priority date: 2023-08-15
Filing date: 2024-08-13
Publication date: 2025-02-20
Anticipated expiration: 2026-02-15

Abstract

The present invention relates to novel c-Met binders and uses thereof. Inter alia, the present invention relates to an antigen-binding peptide binding to c-Met and to a bispecific molecule comprising the antigen-binding peptide. The present invention further relates to an antigen-binding peptide-drug conjugate and to a composition comprising an antigen-binding peptide, a bispecific molecule, or an antigen-binding peptide-drug conjugate. The present invention also relates to an antigen-binding peptide, a bispecific molecule, an antigen-binding peptide-drug conjugate, or a composition, for use in a method of preventing or treating a cancer. Moreover, the present invention relates to an isolated nucleic acid encoding an antigen-binding peptide or a bispecific molecule, and to a recombinant cell comprising the isolated nucleic acid.

Description

Novel c-Met Binders and Uses Thereof

FIELD OF THE INVENTION

The present invention relates to novel c-Met binders and uses thereof. Inter alia, the present invention relates to an antigen-binding peptide binding to c-Met and to a bispecific molecule comprising the antigen-binding peptide. The present invention further relates to an antigenbinding peptide-drug conjugate and to a composition comprising an antigen-binding peptide, a bispecific molecule, or an antigen-binding peptide-drug conjugate. The present invention also relates to an antigen-binding peptide, a bispecific molecule, an antigen-binding peptide- drug conjugate, or a composition, for use in a method of preventing or treating a cancer. Moreover, the present invention relates to an isolated nucleic acid encoding an antigenbinding peptide or a bispecific molecule, and to a recombinant cell comprising the isolated nucleic acid.

BACKGROUND OF THE INVENTION c-MET (Mesenchymal-epithelial transition or hepatocyte growth factor receptor, HGFR) plays a key role as tumor driver in tumor indications of high medical need such as non-small cell lung cancer (NSCLC) and gastrointestinal cancers and as a mechanism of resistance to targeted therapies in NSCLC. c-MET is a tyrosine kinase receptor activated following binding of its ligand, HGF. This interaction leads to activation of several oncogenic signaling pathways, c- MET signaling is deregulated in diverse tumor types, including lung cancer, via c-MET overexpression, genomic amplification, autocrine/paracrine ligand stimulation, translocations, point mutations, and alternative splicing. In addition to small molecule inhibitors such as tepotinib, several monoclonal antibodies (mAbs) or more complex biotherapeutics targeting c-MET have been developed. Amongst those, early monovalent and bivalent mAb approaches relied on antagonism and ADCC as main modes of action and failed in clinical development. Next generation biologies were designed as bispecific antibodies such as amivantamab or MM-131 or focused on target degradation. One molecular mechanism for effective target degradation is high order crosslinking via biparatopic binding (Figure la). The underlying modes of action are enhanced apparent affinity for cellular binding, increased internalization by cross-linking, degradation and with it signal transduction inhibition, as well as higher potency cytotoxicity. Such enhanced target removal also provokes degradation of the therapeutic mAb or a corresponding ADC leading to enhanced intracellular payload delivery. An identification of fully human antibodies to distinct epitopes on SEMA and IPT1 domains of c-MET ECD (extracellular domain), termed B10V5 and CS06, respectively, was reported (Sellmann et al., 2016). Comparative docking studies were guided by experimental epitope binning data (Sellmann et al., 2016) and suggested paratope orientation likely supporting i inter- rather than intra-molecular c-MET ECD binding. Initial biparatopic designs yielded high apparent affinities and strong internalization capacities indicating applicability for MET degradation or biparatopic ADC approaches. While a sequence optimized variant of B10V5 revealed favorable binding and hydrophobicity properties in hydrophobic interaction chromatography (HIC), CS06 revealed a significantly delayed retention time in HIC. In the subsequent workflow, the significantly delayed retention time in HIC impeded conjugation of cytotoxic payloads during ADC generation. Due to their structural complexity, ADCs can generally be challenging to manufacture and prone to aggregation, representing an increased risk for immunogenic reactions and rapid clearance rates, particularly if hydrophobic linkers and payloads are involved. Anti-c-Met antigen-binding peptides, such as anti-c-Met antibodies or antigen-binding fragments thereof, should exhibit various characteristics, such as high affinity, high thermal stability, low hydrophobicity, and high potency. Furthermore, anti-c-Met antigen-binding peptides should ideally exhibit ADC conjugatability, i.e. the antigen-binding peptides can be manufactured into ADCs. However, it has been very difficult to obtain anti-c- Met antigen-binding peptides which exhibit these characteristics, particularly which exhibit a combination of these advantageous characteristics.

Therefore, there is a need for antigen-binding peptides, such as antibodies or antigen-binding fragments thereof, which exhibit these characteristics, particularly which exhibit a combination of these characteristics, for example antigen-binding peptides having high affinity, high thermal stability, low hydrophobicity, and high potency. There is also a need for antigen-binding peptides, such as antibodies or antigen-binding fragments thereof, which have improved capability for payload conjugation. Particularly, there is a need for enhanced antigen-binding peptides binding to c-Met, such as anti-c-Met antibodies or antigen-binding fragments thereof, and for enhanced bispecific molecules binding to c-Met; for example having enhanced thermal stability, enhanced affinity, reduced hydrophobicity, enhanced potency, and/or enhanced ADC conjugatability. For example, there is a need for enhanced antigenbinding peptides, such as antibodies or antigen-binding fragments thereof, which have a reduced hydrophobicity and an acceptable affinity. Furthermore, there is a need for ADCs, particularly anti-c-MET-ADCs. There is also a need to enhance cancer treatments, such as to provide a cancer treatment with enhanced selectivity and/ or specificity. Moreover, there is a continuous need to expand the repertoire of high affinity anti-c-MET antigen-binding peptides, such as anti-c-MET antibodies or antigen-binding fragments thereof, anti-c-MET bispecific molecules, and corresponding antibody-drug conjugates, particularly for use in preventing or treating cancer. SUMMARY OF THE INVENTION

In the following, the elements of the invention will be described. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine two or more of the explicitly described embodiments or which combine the one or more of the explicitly described embodiments with any number of the disclosed and/or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.

In a first aspect, the present invention relates to an antigen-binding peptide binding to c-Met, comprising a first domain, preferably a heavy chain variable domain, comprising a first complementarity-determining region comprising or consisting of an amino acid sequence GGTFSSNA (SEQ ID NO: 1), a second complementarity-determining region comprising or consisting of an amino acid sequence IIPXIX₂GTA (SEQ ID NO: 2), and a third complementarity-determining region comprising or consisting of an amino acid sequence ARDQRGX₃X₄X₅YYYX₆GMDV (SEQ ID NO: 3); wherein

Xi is an amino acid selected from I, E, and S,

X₂ is an amino acid selected from F, H, and Q,

X₃ is an amino acid selected from Y, S, H, and E,

X₄ is an amino acid selected from Y, R, H, Q, and S,

X₅ is an amino acid selected from Y, D, and H, and

X<, is an amino acid selected from Y and S; with the proviso that at least one of the following conditions a)-f) applies: a) Xi is not I, b) X₂ is not F, c) X₃ is not Y, d) X₄ is not Y, e) X₅ is not Y, f) X_(l is not Y.

In one embodiment, said first domain comprises or consists of an amino acid sequence which is at least 90% identical, preferably at least 95 % identical, more preferably at least 98% identical, even more preferably at least 99% identical, optionally identical, to an amino acid sequence

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSNAISWVRQAPGQGLEWMGGIIPX₁X₂

GTAIYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDQRGX₃X₄X₅YYYX₆G MDVWGQGTTVTVSS (SEQ ID NO: 4); wherein

Xi is an amino acid selected from I, E, and S,

X₂ is an amino acid selected from F, H, and Q,

X₃ is an amino acid selected from Y, S, H, and E,

X₄ is an amino acid selected from Y, R, H, Q, and S,

X₅ is an amino acid selected from Y, D, and H, and

In one embodiment, X₄ is not Y and/or X₅ is not Y, wherein, preferably,

wherein, more preferably,

wherein, even more preferably,

Xi is I and X₂ is Q.

In one embodiment, X₅ is an amino acid selected from D and H, wherein, preferably,

Xi is I,

X₂ is H or Q,

X₃ is Y, S, or H,

X₄ is R or H, X₅ is D or H, and

Xe is Y or S; wherein, more preferably,

Xi is I,

X₂ is H or Q,

X₃ is Y, S, or H,

X₄ is H and X₅ is D, or X₄ is R and X₅ is H, and

Xe is Y or S; wherein, even more preferably,

Xi is I, X₂ is H, X₄ is H, and X₅ is D, or

Xi is I, X₂ is Q, X₄ is H, and X₅ is D, or

Xi is I, X₂ is Q, X₄ is R, and X₅ is H.

In one embodiment, said first domain comprises a first complementarity-determining region comprising or consisting of an amino acid sequence GGTFSSNA (SEQ ID NO: 1); a second complementarity-determining region comprising or consisting of an amino acid sequence selected from

IIPIQGTA (SEQ ID NO: 5),

IIPIHGTA (SEQ ID NO: 6),

IIPEFGTA (SEQ ID NO: 7), and

IIPSFGTA (SEQ ID NO: 8); and a third complementarity-determining region comprising or consisting of an amino acid sequence selected from

ARDQRGSHDYYYYGMDV (SEQ ID NO: 9),

ARDQRGYRHYYYSGMDV (SEQ ID NO: 10),

ARDQRGHHDYYYYGMDV (SEQ ID NO: 11),

ARDQRGYHDYYYYGMDV (SEQ ID NO: 12),

ARDQRGSYDYYYYGMDV (SEQ ID NO: 13),

ARDQRGYQDYYYYGMDV (SEQ ID NO: 14),

ARDQRGYRDYYYYGMDV (SEQ ID NO: 15),

ARDQRGESDYYYYGMDV (SEQ ID NO: 16),

ARDQRGYHHYYYYGMDV (SEQ ID NO: 17),

ARDQRGYRHYYYYGMDV (SEQ ID NO: 18), and

ARDQRGYHHYYYSGMDV (SEQ ID NO: 19); wherein, preferably, said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 5 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 9, or said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 5 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 10, or said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 6 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 11, or said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 6 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 17, or said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 6 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 12, or said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 7 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 13, or said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 6 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 14, or said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 5 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 15, or said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 8 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 16, or said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 6 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 18, or said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 5 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 19, or said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 6 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 15; wherein, more preferably, said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 5 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 9, or said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 5 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 10, or said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 6 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 11; wherein, even more preferably, said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 5 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 9, or said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 5 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 10.

In one embodiment, said first domain comprises or consists of an amino acid sequence which is at least 90% identical, preferably at least 95 % identical, more preferably at least 98% identical, even more preferably at least 99% identical, optionally identical, to an amino acid sequence selected from the group consisting of QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSNAISWVRQAPGQGLEWMGGIIPIQGTAIYAQ KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDQRGSHDYYYYGMDVWGQGTTVTVS

S (SEQ ID NO: 20),

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSNAISWVRQAPGQGLEWMGGIIPIQGTAIYAQ KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDQRGYRHYYYSGMDVWGQGTTVTVS S (SEQ ID NO: 21),

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSNAISWVRQAPGQGLEWMGGIIPIHGTAIYAQ KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDQRGHHDYYYYGMDVWGQGTTVTVS S (SEQ ID NO: 22),

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSNAISWVRQAPGQGLEWMGGIIPIHGTAIYAQ KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDQRGYRHYYYYGMDVWGQGTTVTVS S (SEQ ID NO: 23),

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSNAISWVRQAPGQGLEWMGGIIPIQGTAIYAQ KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDQRGYHHYYYSGMDVWGQGTTVTVS S (SEQ ID NO: 24), QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSNAISWVRQAPGQGLEWMGGIIPIHGTAIYAQ KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDQRGYHDYYYYGMDVWGQGTTVTVS S (SEQ ID NO: 25),

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSNAISWVRQAPGQGLEWMGGIIPEFGTAIYAQ KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDQRGSYDYYYYGMDVWGQGTTVTVSS (SEQ ID NO: 26), QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSNAISWVRQAPGQGLEWMGGIIPSFGTAIYAQ KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDQRGESDYYYYGMDVWGQGTTVTVSS (SEQ ID NO: 27), QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSNAISWVRQAPGQGLEWMGGIIPIHGTAIYAQ KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDQRGYQDYYYYGMDVWGQGTTVTVS S (SEQ ID NO: 28),

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSNAISWVRQAPGQGLEWMGGIIPIQGTAIYAQ KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDQRGYRDYYYYGMDVWGQGTTVTVS S (SEQ ID NO: 29),

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSNAISWVRQAPGQGLEWMGGIIPIHGTAIYAQ KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDQRGYHHYYYYGMDVWGQGTTVTVS S (SEQ ID NO: 30), and

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSNAISWVRQAPGQGLEWMGGIIPIHGTAIYAQ KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDQRGYRDYYYYGMDVWGQGTTVTVS S (SEQ ID NO: 31); preferably selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22; more preferably selected from SEQ ID NO: 20 and SEQ ID NO: 21. In one embodiment, said antigen-binding peptide comprises a second domain, preferably a light chain variable domain, comprising a first complementarity-determining region comprising or consisting of an amino acid sequence NIRNVG (SEQ ID NO: 32), a second complementarity-determining region comprising or consisting of an amino acid sequence DDD (SEQ ID NO: 33), and a third complementarity-determining region comprising or consisting of an amino acid sequence QVWDSATDQRV (SEQ ID NO: 34); wherein, optionally, said second domain comprises or consists of an amino acid sequence which is at least 90% identical, preferably at least 95 % identical, more preferably at least 98% identical, even more preferably at least 99% identical, optionally identical, to an amino acid sequence SYVLTQPPSVSVAPGKTARITCGGNNIRNVGVHWYQQKPGQAPVLWYDDDDRPSGIPERFS GSNSGNTATLTISRVEAGDEADYYCQVWDSATDQRVFGGGTKLTVL

(SEQ ID NO: 35)-

In one embodiment, said antigen-binding peptide is an antibody or an antigen-binding fragment thereof; wherein, preferably, said antigen-binding peptide is selected from an antibody, a Fab, Fab', a F(ab')2, a scFv, di-scFv, a VH domain, a single-domain antibody (sdAb), a diabody, a triabody, and a tetrabody; wherein, more preferably, said antigen-binding peptide is selected from an antibody, a Fab, and a scFv.

In one embodiment, said antigen-binding peptide binds to human c-Met ECD with a KD of to nM or less, preferably 8.5 nM or less, as determined using biolayer interferometry; and/or said antigen-binding peptide has a half maximal effective concentration (EC₅₀) for binding to c-MET expressing cells of 2 nM or less, preferably 1.6 nM or less, more preferably 1.2 nM or less, as determined using flow cytometry; and/or said antigen-binding peptide has a hydrophobic interaction chromatography retention time of 20 min or less, preferably 19.6 min or less, even more preferably 19 min or less, as determined using hydrophobic interaction chromatography; wherein, optionally, said antigen-binding peptide has a ratio of KDantigen-binding peptide to KD_wiidtype < to, and/or a ratio of EC50antigen-binding peptide to ECsOwiidtype — 1, preferably — 0.8, and/or a hydrophobic interaction chromatography retention time of < 19.6 min.

In a further aspect, the present invention relates to a bispecific molecule, preferably a bispecific antibody or antigen-binding fragment thereof, comprising or consisting of i) a first antigen-binding peptide, wherein said first antigen-binding peptide is an antigen-binding peptide as defined herein; and ii) a second antigen-binding peptide; wherein, preferably, said second antigen-binding peptide binds to a tumor antigen; wherein, more preferably, said second antigen-binding peptide binds to c-Met.

In one embodiment, said bispecific molecule is a biparatopic molecule, preferably a biparatopic antibody or antigen-binding fragment thereof; wherein, optionally, said second antigen-binding peptide comprises or consists of a heavy chain variable domain comprising or consisting of an amino acid sequence which is at least 90% identical, preferably at least 95 % identical, more preferably at least 98% identical, even more preferably at least 99% identical, optionally identical, to an amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDRRITHTYWGQGTLVTVSSASTKGPS VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTV PSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK (SEQ ID NO: 36), and/or a light chain variable domain comprising or consisting of an amino acid sequence which is at least 90% identical, preferably at least 95 % identical, more preferably at least 98% identical, even more preferably at least 99% identical, optionally identical, to an amino acid sequence SYVLTQPPSVSVAPGQTARITCGGDSLGSKIVHWYQQKPGQAPVLVVYDDAARPSGIPERFSG SNSGNTATLTISRVEAGDEADYYCQVYDYHSDVEVFGGGTKLTVLGQPKAAPSVTLFPPSSEE LQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWK SHKSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 37).

In a further aspect, the present invention relates to an antigen-binding peptide-drug conjugate, preferably an antibody-drug conjugate, wherein said antigen-binding peptide- drug conjugate comprises an antigen-binding peptide as defined herein or a bispecific molecule as defined herein; and further comprises a diagnostic and/or therapeutic agent, preferably a therapeutic agent, more preferably a cytotoxic agent, such as exatecan; wherein, optionally, the antigen-binding peptide-drug conjugate comprises a linker.

In a further aspect, the present invention relates to a composition, preferably a pharmaceutical composition, comprising an antigen-binding peptide as defined herein, a bispecific molecule as defined herein, or an antigen-binding peptide-drug conjugate as defined herein, and a pharmaceutically acceptable excipient.

In a further aspect, the present invention relates to an antigen-binding peptide as defined herein, a bispecific molecule as defined herein, an antigen-binding peptide-drug conjugate as defined herein, or a composition as defined herein, for use in a method of preventing or treating a cancer.

In a further aspect, the present invention relates to an isolated nucleic acid encoding an antigen-binding peptide as defined herein or a bispecific molecule as defined herein. In a further aspect, the present invention relates to a recombinant cell comprising the isolated nucleic acid as defined herein.

In a further aspect, the present invention relates to a method of preventing or treating a cancer comprising administering a therapeutically effective amount of an antigen-binding peptide as defined herein, a bispecific molecule as defined herein, an antigen-binding peptide-drug conjugate as defined herein, and/or a composition as defined herein to a patient in need thereof.

In a further aspect, the present invention relates to a use of an antigen-binding peptide as defined herein, a bispecific molecule as defined herein, an antigen-binding peptide-drug conjugate as defined herein, and/or a composition as defined herein for the manufacture of a medicament for preventing or treating a cancer.

DETAILED DESCRIPTION

The present inventors have surprisingly found anti-c-Met antigen-binding peptides, such as antibodies and antigen-binding fragments thereof, which exhibit a combination of highly advantageous characteristics, particularly antigen-binding peptides having high affinity, high thermal stability, low hydrophobicity, and high potency. Furthermore, the inventors have surprisingly found anti-c-Met antigen-binding peptides, such as antibodies and antigenbinding fragments thereof, which may be manufactured into ADCs. For example, the present inventors have surprisingly identified anti-c-Met antigen-binding peptides, such as anti-c-Met antibodies and antigen-binding fragments thereof, which bind c-MET with high affinity and which exhibit an enhanced thermal stability and a reduced hydrophobicity compared to previous anti-c-Met antigen-binding peptides, such as the previous anti-c-Met antigen-binding peptide CS06 referred to in Sellmann et al. 2016 and in EP 3512882 Bi. The inventors have surprisingly found that the antigen-binding peptides of the invention, as well as bispecific molecules comprising said antigen-binding peptides, exhibit enhanced ADC- manufacturability. The antigen-binding peptides, as well as bispecific molecules and ADCs comprising said antigen-binding peptides, may be used to efficiently inhibit c-MET expressing cancers.

The following numbered items describe various embodiments and aspects of the present invention. Any two or more of the numbered items, embodiments, and aspects described herein may be combined unless context clearly indicates otherwise. The variously described numbered items, embodiments, and aspects should not be construed to limit the present invention to only the explicitly described numbered items, embodiments, and aspects. This description should be understood to support and encompass embodiments and aspects which combine any of the described numbered items, embodiments, and aspects. For example, the present invention also relates to the following embodiments defined by the numbered items below:

Numbered item 1: An antigen-binding peptide binding to c-Met, comprising a first domain comprising a first complementarity-determining region comprising or consisting of an amino acid sequence GGTFSSNA (SEQ ID NO: 1), a second complementarity-determining region comprising or consisting of an amino acid sequence IIPX.X.GTA (SEQ ID NO: 2), and a third complementarity-determining region comprising or consisting of an amino acid sequence ARDQRGX₃X₄X₅YYYX₆GMDV (SEQ ID NO: 3); wherein

Xi is an amino acid selected from I, E, and S,

X₂ is an amino acid selected from F, H, and Q,

X₃ is an amino acid selected from Y, S, H, and E,

X₄ is an amino acid selected from Y, R, H, Q, and S,

X₅ is an amino acid selected from Y, D, and H, and X<, is an amino acid selected from Y and S; with the proviso that at least one of the following conditions a)-f) applies: a) Xi is not I, b) X₂ is not F, c) X₃ is not Y, d) X₄ is not Y, e) X₅ is not Y, f) X_(l is not Y.

Numbered item 2: The antigen-binding peptide according to numbered item 1, wherein said first domain is a heavy chain variable domain.

Numbered item 3: The antigen-binding peptide according to numbered item 1 or 2, wherein said first domain comprises or consists of an amino acid sequence which is at least 90% identical to an amino acid sequence QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSNAISWVRQAPGQGLEWMGGIIPX₁X₂GTAIYA QKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDQRGX₃X₄X₅YYYX₆GMDVWGQGTTV TVSS (SEQ ID NO: 4); wherein

Xi is an amino acid selected from I, E, and S,

X₂ is an amino acid selected from F, H, and Q,

X₃ is an amino acid selected from Y, S, H, and E,

X₄ is an amino acid selected from Y, R, H, Q, and S,

X₅ is an amino acid selected from Y, D, and H, and X is an amino acid selected from Y and S; with the proviso that at least one of the following conditions a)-f) applies: a) Xi is not I, b) X₂ is not F, c) X₃ is not Y, d) X₄ is not Y, e) X₅ is not Y, f) Xe is not Y.

Numbered item 4: The antigen-binding peptide according to any one of numbered items 1 to 3, wherein said first domain comprises or consists of an amino acid sequence which is at least 95 % identical to an amino acid sequence QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSNAISWVRQAPGQGLEWMGGIIPX₁X₂GTAIYA QKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDQRGX₃X₄X₅YYYX₆GMDVWGQGTTV TVSS (SEQ ID NO: 4); wherein

Xi is an amino acid selected from I, E, and S,

X₂ is an amino acid selected from F, H, and Q,

X₃ is an amino acid selected from Y, S, H, and E,

X₄ is an amino acid selected from Y, R, H, Q, and S,

X₅ is an amino acid selected from Y, D, and H, and Xe is an amino acid selected from Y and S; with the proviso that at least one of the following conditions a)-f) applies: a) Xi is not I, b) X₂ is not F, c) X₃ is not Y, d) X₄ is not Y, e) X₅ is not Y, f) Xe is not Y. Numbered item 5: The antigen-binding peptide according to any one of numbered items 1 to

4, wherein said first domain comprises or consists of an amino acid sequence which is at least 98% identical to an amino acid sequence

QVQLVQSGAEVI<I<PGSSVI<VSCI<ASGGTFSSNAISWVRQAPGQGLEWMGGIIPXN GTAIYA QKFQGRVTITADESTSTA¥MELSSLRSEDTAVYYCARDQRGX3X₄X₅YYYX6GMDVWGQGTTV TVSS (SEQ ID NO: 4); wherein

Xi is an amino acid selected from I, E, and S,

X₂ is an amino acid selected from F, H, and Q,

X₃ is an amino acid selected from Y, S, H, and E,

X₄ is an amino acid selected from Y, R, H, Q, and S,

X₅ is an amino acid selected from Y, D, and H, and

X is an amino acid selected from Y and S; with the proviso that at least one of the following conditions a)-f) applies: a) Xi is not I, b) X₂ is not F, c) X₃ is not Y, d) X₄ is not Y, e) X₅ is not Y, f) X_(l is not Y.

Numbered item 6: The antigen-binding peptide according to any one of numbered items 1 to

5, wherein said first domain comprises or consists of an amino acid sequence which is at least 99% identical to an amino acid sequence

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSNAISWVRQAPGQGLEWMGGIIPX₁X₂GTAIYA QKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDQRGX₃X₄X₅YYYX₆GMDVWGQGTT TVSS (SEQ ID NO: 4); wherein

Xi is an amino acid selected from I, E, and S,

X₂ is an amino acid selected from F, H, and Q,

X₃ is an amino acid selected from Y, S, H, and E,

X₄ is an amino acid selected from Y, R, H, Q, and S,

X₅ is an amino acid selected from Y, D, and H, and

X is an amino acid selected from Y and S; with the proviso that at least one of the following conditions a)-f) applies: a) Xi is not I, b) X₂ is not F, c) X₃ is not Y, d) X₄ is not Y, e) X₅ is not Y, f) Xe is not Y.

Numbered item 7: The antigen-binding peptide according to any one of numbered items 1 to

6, wherein said first domain comprises or consists of an amino acid sequence which is identical to an amino acid sequence

QVQLVQSGAEVI<I<PGSSVI<VSCI<ASGGTFSSNAISWVRQAPGQGLEWMGGIIPXX2GTAIYA QKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDQRGX₃X₄X₅YYYX6GMDVWGQGTTV TVSS (SEQ ID NO: 4); wherein

Xi is an amino acid selected from I, E, and S,

X₂ is an amino acid selected from F, H, and Q,

X₃ is an amino acid selected from Y, S, H, and E,

X₄ is an amino acid selected from Y, R, H, Q, and S,

X₅ is an amino acid selected from Y, D, and H, and

Xe is an amino acid selected from Y and S; with the proviso that at least one of the following conditions a)-f) applies: a) Xi is not I, b) X₂ is not F, c) X₃ is not Y, d) X₄ is not Y, e) X₅ is not Y, f) Xe is not Y.

Numbered item 8: The antigen-binding peptide according to any one of numbered items 1 to

7, wherein X₄ is not Y and/or X₅ is not Y.

Numbered item 9: The antigen-binding peptide according to any one of numbered items 1 to

8, wherein

Numbered item 10: The antigen-binding peptide according to any one of numbered items 1 to

9, wherein

Xi is I and X₂ is H, or Xi is I and X₂ is Q;

Numbered item 11: The antigen-binding peptide according to any one of numbered items 1 to

10, wherein

Xi is I and X₂ is Q.

Numbered item 12: The antigen-binding peptide according to any one of numbered items 1 to

11, wherein

X₅ is an amino acid selected from D and H.

Numbered item 13: The antigen-binding peptide according to any one of numbered items 1 to

12, wherein

Xi is I,

X₂ is H or Q,

X₃ isY, S, or H,

X₄ is R or H,

X₅ is D or H, and

Xe is Y or S;

Numbered item 14: The antigen-binding peptide according to any one of numbered items 1 to

13, wherein

Xi is I,

X₂ is H or Q,

X₃ isY, S, or H,

X₄ is H and X₅ is D, or X₄ is R and X₅ is H, and

Xe is Y or S.

Numbered item 15: The antigen-binding peptide according to any one of numbered items 1 to

14, wherein

Xi is I, X₂ is H, X₄ is H, and X₅ is D, or

Xi is I, X₂ is Q, X₄ is H, and X₅ is D, or

Xi is I, X₂ is Q, X₄ is R, and X₅ is H.

Numbered item 16: The antigen-binding peptide according to any one of numbered items 1 to

15, wherein said first domain comprises a first complementarity-determining region comprising or consisting of an amino acid sequence GGTFSSNA (SEQ ID NO: 1); a second complementarity-determining region comprising or consisting of an amino acid sequence selected from

IIPIQGTA (SEQ ID NO: 5),

IIPIHGTA (SEQ ID NO: 6),

IIPEFGTA (SEQ ID NO: 7), and

ARDQRGSHDYYYYGMDV (SEQ ID NO: 9),

ARDQRGYRHYYYSGMDV (SEQ ID NO: 10),

ARDQRGHHDYYYYGMDV (SEQ ID NO: 11),

ARDQRGYHDYYYYGMDV (SEQ ID NO: 12),

ARDQRGSYDYYYYGMDV (SEQ ID NO: 13),

ARDQRGYQDYYYYGMDV (SEQ ID NO: 14),

ARDQRGYRDYYYYGMDV (SEQ ID NO: 15),

ARDQRGESDYYYYGMDV (SEQ ID NO: 16),

ARDQRGYHHYYYYGMDV (SEQ ID NO: 17),

ARDQRGYRHYYYYGMDV (SEQ ID NO: 18), and

ARDQRGYHHYYYSGMDV (SEQ ID NO: 19).

Numbered item 17: The antigen-binding peptide according to any one of numbered items 1 to 16, wherein said first domain comprises a first complementarity-determining region comprising or consisting of an amino acid sequence GGTFSSNA (SEQ ID NO: 1); a second complementarity-determining region comprising or consisting of an amino acid sequence selected from

IIPIQGTA (SEQ ID NO: 5),

IIPIHGTA (SEQ ID NO: 6); and a third complementarity-determining region comprising or consisting of an amino acid sequence selected from

ARDQRGSHDYYYYGMDV (SEQ ID NO: 9),

ARDQRGYRHYYYSGMDV (SEQ ID NO: 10),

ARDQRGHHDYYYYGMDV (SEQ ID NO: 11),

ARDQRGYHDYYYYGMDV (SEQ ID NO: 12),

ARDQRGSYDYYYYGMDV (SEQ ID NO: 13),

ARDQRGYQDYYYYGMDV (SEQ ID NO: 14),

ARDQRGYRDYYYYGMDV (SEQ ID NO: 15),

ARDQRGESDYYYYGMDV (SEQ ID NO: 16), ARDQRGYHHYYYYGMDV (SEQ ID NO: 17),

ARDQRGYRHYYYYGMDV (SEQ ID NO: 18), and

ARDQRGYHHYYYSGMDV (SEQ ID NO: 19).

Numbered item 18: The antigen-binding peptide according to any one of numbered items 1 to 17, wherein said antigen-binding peptide comprises a first domain comprising a first complementarity-determining region comprising or consisting of an amino acid sequence of SEQ ID NO: 1, a second complementarity-determining region, and a third complementaritydetermining region; wherein said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 5 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 9, or said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 5 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 10, or said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 6 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 11, or said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 6 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 17, or said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 6 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 12, or said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 7 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 13, or said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 6 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 14, or said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 5 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 15, or said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 8 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 16, or said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 6 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 18, or said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 5 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 19, or said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 6 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 15.

Numbered item 19: The antigen-binding peptide according to any one of numbered items 1 to 18, wherein said antigen-binding peptide comprises a first domain comprising a first complementarity-determining region comprising or consisting of an amino acid sequence of SEQ ID NO: 1, a second complementarity-determining region, and a third complementaritydetermining region, wherein said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 5 and said third complementaritydetermining region comprises or consists of an amino acid sequence of SEQ ID NO: 9.

Numbered item 20: The antigen-binding peptide according to any one of numbered items 1 to 18, wherein said antigen-binding peptide comprises a first domain comprising a first complementarity-determining region comprising or consisting of an amino acid sequence of SEQ ID NO: 1, a second complementarity-determining region, and a third complementaritydetermining region, wherein said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 5 and said third complementaritydetermining region comprises or consists of an amino acid sequence of SEQ ID NO: 10.

Numbered item 21: The antigen-binding peptide according to any one of numbered items 1 to 18, wherein said antigen-binding peptide comprises a first domain comprising a first complementarity-determining region comprising or consisting of an amino acid sequence of SEQ ID NO: 1, a second complementarity-determining region, and a third complementaritydetermining region, wherein second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 6 and said third complementaritydetermining region comprises or consists of an amino acid sequence of SEQ ID NO: 11.

Numbered item 22: The antigen-binding peptide according to any one of numbered items 1 to 18, wherein said antigen-binding peptide comprises a first domain comprising a first complementarity-determining region comprising or consisting of an amino acid sequence of SEQ ID NO: 1, a second complementarity-determining region, and a third complementarity- determining region, wherein said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 6 and said third complementaritydetermining region comprises or consists of an amino acid sequence of SEQ ID NO: 17.

Numbered item 23: The antigen-binding peptide according to any one of numbered items 1 to 18, wherein said antigen-binding peptide comprises a first domain comprising a first complementarity-determining region comprising or consisting of an amino acid sequence of SEQ ID NO: 1, a second complementarity-determining region, and a third complementaritydetermining region, wherein said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 6 and said third complementaritydetermining region comprises or consists of an amino acid sequence of SEQ ID NO: 12.

Numbered item 24: The antigen-binding peptide according to any one of numbered items 1 to 18, wherein said antigen-binding peptide comprises a first domain comprising a first complementarity-determining region comprising or consisting of an amino acid sequence of SEQ ID NO: 1, a second complementarity-determining region, and a third complementaritydetermining region, wherein said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 7 and said third complementaritydetermining region comprises or consists of an amino acid sequence of SEQ ID NO: 13.

Numbered item 25: The antigen-binding peptide according to any one of numbered items 1 to 18, wherein said antigen-binding peptide comprises a first domain comprising a first complementarity-determining region comprising or consisting of an amino acid sequence of SEQ ID NO: 1, a second complementarity-determining region, and a third complementaritydetermining region, wherein said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 6 and said third complementaritydetermining region comprises or consists of an amino acid sequence of SEQ ID NO: 14.

Numbered item 26: The antigen-binding peptide according to any one of numbered items 1 to 18, wherein said antigen-binding peptide comprises a first domain comprising a first complementarity-determining region comprising or consisting of an amino acid sequence of SEQ ID NO: 1, a second complementarity-determining region, and a third complementaritydetermining region, wherein said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 5 and said third complementaritydetermining region comprises or consists of an amino acid sequence of SEQ ID NO: 15.

Numbered item 27: The antigen-binding peptide according to any one of numbered items 1 to 18, wherein said antigen-binding peptide comprises a first domain comprising a first complementarity-determining region comprising or consisting of an amino acid sequence of SEQ ID NO: 1, a second complementarity-determining region, and a third complementaritydetermining region, wherein said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 8 and said third complementaritydetermining region comprises or consists of an amino acid sequence of SEQ ID NO: 16.

Numbered item 28: The antigen-binding peptide according to any one of numbered items 1 to 18, wherein said antigen-binding peptide comprises a first domain comprising a first complementarity-determining region comprising or consisting of an amino acid sequence of SEQ ID NO: 1, a second complementarity-determining region, and a third complementaritydetermining region, wherein said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 6 and said third complementaritydetermining region comprises or consists of an amino acid sequence of SEQ ID NO: 18.

Numbered item 29: The antigen-binding peptide according to any one of numbered items 1 to 18, wherein said antigen-binding peptide comprises a first domain comprising a first complementarity-determining region comprising or consisting of an amino acid sequence of SEQ ID NO: 1, a second complementarity-determining region, and a third complementaritydetermining region, wherein said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 5 and said third complementaritydetermining region comprises or consists of an amino acid sequence of SEQ ID NO: 19.

Numbered item 30: The antigen-binding peptide according to any one of numbered items 1 to 18, wherein said antigen-binding peptide comprises a first domain comprising a first complementarity-determining region comprising or consisting of an amino acid sequence of SEQ ID NO: 1, a second complementarity-determining region, and a third complementaritydetermining region, wherein said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 6 and said third complementaritydetermining region comprises or consists of an amino acid sequence of SEQ ID NO: 15.

Numbered item 31: The antigen-binding peptide according to any one of numbered items 1 to 18, wherein said antigen-binding peptide comprises a first domain comprising a first complementarity-determining region comprising or consisting of an amino acid sequence of SEQ ID NO: 1, a second complementarity-determining region, and a third complementaritydetermining region, wherein said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 5 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 9, or said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 5 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 10, or said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 6 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 11.

Numbered item 32: The antigen-binding peptide according to any one of numbered items 1 to 18, wherein said antigen-binding peptide comprises a first domain comprising a first complementarity-determining region comprising or consisting of an amino acid sequence of SEQ ID NO: 1, a second complementarity-determining region, and a third complementaritydetermining region, wherein said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 5 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 9, or said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 5 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 10.

Numbered item 33: The antigen-binding peptide according to any one of numbered items 1 to 18, wherein said antigen-binding peptide comprises a first domain comprising a first complementarity-determining region comprising or consisting of an amino acid sequence of SEQ ID NO: 1, a second complementarity-determining region, and a third complementaritydetermining region, wherein said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 5 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 9, or said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 5 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 10, or said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 6 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 11, or said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 6 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 17, or said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 6 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 12, or said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 7 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 13, or said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 6 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 14, or said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 5 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 15, or said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 8 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 16, or said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 5 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 19, or said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 6 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 15.

Numbered item 34: The antigen-binding peptide according to any one of numbered items 1 to 18, wherein said antigen-binding peptide comprises a first domain comprising a first complementarity-determining region comprising or consisting of an amino acid sequence of SEQ ID NO: 1, a second complementarity-determining region, and a third complementaritydetermining region, wherein said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 5 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 9, or said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 5 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 10, or said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 6 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 11, or said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 5 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 15, or said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 6 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 18.

Numbered item 35: The antigen-binding peptide according to any one of numbered items 1 to 34, wherein said first domain comprises or consists of an amino acid sequence which is at least 90% identical to an amino acid sequence selected from the group consisting of QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSNAISWVRQAPGQGLEWMGGIIPIQGTAIYAQ KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDQRGSHDYYYYGMDVWGQGTTVTVS S (SEQ ID NO: 20),

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSNAISWVRQAPGQGLEWMGGIIPIQGTAIYAQ KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDQRGYHHYYYSGMDVWGQGTTVTVS S (SEQ ID NO: 24),

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSNAISWVRQAPGQGLEWMGGIIPIHGTAIYAQ KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDQRGYHDYYYYGMDVWGQGTTVTVS S (SEQ ID NO: 25),

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSNAISWVRQAPGQGLEWMGGIIPEFGTAIYAQ KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDQRGSYDYYYYGMDVWGQGTTVTVSS (SEQ ID NO: 26), QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSNAISWVRQAPGQGLEWMGGIIPSFGTAIYAQ KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDQRGESDYYYYGMDVWGQGTTVTVSS (SEQ ID NO: 27), QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSNAISWVRQAPGQGLEWMGGIIPIHGTAIYAQ KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDQRGYQDYYYYGMDVWGQGTTVTVS S (SEQ ID NO: 28), QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSNAISWVRQAPGQGLEWMGGIIPIQGTAIYAQ KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDQRGYRDYYYYGMDVWGQGTTVTVS S (SEQ ID NO: 29),

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSNAISWVRQAPGQGLEWMGGIIPIHGTAIYAQ KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDQRGYRDYYYYGMDVWGQGTTVTVS S (SEQ ID NO: 31).

Numbered item 36: The antigen-binding peptide according to any one of numbered items 1 to 35, wherein said first domain comprises or consists of an amino acid sequence which is at least 95 % identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31.

Numbered item 37: The antigen-binding peptide according to any one of numbered items 1 to 36, wherein said first domain comprises or consists of an amino acid sequence which is at least 98% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31.

Numbered item 38: The antigen-binding peptide according to any one of numbered items 1 to 37, wherein said first domain comprises or consists of an amino acid sequence which is at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31.

Numbered item 39: The antigen-binding peptide according to any one of numbered items 1 to 38, wherein said first domain comprises or consists of an amino acid sequence which is identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31- Numbered item 40: The antigen-binding peptide according to any one of numbered items 35 to 39, wherein said group consists of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31.

Numbered item 41: The antigen-binding peptide according to any one of numbered items 35 to 39, wherein said group consists of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 29

Numbered item 42: The antigen-binding peptide according to any one of numbered items 1 to 41, wherein said first domain comprises or consists of an amino acid sequence which is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22.

Numbered item 43: The antigen-binding peptide according to any one of numbered items 1 to 42, wherein said first domain comprises or consists of an amino acid sequence which is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22.

Numbered item 44: The antigen-binding peptide according to any one of numbered items 1 to 43, wherein said first domain comprises or consists of an amino acid sequence which is at least 98% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22.

Numbered item 45: The antigen-binding peptide according to any one of numbered items 1 to 44, wherein said first domain comprises or consists of an amino acid sequence which is at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22.

Numbered item 46: The antigen-binding peptide according to any one of numbered items 1 to 45, wherein said first domain comprises or consists of an amino acid sequence which is identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22.

Numbered item 47: The antigen-binding peptide according to any one of numbered items 1 to 46, wherein said first domain comprises or consists of an amino acid sequence which is at least 90% identical to an amino acid sequence selected from SEQ ID NO: 20 and SEQ ID NO: 21. Numbered item 48: The antigen-binding peptide according to any one of numbered items 1 to 47, wherein said first domain comprises or consists of an amino acid sequence which is at least 95% identical to an amino acid sequence selected from SEQ ID NO: 20 and SEQ ID NO: 21.

Numbered item 49: The antigen-binding peptide according to any one of numbered items 1 to 48, wherein said first domain comprises or consists of an amino acid sequence which is at least 98% identical to an amino acid sequence selected from SEQ ID NO: 20 and SEQ ID NO: 21.

Numbered item 50: The antigen-binding peptide according to any one of numbered items 1 to 49, wherein said first domain comprises or consists of an amino acid sequence which is at least 99% identical to an amino acid sequence selected from SEQ ID NO: 20 and SEQ ID NO: 21.

Numbered item 51: The antigen-binding peptide according to any one of numbered items 1 to

50, wherein said first domain comprises or consists of an amino acid sequence which is identical to an amino acid sequence selected from SEQ ID NO: 20 and SEQ ID NO: 21.

Numbered item 52: The antigen-binding peptide according to any one of numbered items 1 to

51, wherein said antigen-binding peptide comprises a second domain comprising a first complementarity-determining region comprising or consisting of an amino acid sequence NIRNVG (SEQ ID NO: 32), a second complementarity-determining region comprising or consisting of an amino acid sequence DDD (SEQ ID NO: 33), and a third complementarity-determining region comprising or consisting of an amino acid sequence QVWDSATDQRV (SEQ ID NO: 34).

Numbered item 53: The antigen-binding peptide according to numbered item 52, wherein said second domain is a light chain variable domain comprising a first complementarity-determining region comprising or consisting of an amino acid sequence NIRNVG (SEQ ID NO: 32), a second complementarity-determining region comprising or consisting of an amino acid sequence DDD (SEQ ID NO: 33), and a third complementarity-determining region comprising or consisting of an amino acid sequence QVWDSATDQRV (SEQ ID NO: 34). Numbered item 54: The antigen-binding peptide according to any one of numbered items 1 to

53, wherein said antigen-binding peptide comprises a second domain, wherein said second domain comprises or consists of an amino acid sequence which is at least 90% identical to an amino acid sequence

SYVLTQPPSVSVAPGKTARITCGGNNIRNVGVHWYQQKPGQAPVLWYDDDDRPSGIPERFS GSNSGNTATLTISRVEAGDEADYYCQVWDSATDQRVFGGGTKLTVL (SEQ ID NO: 35).

Numbered item 55: The antigen-binding peptide according to any one of numbered items 1 to

54, wherein said antigen-binding peptide comprises a second domain, wherein said second domain comprises or consists of an amino acid sequence which is at least 95 % identical to an amino acid sequence of SEQ ID NO: 35.

Numbered item 56: The antigen-binding peptide according to any one of numbered items 1 to

55, wherein said antigen-binding peptide comprises a second domain, wherein said second domain comprises or consists of an amino acid sequence which is at least 98 % identical to an amino acid sequence of SEQ ID NO: 35.

Numbered item 57: The antigen-binding peptide according to any one of numbered items 1 to

56, wherein said antigen-binding peptide comprises a second domain, wherein said second domain comprises or consists of an amino acid sequence which is at least 99 % identical to an amino acid sequence of SEQ ID NO: 35.

Numbered item 58: The antigen-binding peptide according to any one of numbered items 1 to 57, wherein said antigen-binding peptide comprises a second domain, wherein said second domain comprises or consists of an amino acid sequence which is identical to an amino acid sequence of SEQ ID NO: 35.

Numbered item 59: The antigen-binding peptide according to any one of numbered items 54 to 58, wherein said second domain is a light chain variable domain.

Numbered item 60: The antigen-binding peptide according to any one of numbered items 1 to 59, wherein said antigen-binding peptide is an antibody or an antigen-binding fragment thereof.

Numbered item 61: The antigen-binding peptide according to any one of numbered items 1 to 60, wherein said antigen-binding peptide is selected from an antibody, a Fab, Fab', a F(ab')2, a scFv, di-scFv, a VH domain, a single-domain antibody (sdAb), a diabody, a triabody, and a tetrabody. Numbered item 62: The antigen-binding peptide according to any one of numbered items 1 to 61, wherein said antigen-binding peptide is selected from an antibody, a Fab, and a scFv.

Numbered item 63: The antigen-binding peptide according to any one of numbered items 1 to 62, wherein said antigen-binding peptide binds to human c-Met ECD with a KD of 10 nM or less, as determined using biolayer interferometry.

Numbered item 64: The antigen-binding peptide according to any one of numbered items 1 to 63, wherein said antigen-binding peptide binds to human c-Met ECD with a KD of 8.5 nM or less, as determined using biolayer interferometry.

Numbered item 65: The antigen-binding peptide according to any one of numbered items 1 to 64, wherein said antigen-binding peptide has a half maximal effective concentration (EC₅₀) for binding to c-MET expressing cells of 2 nM or less, as determined using flow cytometry.

Numbered item 66: The antigen-binding peptide according to any one of numbered items 1 to 65, wherein said antigen-binding peptide has a half maximal effective concentration (EC₅₀) for binding to c-MET expressing cells of 1.6 nM or less, as determined using flow cytometry.

Numbered item 67: The antigen-binding peptide according to any one of numbered items 1 to 66, wherein said antigen-binding peptide has a half maximal effective concentration (EC₅₀) for binding to c-MET expressing cells of 1.2 nM or less, as determined using flow cytometry.

Numbered item 68: The antigen-binding peptide according to any one of numbered items 65 to 67, wherein said c-MET expressing cells are human lung carcinoma EBC-1 cells.

Numbered item 69: The antigen-binding peptide according to any one of numbered items 1 to 68, wherein said antigen-binding peptide has a hydrophobic interaction chromatography retention time of 20 min or less, as determined using hydrophobic interaction chromatography.

Numbered item 70: The antigen-binding peptide according to any one of numbered items 1 to 69, wherein said antigen-binding peptide has a hydrophobic interaction chromatography retention time of 19.6 min or less, as determined using hydrophobic interaction chromatography. Numbered item 71: The antigen-binding peptide according to any one of numbered items 1 to

70, wherein said antigen-binding peptide has a hydrophobic interaction chromatography retention time of 19 min or less, as determined using hydrophobic interaction chromatography.

Numbered item 72: The antigen-binding peptide according to any one of numbered items 1 to

71, wherein said antigen-binding peptide has a ratio of KDantigen-binding peptide to KD_wiidty_Pe < 10.

Numbered item 73: The antigen-binding peptide according to any one of numbered items 1 to

72, wherein said antigen-binding peptide has a ratio of ECsOantigen-binding peptide to EC5O_wiidty_Pe < 1.

Numbered item 74: The antigen-binding peptide according to any one of numbered items 1 to

73, wherein said antigen-binding peptide has a ratio of ECsOantigen-binding peptide to ECsOwiidtype < 0.8.

Numbered item 75: The antigen-binding peptide according to any one of numbered items 1 to

74, wherein said antigen-binding peptide has a hydrophobic interaction chromatography retention time of < 19.6 min, as determined using hydrophobic interaction chromatography.

Numbered item 76: An anti-c-Met antibody or antigen-binding fragment thereof comprising or consisting of an antigen-binding peptide according to any one of numbered items 1 to 75.

Numbered item 77: A bispecific molecule comprising or consisting of iii) a first antigen-binding peptide, wherein said first antigen-binding peptide is an antigen-binding peptide as defined in any one of numbered items 1-75 or an anti-c-Met antibody or fragment thereof as defined in numbered item 76; and iv) a second antigen-binding peptide.

Numbered item 78: The bispecific molecule according to numbered item 77, wherein said bispecific molecule is a bispecific antibody or antigen-binding fragment thereof.

Numbered item 79: The bispecific molecule according to numbered item 77 or 78, wherein said second antigen-binding peptide binds to a tumor antigen.

Numbered item 80: The bispecific molecule according to any one of numbered items 77 to 79, wherein said second antigen-binding peptide binds to c-Met. Numbered item 81: The bispecific molecule according to any one of numbered items 77 to 80, wherein said bispecific molecule is a biparatopic molecule.

Numbered item 82: The bispecific molecule according to any one of numbered items 77 to 81, wherein said bispecific molecule is a biparatopic antibody or antigen-binding fragment thereof.

Numbered item 83: The bispecific molecule according to any one of numbered items 77 to 82, wherein said second antigen-binding peptide comprises or consists of a heavy chain variable domain comprising or consisting of an amino acid sequence which is at least 90% identical to an amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDRRITHTYWGQGTLVTVSSASTKGPS VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTV PSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK (SEQ ID NO: 36), and/or a light chain variable domain comprising or consisting of an amino acid sequence which is at least 90% identical to an amino acid sequence SYVLTQPPSVSVAPGQTARITCGGDSLGSKIVHWYQQKPGQAPVLVVYDDAARPSGIPERFSG SNSGNTATLTISRVEAGDEADYYCQVYDYHSDVEVFGGGTKLTVLGQPKAAPSVTLFPPSSEE LQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWK SHKSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 37).

Numbered item 84: The bispecific molecule according to any one of numbered items 77 to 83, wherein said second antigen-binding peptide comprises or consists of a heavy chain variable domain comprising or consisting of an amino acid sequence which is at least 95 % identical to an amino acid sequence of SEQ ID NO: 36, and/or a light chain variable domain comprising or consisting of an amino acid sequence which is at least 95 % identical to an amino acid sequence of SEQ ID NO: 37.

Numbered item 85: The bispecific molecule according to any one of numbered items 77 to 84, wherein said second antigen-binding peptide comprises or consists of a heavy chain variable domain comprising or consisting of an amino acid sequence which is at least 98 % identical to an amino acid sequence of SEQ ID NO: 36, and/or a light chain variable domain comprising or consisting of an amino acid sequence which is at least 98 % identical to an amino acid sequence of SEQ ID NO: 37.

Numbered item 86: The bispecific molecule according to any one of numbered items 77 to 85, wherein said second antigen-binding peptide comprises or consists of a heavy chain variable domain comprising or consisting of an amino acid sequence which is at least 99 % identical to an amino acid sequence of SEQ ID NO: 36, and/or a light chain variable domain comprising or consisting of an amino acid sequence which is at least 99 % identical to an amino acid sequence of SEQ ID NO: 37.

Numbered item 87: The bispecific molecule according to any one of numbered items 77 to 86, wherein said second antigen-binding peptide comprises or consists of a heavy chain variable domain comprising or consisting of an amino acid sequence which is identical to an amino acid sequence of SEQ ID NO: 36, and/or a light chain variable domain comprising or consisting of an amino acid sequence which is identical to an amino acid sequence of SEQ ID NO: 37.

Numbered item 88: The bispecific molecule according to any one of numbered items 77 to 87, wherein said second antigen-binding peptide comprises or consists of a heavy chain variable domain comprising or consisting of an amino acid sequence of SEQ ID NO: 36.

Numbered item 89: The bispecific molecule according to any one of numbered items 77 to 87, wherein said second antigen-binding peptide comprises or consists of a light chain variable domain comprising or consisting of an amino acid sequence of SEQ ID NO: 37.

Numbered item 90: The bispecific molecule according to any one of numbered items 77 to 87, wherein said second antigen-binding peptide comprises or consists of a heavy chain variable domain comprising or consisting of an amino acid sequence of SEQ ID NO: 36, and a light chain variable domain comprising or consisting of an amino acid sequence of SEQ ID NO: 37.

Numbered item 91: An antigen-binding peptide-drug conjugate, wherein said antigenbinding peptide-drug conjugate comprises an antigen-binding peptide according to any one of numbered items 1-75, an anti-c-Met antibody or antigen-binding fragment thereof according to numbered item 76, or a bispecific molecule according to any one of numbered items 77-90; and further comprises a diagnostic and/or therapeutic agent. Numbered item 92: The antigen-binding peptide-drug conjugate according to numbered item 91, wherein said antigen-binding peptide-drug conjugate is an antibody-drug conjugate.

Numbered item 93: The antigen-binding peptide-drug conjugate according to numbered item 91 or 92, wherein said diagnostic and/or therapeutic agent is a therapeutic agent.

Numbered item 94: The antigen-binding peptide-drug conjugate according to any one of numbered items 91 to 93, wherein said diagnostic and/or therapeutic agent is a cytotoxic agent.

Numbered item 95: The antigen-binding peptide-drug conjugate according to any one of numbered items 91 to 94, wherein said diagnostic and/or therapeutic agent is exatecan.

Numbered item 96: The antigen-binding peptide-drug conjugate according to any one of numbered items 91 to 95, wherein the antigen-binding peptide-drug conjugate comprises a linker.

Numbered item 97: A composition comprising an antigen-binding peptide according to any one of numbered items 1-75, an anti-c-Met antibody or antigen-binding fragment thereof according to numbered item 76, a bispecific molecule according to any one of numbered items 77-90, or an antigen-binding peptide-drug conjugate according to any one of numbered items 91 to 96, and a pharmaceutically acceptable excipient.

Numbered item 98: The composition according to numbered item 97, wherein said composition is a pharmaceutical composition.

Numbered item 99: An antigen-binding peptide according to any one of numbered items 1-

75, an anti-c-Met antibody or antigen-binding fragment thereof according to numbered item

76, a bispecific molecule according to any one of numbered items 77-90, an antigen-binding peptide-drug conjugate according to any one of numbered items 91 to 96, or a composition according to numbered item 97 or 98, for use in a method of preventing or treating a cancer.

Numbered item too: An isolated nucleic acid encoding an antigen-binding peptide according to any one of numbered items 1-75, an anti-c-Met antibody or antigen-binding fragment thereof according to numbered item 76, or a bispecific molecule according to any one of numbered items 77-90. Numbered item 101: A recombinant cell comprising the isolated nucleic acid according to numbered item too.

Numbered item 102: A method of preventing or treating a cancer comprising administering a therapeutically effective amount of an antigen-binding peptide according to any one of numbered items 1-75, an anti-c-Met antibody or antigen-binding fragment thereof according to numbered item 76, a bispecific molecule according to any one of numbered items 77-90, an antigen-binding peptide-drug conjugate according to any one of numbered items 91 to 96, or a composition according to numbered item 97 or 98, to a patient in need thereof.

Numbered item 103: Use of an antigen-binding peptide according to any one of numbered items 1-75, an anti-c-Met antibody or antigen-binding fragment thereof according to numbered item 76, a bispecific molecule according to any one of numbered items 77-90, an antigen-binding peptide-drug conjugate according to any one of numbered items 91 to 96, or a composition according to numbered item 97 or 98 for the manufacture of a medicament for preventing or treating a cancer.

The inventors have found that there is an obstacle in manufacturing ADCs from previous anti- c-Met antigen-binding peptides, such as the antigen-binding peptide CS06 referred to in Sellmann et al. 2016 and EP 3512882 Bi. It appeared to the inventors that providing stable ADCs from the previous anti-c-Met antigen-binding peptides such as CS06 may be hindered by hydrophobic patches, e.g. in the HCDR2 and HCDR3 of CS06. The inventors surprisingly found antigen-binding peptides with enhanced thermal stability and significantly lowered hydrophobicity compared to previous antigen-binding peptides, such as the previous antigenbinding peptide CS06 referred to in Sellmann et al. 2016 and EP 3512882 Bi. Advantageously, the antigen-binding peptides of the invention exhibit enhanced thermal stability, significantly lowered hydrophobicity, and enhanced conjugatability so that the antigen-peptides may be provided as ADCs. Surprisingly, the antigen-binding peptides of the invention disclosed herein, such as CS06 VH6.18 & VH6.21, showed strongly reduced hydrophobicity while maintaining pico-molar affinities and elevated cell binding properties as biparatopic antibodies, appropriate in silico developability profiles and, most relevant, improved capability for payload conjugation. Advantageously, in vitro potency of resulting biparatopic ADCs on several c-MET expressing tumor cell lines was in the sub-nanomolar to one-digit nanomolar range.

The term “antigen-binding peptide”, as used herein, relates to a peptide which binds, particularly specifically binds, to an antigen, such as c-Met. An antigen-binding molecule may be based on an immunoglobulin, such as an antibody or an antigen-binding fragment thereof, or on a protein scaffold structure having antigen-binding capacity, such as an anticalin protein, an Affilin, an Affimer, an Affitin, an Alphabody, or a DARPin. In a preferred embodiment, the antigen-binding peptide is an antibody or antigen-binding fragment thereof. In a preferred embodiment, the antigen-binding peptide of the present invention is an anti-c-Met antibody or an antigen-binding fragment thereof. For example, the antigen-binding peptide maybe an antibody or antigen-binding fragment thereof, wherein said antigen-binding fragment may be part of an antibody, e.g. part of a bispecific antibody.

There are several classes of antibodies, particularly human antibodies, such as IgA, IgG, IgM, IgD and IgE, as well as subclasses, such as IgGi, IgG2, IgGs, and IgG4. Antibodies are typically grouped into classes, also referred to as isotypes, as determined genetically by the constant region. Human constant light chains are classified as kappa (Ck) and lambda (Cl) light chains. Human heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody’s isotype as IgM, IgD, IgG, IgA, and IgE, respectively. The IgG class is the most commonly used for therapeutic purposes. "IgG", as used herein, relates to a polypeptide belonging to the class of antibodies that are substantially encoded by a recognized immunoglobulin gamma gene. In humans this class comprises subclasses IgGi, IgG2, IgG3, and IgG4. In mice this class comprises subclasses IgGi, IgG2a, IgG2b and IgG3. IgA has several subclasses, including but not limited to IgAi and IgA2. Thus, “isotype”, as used herein, relates to any of the classes or subclasses of immunoglobulins defined by the chemical and antigenic characteristics of their constant regions. The known human immunoglobulin isotypes are IgGi, IgG2, IgG3, IgG4, IgAi, IgA2, IgM, IgD, and IgE. Also useful for the invention maybe IgGs that are hybrid compositions of the natural human IgG isotypes. The antigen-binding peptide of the present invention, the bispecific molecule of the present invention, and the antigen-binding peptide-drug conjugate of the present invention may comprise a domain of any of the above mentioned antibody classes, subclasses, and/or chains. The amino acid sequence of the antigen-binding peptide of the present invention, the bispecific molecule of the present invention, and the antigen-binding peptide-drug conjugate of the present invention may be modified by protein engineering, e.g. to comprise constant regions from other immunoglobulin classes mediating improved effector function properties. Such engineered hybrid IgG compositions may provide improved effector function properties, such as improved serum half-life.

An antibody maybe, for example, a human antibody, a human-chimeric antibody, a humanized antibody, a chimeric antibody, or a CDR-grafted antibody. In a preferred embodiment, the antibody is an IgG type antibody. The term “IgG type antibody”, as used herein, refers to IgG class antibodies, which in humans includes four subclasses (IgGi, IgG2, IgG3, and IgG4). The antibody may thus be an IgGi, IgG2, IgGs, or IgG4 type antibody, preferably is an IgGi type antibody. The term “peptide”, as used herein, may refer to, for example, an oligopeptide, polypeptide, or protein.

Standard techniques of antibody design and preparation are known to a skilled person (see e.g. Antibodies: A Laboratory Manual, 2nd edition (2014), editor Greenfield, Cold Spring Harbor Laboratory Press (U.S.); Antibody Engineering - Methods and Protocols, 2nd edition (2010), editors Nevoltris and Chames, publisher Springer (Germany); Handbook of Therapeutic Antibodies (2014), editors Diibel and Reichert, publisher Wiley-VCH Verlag GmbH & Co. KGaA (Germany); Harper, Methods in Molecular Biology (2013), vol. 1045, p. 41-49).

In one embodiment, the antigen-binding peptide of the present invention is selected from an antibody, a Fab, Fab', a F(ab')2, a scFv, di-scFv, a VH domain, a single-domain antibody (sdAb), a diabody, a triabody, and a tetrabody. For example, said antigen-binding peptide may be selected from an antibody, a Fab, a F(ab')2 , and a scFv. In a preferred embodiment, said antigen-binding peptide is selected from an antibody, a Fab, and a scFv. In one embodiment, the term “antigen-binding peptide” relates to an anti-c-Met antibody or an antigen-binding fragment thereof.

The term “antigen-binding fragment”, as used herein, may refer to, for example, a Fab, a Fab', a F(ab')2, a scFv, a di-scFv, a VH domain, a single-domain antibody (sdAb), a diabody, a triabody, or a tetrabody. In one embodiment, the antigen-binding fragment of an antibody has the same binding properties as the antibody. In one embodiment, the antigen-binding fragment is selected from a Fab, a Fab', a F(ab')2, a scFv, a di-scFv, a VH domain, a singledomain antibody (sdAb), a diabody, a triabody, and a tetrabody.

For example, Fab fragments can be obtained by papain treatment of IgG type immunoglobulins, which results in two Fab fragment and an Fc domain. The term “scFv”, as used herein, preferably refers to a molecule comprising an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) connected by a linker, and lacks constant domains. For example, a scFv may include binding molecules which consist of one light chain variable domain (VL) or portion thereof, and one heavy chain variable domain (VH) or portion thereof, wherein each variable domain (or portion thereof) is derived from the same or different antibodies. scFv molecules preferably comprise a linker interposed between the VH domain and the VL domain, for example a peptide sequence comprising or consisting of the amino acids glycine and serine. For example, the peptide sequence may comprise the amino acid sequence (Gly4 Ser)_n, whereby n is an integer from 1- 6, e.g. n maybe 1, 2, 3, 4, 5, or 6, preferably n=4. The term “di-scFv”, as used herein, preferably refers to two scFv fragments which are coupled to each other via a linker. The term “diabody”, as used herein, preferably refers to an antibody fragment with two antigen-binding sites, said fragments comprising a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). For example, antigen-binding fragments may be obtained by digestion with peptidases such as pepsin, or papain: pepsin results in proteolytic cleavage below the disulfide linkages and results in a F(ab')2 antibody fragment, while proteolytic cleavage by papain, which cleaves above the disulfide linkages, results in two Fab fragments.

In one embodiment, the antigen-binding peptide of the invention is an anti-c-Met antibody or antigen-binding fragment thereof, comprising said first domain, preferably said heavy chain variable domain, comprising said first complementarity-determining region comprising or consisting of an amino acid sequence GGTFSSNA (SEQ ID NO: 1), said second complementarity-determining region comprising or consisting of an amino acid sequence IIPX1X2GTA (SEQ ID NO: 2), and said third complementarity-determining region comprising or consisting of an amino acid sequence ARDQRGX₃X₄X₅YYYX₆GMDV (SEQ ID NO: 3); wherein

Xi is an amino acid selected from I, E, and S,

X₂ is an amino acid selected from F, H, and Q,

X₃ is an amino acid selected from Y, S, H, and E,

X₄ is an amino acid selected from Y, R, H, Q, and S,

X₅ is an amino acid selected from Y, D, and H, and

X<, is an amino acid selected from Y and S; with the proviso that at least one of the following conditions a)-f) applies: a) Xi is not I, b) X₂ is not F, c) X₃ is not Y, d) X₄ is not Y, e) X₅ is not Y, f) Xe is not Y; wherein, optionally, said anti-c-Met antibody or antigen-binding fragment thereof comprises said second domain, preferably said light chain variable domain.

The inventors have surprisingly found that, advantageously, the antigen-binding peptide of the invention, for example variants CS06 VH6.18 & VH6.21 described herein, show strongly reduced hydrophobicity while maintaining pico-molar affinities and elevated cell binding properties as biparatopic antibodies, appropriate in silico developability profiles and improved capability for payload conjugation.

The term “c-Met”, as used herein, refers to MET Proto-Oncogene, Receptor Tyrosine Kinase (UniProtKB database entry P08581), which may also be referred to as Hepatocyte Growth Factor Receptor. In one embodiment, the antigen-binding peptide of the present invention binds to human c-MET variant N375S. In one embodiment, the antigen-binding peptide of the present invention binds to an epitope comprised in the IPT1 domain of human c-MET and inhibits c-MET signaling. In one embodiment, the antigen-binding peptide of the present invention binds to an epitope comprised in IPT domains 1-4 of human c-MET and inhibits c- MET signaling. In one embodiment, the first antigen-binding peptide of the bispecific molecule of the present invention binds to an epitope comprised in the IPT1 domain of human c-MET, and, optionally, the second antigen-binding peptide of the bispecific molecule of the present invention binds to an epitope comprised in the SEMA domain of human c-MET.

The antigen-binding peptide of the invention may bind to, for example, murine or human c- MET, preferably to human c-MET having the amino acid sequence according to SEQ ID NO: 38. For example, the antigen-binding peptide of the invention may bind to human c-MET in the form of full-length c-MET or in the form of c-MET with its signal peptide removed, e.g. with amino acids 1-24 cleaved off, including c-MET variant N375S. For example, the antigenbinding peptide of the invention may specifically bind to c-MET variant N375S. In one embodiment, the antigen-binding peptide of the invention binds to human c-MET. In one embodiment, the antigen-binding peptide of the invention binds to an epitope in the IPT1 domain of human c-MET and inhibits c-MET signaling. For example, the bispecific molecule of the invention may comprise a first antigen-binding peptide binding to an epitope in the IPT1 domain of human c-Met; and a second antigen-binding peptide binding to an epitope in the SEMA domain of human c-Met, such as to an epitope within amino acids 52-496 of mature human c-MET (UniProtKB P08581), e.g. to a linear or conformational epitope within amino acids 52 - 496 or within amino acids 27-515 of human c-MET; thereby inhibiting c-MET signaling.

In one embodiment, the antigen-binding peptide of the invention binds to an epitope within immunoglobulin-pl exin-transcription (IPT) domains 1-4 of human c-MET and inhibits c-MET signaling. For example, the IPT domains of human c-Met (UniProtKB P08581) comprise amino acids 562-655 (IPT domain 1), 656-739 (IPT domain 2), 741-842 (IPT domain 3) and amino acids 856-952 (IPT domain 4). In one embodiment, the antigen-binding peptide of the invention binds to IPT domain 1 and inhibits c-Met signaling. In one embodiment, the antigenbinding peptide of the invention binds to more than one IPT domain if the epitope is a conformational epitope. For example, the antigen-binding peptide of the invention may bind to an epitope which is comprised in IPT domains 1 and 2, or 2 and 3, or 3 and 4, or 1 and 4, or 1 and 3, or 2 and 4, or e.g. in IPT domains 1, 2, 3 and 4.

In a preferred embodiment, said antigen-binding peptide, for example said antibody or antigen-binding fragment thereof, specifically binds to c-Met. The term “specific” or “specifically binding”, as used herein, means that the antigen-binding peptide is capable of binding to an antigen of interest, such as c-Met, and does not essentially bind to other antigens; for example, the antigen-binding peptide maybe capable of binding to c-Met with a KD that is at least ten-fold, preferably at least 100-fold stronger than the KD for binding to other antigens in the human body. Such binding may be exemplified by the specificity of a lock-and-key- principle.

The term “first domain”, as used herein, relates to a domain comprising a first complementarity-determining region, a second complementarity-determining region, and a third complementarity-determining region. For example, said domain may comprise HCDR1, HCDR2, and HCDR3. In a preferred embodiment, the first domain of the antigen-binding peptide comprises or consists of a heavy chain variable domain. The term “heavy chain variable domain”, as used herein, preferably relates to a heavy chain variable domain of an antibody. In one embodiment, the antigen-binding peptide comprises or consists of the first domain. For example, an antigen-binding peptide comprising the first domain may be an antibody or antigen-binding fragment thereof, such as an antibody or an antigen-binding fragment selected from a Fab, Fab', a F(ab')2, a scFv, di-scFv, a diabody, a triabody, and a tetrabody. For example, an antigen-binding peptide consisting of the first domain maybe an antigen-binding fragment such as a VH domain or a single-domain antibody (sdAb). In one embodiment, the antigen-binding peptide comprises the first domain and optionally further comprises a second domain.

The inventors have surprisingly found that antigen-binding peptides comprising, for example, any of the following combinations of complementarity-determining regions have an enhanced thermal stability and significantly lowered hydrophobicity:

In one embodiment, said second complementarity-determining region of said first domain comprises or consists of an amino acid sequence of SEQ ID NO: 5 and said third complementarity-determining region of said first domain comprises or consists of an amino acid sequence of SEQ ID NO: 9.

In one embodiment, said second complementarity-determining region of said first domain comprises or consists of an amino acid sequence of SEQ ID NO: 5 and said third complementarity-determining region of said first domain comprises or consists of an amino acid sequence of SEQ ID NO: to.

In one embodiment, said second complementarity-determining region of said first domain comprises or consists of an amino acid sequence of SEQ ID NO: 6 and said third complementarity-determining region of said first domain comprises or consists of an amino acid sequence of SEQ ID NO: 11.

In one embodiment, said second complementarity-determining region of said first domain comprises or consists of an amino acid sequence of SEQ ID NO: 6 and said third complementarity-determining region of said first domain comprises or consists of an amino acid sequence of SEQ ID NO: 17.

In one embodiment, said second complementarity-determining region of said first domain comprises or consists of an amino acid sequence of SEQ ID NO: 6 and said third complementarity-determining region of said first domain comprises or consists of an amino acid sequence of SEQ ID NO: 12.

In one embodiment, said second complementarity-determining region of said first domain comprises or consists of an amino acid sequence of SEQ ID NO: 7 and said third complementarity-determining region of said first domain comprises or consists of an amino acid sequence of SEQ ID NO: 13.

In one embodiment, said second complementarity-determining region of said first domain comprises or consists of an amino acid sequence of SEQ ID NO: 6 and said third complementarity-determining region of said first domain comprises or consists of an amino acid sequence of SEQ ID NO: 14.

In one embodiment, said second complementarity-determining region of said first domain comprises or consists of an amino acid sequence of SEQ ID NO: 5 and said third complementarity-determining region of said first domain comprises or consists of an amino acid sequence of SEQ ID NO: 15.

In one embodiment, said second complementarity-determining region of said first domain comprises or consists of an amino acid sequence of SEQ ID NO: 8 and said third complementarity-determining region of said first domain comprises or consists of an amino acid sequence of SEQ ID NO: 16.

In one embodiment, said second complementarity-determining region of said first domain comprises or consists of an amino acid sequence of SEQ ID NO: 6 and said third complementarity-determining region of said first domain comprises or consists of an amino acid sequence of SEQ ID NO: 18.

In one embodiment, said second complementarity-determining region of said first domain comprises or consists of an amino acid sequence of SEQ ID NO: 5 and said third complementarity-determining region of said first domain comprises or consists of an amino acid sequence of SEQ ID NO: 19. In one embodiment, said second complementarity-determining region of said first domain comprises or consists of an amino acid sequence of SEQ ID NO: 6 and said third complementarity-determining region of said first domain comprises or consists of an amino acid sequence of SEQ ID NO: 15.

Advantageously, the antigen-binding peptides of the invention, for example antigen-binding peptides comprising or consisting of any one of SEQ ID NO: 20-31, have an enhanced thermal stability and significantly lowered hydrophobicity, e.g. compared to an antigenbinding peptide comprising or consisting of SEQ ID NO:45.

The term “second domain”, as used herein, relates to a domain comprising a first complementarity-determining region, a second complementarity-determining region, and a third complementarity-determining region. For example, said domain may comprise LCDR1, LCDR2, and LCDR3. In a preferred embodiment, the second domain of the antigen-binding peptide, if present, comprises or consists of a light chain variable domain. The term “light chain variable domain”, as used herein, preferably relates to a light chain variable domain of an antibody. In one embodiment, the antigen-binding peptide comprises or consists of the first domain and the second domain. For example, an antigen-binding peptide comprising the first domain and the second domain may be an antibody or antigen-binding fragment thereof, such as an antibody or an antigen-binding fragment selected from a Fab, Fab', a F(ab')2, a scFv, di- scFv, a diabody, a triabody, and a tetrabody. In one embodiment, the antigen-binding peptide comprises a second domain comprising one or more, optionally all, amino acid sequences selected from SEQ ID NO: 32-35.

The usage of the ordinal adjectives “first” and “second” in the context of the domain(s) of an antigen-binding peptide simply serves to distinguish the different domains from each other, and any such (first or second) domain(s) can also simply be referred to as “domain(s)”, without specifying them to be “first” or “second”. In particular, usage of the ordinal adjectives “first” and “second” does not imply any order or quality attributed to the different domains thus referred to, other than that these domains are not the same entities and can be distinguished from each other, e.g. by their sequences. For example, the antigen-binding peptide may comprise only the first domain. In one embodiment, the antigen-binding peptide binding to c- Met consists of the first domain.

The terms “complementarity determining region” and “CDR”, as used herein, relate to hypervariable or complementarity determining regions (CDRs) of antigen-binding peptides, such as hypervariable or complementarity determining regions (CDRs) found in the variable regions of light or heavy chains of antibodies and antigen-binding fragments thereof. In one embodiment, the terms “hypervariable region” and “complementarity determining region” are used interchangeably. The CDRs in each chain are typically held in close proximity by framework regions and contribute to the formation of the antigen-binding site, for example together with CDRs of another domain. In one embodiment, the antigen-binding peptide comprises the first domain and the second domain, and the CDRs of the first and second domains together form an anti-C-Met antigen-binding site. The CDRs may be annotated using IMGT (e.g. as described in Lefranc, M.-P., Immunology Today, 18, 509 (1997); Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); and Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55- 77 (2003)). The CDRs are determined according to the standard definitions of IMGT, for example as described in Lefranc et al. 2003.

The terms “first complementarity-determining region”, “second complementaritydetermining region”, and “third complementarity-determining region”, as used herein, relate to three CDRs of an antigen-binding peptide and/or of a first or second domain. For example, in the context of the first domain of the antigen-binding peptide, the first CDR may be HCDR1, the second CDR may be HCDR2, and the third CDR may be HCDR3. For example, in the context of the second domain of the antigen-binding peptide, the first CDR may be LCDR1, the second CDR maybe LCDR2, and the third CDR maybe LCDR3.

The term “at least one of the following conditions a)-f) applies”, as used herein, is meant to be understood as referring to the proviso that one or more of conditions a)-f) apply, particularly one or more of conditions a)-f) apply to the definition of sequences comprising residues X_x - X_(l; wherein conditions a)-f) are as follows: a) Xi is not I, b) X₂ is not F, c) X₃ is not Y, d) X₄ is not Y, e) X₅ is not Y, f) X_(l is not Y.

For example, condition a), b), c), d), e), or f); or conditions d) and e); or conditions b), d), and e); or conditions c), d), and e); or conditions b), c), d), and e) may apply. Other combinations of conditions a)-f) than the explicitly mentioned combinations of conditions may apply. In a preferred embodiment, condition d) and/or condition e), preferably condition e), applies. It is meant to be understood that, for example, an antigen-binding peptide fulfilling at least one of conditions a)-f) has at least one mutation compared to VH1.0, particularly compared to an antigen-binding peptide as defined in SEQ ID NO: 45. Furthermore, it is meant to be understood that, for example in a case in which condition a) applies, since X_x is not amino acid I in this case, X_x is an amino acid selected from E and S in this case; or for example in a case in which condition b) applies, since X₂ is not amino acid F in this case, X₂ is an amino acid selected from H and Q in this case. For example, if condition a) applies, X_x is an amino acid selected from E and S.

For example, if condition b) applies, X₂ is an amino acid selected from H and Q.

For example, if condition c) applies, X₃ is an amino acid selected from S, H, and E.

For example, if condition d) applies, X₄ is an amino acid selected from R, H, Q, and S.

For example, if condition e) applies, X₅ is an amino acid selected from D and H.

For example, if condition f) applies, X<> is amino acid S.

Particularly, if condition a) applies, then

Xi is an amino acid selected from E and S,

X₂ is an amino acid selected from F, H, and Q,

X₃ is an amino acid selected from Y, S, H, and E,

X₄ is an amino acid selected from Y, R, H, Q, and S,

X₅ is an amino acid selected from Y, D, and H, and

Xe is an amino acid selected from Y and S.

Particularly, if condition b) applies, then

Xi is an amino acid selected from I, E, and S,

X₂ is an amino acid selected from H and Q,

X₃ is an amino acid selected from Y, S, H, and E,

X₄ is an amino acid selected from Y, R, H, Q, and S,

X₅ is an amino acid selected from Y, D, and H, and

Xe is an amino acid selected from Y and S.

Particularly, if condition c) applies, then

Xi is an amino acid selected from I, E, and S,

X₂ is an amino acid selected from F, H, and Q,

X₃ is an amino acid selected from S, H, and E,

X₄ is an amino acid selected from Y, R, H, Q, and S,

X₅ is an amino acid selected from Y, D, and H, and

Xe is an amino acid selected from Y and S.

Particularly, if condition d) applies, then

Xi is an amino acid selected from I, E, and S,

X₂ is an amino acid selected from F, H, and Q,

X₃ is an amino acid selected from Y, S, H, and E,

X₄ is an amino acid selected from R, H, Q, and S,

X₅ is an amino acid selected from Y, D, and H, and

Xe is an amino acid selected from Y and S.

Particularly, if condition e) applies, then

Xi is an amino acid selected from I, E, and S,

X₂ is an amino acid selected from F, H, and Q, X₃ is an amino acid selected from Y, S, H, and E,

X₄ is an amino acid selected from Y, R, H, Q, and S,

X₅ is an amino acid selected from D and H, and

X<, is an amino acid selected from Y and S.

Particularly, if condition f) applies, then

Xi is an amino acid selected from I, E, and S,

X₂ is an amino acid selected from F, H, and Q,

X₃ is an amino acid selected from Y, S, H, and E,

X₄ is an amino acid selected from Y, R, H, Q, and S, X₅ is an amino acid selected from Y, D, and H, and Xe is amino acid S.

The terms “(X) % sequence identity” and “at least (X) % identical to”, as used herein, preferably relate to the percentage of pair-wise identical residues with regard to a homology alignment of a sequence of a polypeptide/nucleic acid of the present invention with a sequence in question. The terms “identity” and “identical”, as used herein, refer to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. Particularly, the expressions “% identity” or “% identical” may refer to the percent of identical residues between the amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest of the molecules being compared. For these calculations, gaps in alignments (if any) are preferably addressed by a particular mathematical model or computer program (i.e. an algorithm). In calculating percent identity, the sequences being compared are typically aligned in a way that gives the largest match between the sequences. The sequences are typically aligned for optimal matching of their respective amino acid or nucleotide. A gap opening penalty and a gap extension penalty, as well as a comparison matrix such as PAM 250 or BLOSUM 62, maybe used in conjunction with the algorithm.

The term “KD”, as used herein, relates to the dissociation constant, which is the inverse of the association constant. In one embodiment, said antigen-binding peptide binds to human c-Met ECD with a KD of 10 nM or less, preferably 8.5 nM or less, as determined using biolayer interferometry. In a preferred embodiment, the KD is determined using biolayer interferometry. In one embodiment, said determination using biolayer interferometry comprises performing biolayer interferometry at 30 °C and 1000 rpm in kinetics buffer; wherein, preferably, said kinetics buffer comprises phosphate-buffered saline, 0.1% bovine serum albumin, and 0.02% Polyoxyethylene (20) sorbitan monolaurate, and has a pH of pH 7.4. In one embodiment, said determination using biolayer interferometry is performed analogously to the biolayer interferometry as described in Sellmann et al., JBC (2016). In one embodiment, said antigen-binding peptide has a half maximal effective concentration (EC₅o) for binding to c-MET expressing cells of 2 nM or less, preferably 1.6 nM or less, more preferably 1.2 nM or less, as determined using flow cytometry. In one embodiment, said c-MET expressing cells are EBC-1 tumor cells, particularly human lung carcinoma EBC-1 cells. In one embodiment, said half maximal effective concentration is determined on EBC-i tumor cells by titration of the antigen-binding peptide and performing flow cytometry (for example using iQue 3 screener, Sartorius). In one embodiment, said half maximal effective concentration is determined using flow cytometry in 1:2 titration series from 500 nM to 0.01 nM of the molecule to be analyzed, such as the antigen-binding peptide of the invention or the bispecific molecule of the invention, on c-MET expressing human lung carcinoma EBC-1 cells (for example, human lung carcinoma EBC-1 cells of Riken Bioresource Center Cell Bank JCRB0920 031496, cultured under recommended conditions); optionally comprising detection using AF-488 AffiniPure Fab Fragment Goat-anti-Human IgG and a Fey fragment specific detection antibody (for example, Jackson Immuno Research). In one embodiment, said half maximal effective concentration is determined using flow cytometry in 1:2 titration series from 500 nM to 0.01 nM of the molecule to be analyzed, such as the antigen-binding peptide of the invention or the bispecific molecule of the invention, on c-MET expressing human lung carcinoma EBC- 1 cells.

The terms “half maximal effective concentration”, “EC50”, and “EC₅₀”, as used herein, particularly as used herein interchangeably, preferably relate to a measure of the concentration of a molecule, such as an antigen-binding peptide, a bispecific molecule, an ADC, or a drug, which induces a biological response halfway between the baseline and maximum after a specified exposure time. It is commonly used as a measure of potency. The EC₅₀ of a graded dose response curve may represent the concentration of a molecule where 50% of its maximal effect is observed. In one embodiment, said antigen-binding peptide has a half maximal effective concentration for binding to c-MET expressing cells of 2 nM or less, preferably 1.6 nM or less, more preferably 1.2 nM or less, as determined using flow cytometry.

In one embodiment, said antigen-binding peptide has a hydrophobic interaction chromatography retention time of 20 min or less, preferably 19.6 min or less, even more preferably 19 min or less, as determined using hydrophobic interaction chromatography. In a preferred embodiment, said determination using hydrophobic interaction chromatography comprises using hydrophobic interaction chromatography which yields a hydrophobic interaction chromatography retention time of 19.6 min for reference Cetuximab, such as Erbitux®. In one embodiment, said determination of said hydrophobic interaction chromatography retention time using hydrophobic interaction chromatography comprises providing a hydrophobic interaction chromatography method which yields a hydrophobic interaction chromatography retention time of 19.6 min for reference Cetuximab, such as Erbitux®, and determining the hydrophobic interaction chromatography retention time of the molecule to be analyzed, such as the antigen-binding peptide of the invention, the bispecific molecule of the invention, or the antigen-binding peptide-drug conjugate of the invention, using said hydrophobic interaction chromatography method. In one embodiment, said determination using hydrophobic interaction chromatography comprises loading the molecule to be analyzed, such as the antigen-binding peptide of the invention, the bispecific molecule of the invention, or the antigen-binding peptide-drug conjugate of the invention, on a Butyl-NPR column, particularly a Butyl-NPR 2.5 pm 4.6 mm x too mm column, and running a linear gradient, preferably a linear gradient of from 50 mM sodium phosphate and 1.5 M ammonium sulfate at pH 7.0 to 50 mM sodium phosphate and 5% isopropanol at pH 7.0, e.g. at a temperature of 25°C for 33 min. In one embodiment, said linear gradient comprises or consists of a linear gradient from a starting mixture which comprises 50 mM sodium phosphate and 1.5 M ammonium sulfate having pH 7.0 to a final mixture which comprises 50 mM sodium phosphate and 5% isopropanol having pH 7.0.

In one embodiment, said determination using hydrophobic interaction chromatography comprises providing the molecule to be analyzed, such as the antigen-binding peptide of the invention or the bispecific molecule of the invention, in PBS with a pH of pH 7.4, and adding ammonium sulfate thereto to obtain a final concentration of 1M ammonium sulfate; wherein, optionally, the molecule to be analyzed is provided in an amount of 20 pg and/or is provided in a concentration of 0.4 mg/ml; providing a Butyl-NPR column, particularly a Butyl-NPR 2.5 pm 4.6 mm x too mm column; running a gradient at a flow rate of 0.75 ml/min using a linear gradient, particularly a linear gradient of 50 mM sodium phosphate and 1.5 M ammonium sulfate at pH 7.0 to 50 mM sodium phosphate and 5% isopropanol at pH 7.0 at a temperature of 25°C for 33 min; and monitoring absorbance at 214 nm using a multi-wavelength detector.

The term “hydrophobic interaction chromatography retention time”, as used herein, preferably relates to a retention time determined for a molecule to be analyzed, such as the antigenbinding peptide of the invention, the bispecific molecule of the invention, or the antigenbinding peptide-drug conjugate of the invention, using said hydrophobic interaction chromatography. The retention time determined for a molecule to be analyzed is typically compared to a retention time determined for a reference, for example Cetuximab, particularly a retention time determined for a reference using HIC with the same conditions. For example, the hydrophobic interaction chromatography retention time of a molecule to be analyzed, such as the antigen-binding peptide of the invention, the bispecific molecule of the invention, or the antigen-binding peptide-drug conjugate of the invention, may be determined using hydrophobic interaction chromatography and may subsequently be compared to a hydrophobic interaction chromatography retention time of a reference, such as Cetuximab. For example, the hydrophobic interaction chromatography retention time may be used as a measure of hydrophobicity of a molecule to be analyzed.

In a preferred embodiment, said antigen-binding peptide has a hydrophobic interaction chromatography retention time of < 19.6 min. In one embodiment, said antigen-binding peptide has a hydrophobic interaction chromatography retention time of 20 min or less, preferably 19.6 min or less, more preferably of < 19.6 min.

In one embodiment, said antigen-binding peptide has a hydrophobic interaction chromatography retention time of 19 min or less. The inventors have surprisingly found that, for example, an antigen-binding peptide of the present invention having any of the following sequences has a hydrophobic interaction chromatography retention time of 19 min or less: In one embodiment, said antigen-binding peptide comprises a first domain comprising a first complementarity-determining region comprising or consisting of an amino acid sequence of SEQ ID NO: 1, a second complementarity-determining region and a third complementaritydetermining region; wherein said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 5 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 9, or said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 5 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 10, or said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 6 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 11, or said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 5 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 15, or said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 6 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 18.

In one embodiment, said antigen-binding peptide comprises a first domain comprising or consisting of an amino acid sequence which is at least 90% identical, preferably at least 95 % identical, more preferably at least 98% identical, even more preferably at least 99% identical, optionally identical, to an amino acid sequence selected from the group consisting of QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSNAISWVRQAPGQGLEWMGGIIPIQGTAIYAQ KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDQRGSHDYYYYGMDVWGQGTTVTVS S (SEQ ID NO: 20),

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSNAISWVRQAPGQGLEWMGGIIPIHGTAIYAQ KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDQRGYRHYYYYGMDVWGQGTTVTVS S (SEQ ID NO: 23), and

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSNAISWVRQAPGQGLEWMGGIIPIQGTAIYAQ KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDQRGYRDYYYYGMDVWGQGTTVTVS S (SEQ ID NO: 29).

In one embodiment, said antigen-binding peptide binds to human c-Met ECD with a KD of 10 nM or less, preferably 8.5 nM or less, as determined using biolayer interferometry, and said antigen-binding peptide has a hydrophobic interaction chromatography retention time of 20 min or less, preferably 19.6 min or less, even more preferably 19 min or less, as determined using hydrophobic interaction chromatography; wherein, optionally, said antigen-binding peptide has a half maximal effective concentration (EC₅₀) for binding to c-MET expressing cells of 2 nM or less, preferably 1.6 nM or less, more preferably 1.2 nM or less, as determined using flow cytometry. In one embodiment, said antigen-binding peptide has a half maximal effective concentration (EC₅₀) for binding to c-MET expressing cells of 2 nM or less, preferably 1.6 nM or less, more preferably 1.2 nM or less, as determined using flow cytometry, and said antigenbinding peptide has a hydrophobic interaction chromatography retention time of 20 min or less, preferably 19.6 min or less, even more preferably 19 min or less, as determined using hydrophobic interaction chromatography; wherein, optionally, said antigen-binding peptide binds to human c-Met ECD with a KD of 10 nM or less, preferably 8.5 nM or less, as determined using biolayer interferometry.

In one embodiment, said antigen-binding peptide has a ratio of KDantigen-binding peptide to KD_wiidty_Pe < 10. The term “KDantigen-binding peptide”, as used herein, relates to a KD of a molecule to be analyzed, particularly a KD of the antigen-binding peptide of the invention. In one embodiment, said KDantigen-binding peptide designates the KD for binding of the antigen-binding peptide according to the present invention to human c-Met ECD. The term “KD_wiidty_Pe”, as used herein, relates to a KD of a reference molecule, such as an antigen-binding peptide comprising or consisting of a sequence of SEQ ID NO: 45; particularly a KD of variant VH1.0 as disclosed herein. In one embodiment, said KD_wiidty_Pe designates the KD for binding of a reference molecule, preferably an antigen-binding peptide comprising or consisting of a sequence of SEQ ID NO: 45, to human c-Met ECD. The term “ratio of KD_antigen-binding_Pe_Ptideto KD_wiidty_Pe”, as used herein, relates to a ratio of a KD of a molecule to be analyzed, particularly a KD of the antigenbinding peptide of the invention, to a reference KD, particularly KD_wiidty_Pe which is preferably a KD of variant VH1.0 as disclosed herein. The term “wildtype”, as used herein, preferably refers to an antigen-binding peptide comprising or consisting of a sequence of SEQ ID NO: 45, particularly to VH1.0 as disclosed herein. The term “VH1.0”, as used herein, refers to a reference antigen-binding peptide, preferably comprising or consisting of a sequence of SEQ ID NO: 45; particularly to a CS06 variant with mutations Q5V and A18V, wherein CS06 is as disclosed in Sellmann et al. 2016 and EP 3512882 Bi. In one embodiment, the terms “VH1.0”, ’’variant VH1.0”, and “antigen-binding peptide comprising or consisting of a sequence of SEQ ID NO: 45” are used interchangeably.

In one embodiment, said antigen-binding peptide binds to human c-Met ECD with a KD of 10 nM or less, preferably 8.5 nM or less, as determined using biolayer interferometry, and has a ratio Of KDantigen-binding _Pe_Ptide tO KD_wildty_Pe < 10.

In one embodiment, said antigen-binding peptide has a ratio of ECsOantigen-binding _peptide to EC5O_wiidty_Pe < 1, preferably < 0.8.

The term “ECsOantigen-binding _peptide”, as used herein, relates to an EC50 of a molecule to be analyzed, particularly an EC50 of the antigen-binding peptide of the invention. In one embodiment, said ECsOantigen-binding _peptide designates a half maximal effective concentration (EC50) of the antigen-binding peptide according to the present invention for binding to c-MET expressing cells. The term “EC5O_wiidty_Pe”, as used herein, relates to an EC50 of a reference molecule, such as an antigen-binding peptide comprising or consisting of a sequence of SEQ ID NO: 45; particularly an EC50 of variant VH 1.0 as disclosed herein. In one embodiment, said EC5O_wiidty_Pe designates a half maximal effective concentration (EC50) of a reference molecule, preferably an antigen-binding peptide comprising or consisting of a sequence of SEQ ID NO: 45, for binding to c-MET expressing cells. The term “ratio of ECsOantigen-binding _peptide to EC5O_wiidty_Pe”, as used herein, relates to a ratio of an EC50 of a molecule to be analyzed, particularly an EC50 of the antigen-binding peptide of the invention, to a reference EC50, particularly EC5O_wiidty_Pe which is preferably an EC50 of variant VH1.0 as disclosed herein. When determining the ratio of EC5Oanti_gen-bindin_{g P}e_Ptideto EC5O_wiidty_Pe and/or the ratio of KDantigen- binding peptide to KD_wiidtype, the molecule to be analyzed, particularly the antigen-binding peptide of the invention, and the reference molecule, particularly variant VHi.o as disclosed herein, are preferably provided in the same format, for example both are provided in the form of the same antibody type, e.g. IGgi, or in the form of the same antibody fragment, e.g. Fab or scFv.

In one embodiment, said antigen-binding peptide has a half maximal effective concentration (EC₅o) for binding to c-MET expressing cells of 2 nM or less, preferably 1.6 nM or less, more preferably 1.2 nM or less, as determined using flow cytometry, and has a ratio of ECsOantigen- binding peptide to EC,5Owii<iivpe — i, preferably — 0.8.

In one embodiment, said antigen-binding peptide has a ratio of KDantigen-binding peptide to KDwiidtype < 10, and said antigen-binding peptide has a ratio of ECsOantigen-binding peptide to EC50_wiidty_Pe < 1, preferably < 0.8. In one embodiment, said antigen-binding peptide has a ratio of KDantigen-binding peptide to KDwiidtype < 10, and said antigen-binding peptide has a hydrophobic interaction chromatography retention time of < 19.6 min. In one embodiment, said antigen-binding peptide has a ratio of ECsOantigen-binding peptide to EC50wiidty_Pe < 1, preferably < 0.8, and said antigen-binding peptide has a hydrophobic interaction chromatography retention time of < 19.6 min. In a preferred embodiment, said antigen-binding peptide has a ratio of KDantigen-binding peptide to KDwiidtype < 10, said antigen-binding peptide has a ratio of ECsOantigen-binding peptide to EC50_wiidtype < i, preferably < 0.8, and said antigen-binding peptide has a hydrophobic interaction chromatography retention time of < 19.6 min.

For example, the ratio of KDantigen-binding peptide to KDwiidtype and/or the ratio of ECsOantigen-binding peptide to ECsOwikiivpe may be analyzed by analyzing the antigen-binding peptide and the wildtype each in the form of an IgGi.

In one embodiment, said antigen-binding peptide is selected from a Fab comprising an amino acid sequence of SEQ ID NO: 20, a Fab comprising an amino acid sequence of SEQ ID NO: 21, a scFv comprising an amino acid sequence of SEQ ID NO: 20, and a scFv comprising an amino acid sequence of SEQ ID NO: 21; preferably selected from a Fab comprising an amino acid sequence of SEQ ID NO: 20, a Fab comprising an amino acid sequence of SEQ ID NO: 21, and a scFv comprising an amino acid sequence of SEQ ID NO: 21.

The inventors have surprisingly found that the antigen-binding peptides of the invention have an enhanced thermal stability compared to previous antigen-binding peptide CS06 VHi.o. In one embodiment, the antigen-binding peptide of the invention has a melting temperature, particularly a melting temperature Tmi, of at least 68°C, preferably at least 68.5°C, more preferably at least 69°C. The melting temperature, particularly a melting temperature Tmi, may be determined by differential scanning fluorometry. For example, said differential scanning fluorometry (DSF) may be nano-DSF, e.g. using a Prometheus device. For example, the melting temperature, particularly a melting temperature Tmi, may be determined by subjecting the antigen-binding peptide, e.g. an antibody, to a linear thermal ramp (e.g. i °C/ min, from 25 to 90 °C), and collecting tryptophan fluorescence at 350 and 330 nm at a rate of 10 datapoints per minute; wherein, preferably, unfolding transition midpoints are determined from a 2nd derivative of the fluorescence ratio (F350/F330).

The term “Tmi”, as used herein, relates to a melting temperature; particularly, if a molecule such as an antigen-binding peptide, displays more than one transition, “Tmi” relates to a lowest melting temperature of said molecule.

In one embodiment, the antigen-binding peptide of the invention is selected from antigenbinding peptides comprising a first domain which comprises a first complementaritydetermining region comprising or consisting of an amino acid sequence GGTFSSNA (SEQ ID NO: 1), and which comprises a second complementarity-determining region and a third complementarity-determining region; wherein said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 5 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 9, or said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 5 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 10, or said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 6 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 11, or said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 6 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 17, or said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 6 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 12, or said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 7 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 13, or said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 6 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 14, or said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 5 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 15, or said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 8 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 16, or said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 5 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 19, or said second complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 6 and said third complementarity-determining region comprises or consists of an amino acid sequence of SEQ ID NO: 15. Advantageously, the antigen-binding peptides of the invention, for example antigen-binding peptides selected from these exemplary antigen-binding peptides, have a greatly enhanced thermal stability.

In one embodiment, the first domain of the antigen-binding of the invention comprises or consists of an amino acid sequence which is at least 90% identical, preferably at least 95 % identical, more preferably at least 98% identical, even more preferably at least 99% identical, optionally identical, to an amino acid sequence selected from the group consisting of QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSNAISWVRQAPGQGLEWMGGIIPIQGTAIYAQ KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDQRGSHDYYYYGMDVWGQGTTVTVS S (SEQ ID NO: 20),

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSNAISWVRQAPGQGLEWMGGIIPEFGTAIYAQ KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDQRGSYDYYYYGMDVWGQGTTVTVSS (SEQ ID NO: 26), QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSNAISWVRQAPGQGLEWMGGIIPSFGTAIYAQ KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDQRGESDYYYYGMDVWGQGTTVTVSS (SEQ ID NO: 27),

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSNAISWVRQAPGQGLEWMGGIIPIHGTAIYAQ KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDQRGYQDYYYYGMDVWGQGTTVTVS S (SEQ ID NO: 28),

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSNAISWVRQAPGQGLEWMGGIIPIHGTAIYAQ KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDQRGYRDYYYYGMDVWGQGTTVTVS

S (SEQ ID NO: 31). The inventors have found that, advantageously, antigen-peptides of the invention, such as these exemplary antigen-binding peptides of the invention, have an enhanced thermal stability, particularly a melting temperature of 69°C or more.

In one embodiment, the antigen-binding peptide is coupled to a diagnostic and/or therapeutic agent, preferably a therapeutic agent, more preferably a cytotoxic agent, such as exatecan.

The term “bispecific molecule”, as used herein, relates to a molecule, preferably an antibody or an antigen-binding fragment thereof, that can simultaneously bind to two different types of antigen or two different epitopes on the same antigen. In a preferred embodiment, the bispecific molecule is a bispecific antibody or antigen-binding fragment thereof. For example, the bispecific molecule may bind to c-Met and a tumor antigen other than c-Met, such as EGFR or CEA5; or the bispecific molecule may bind to two different epitopes of c-Met. In one embodiment, the bispecific molecule comprises a first antigen-binding peptide binding to c- Met; and comprises a second antigen-binding peptide binding to c-Met, particularly binding to an epitope of c-Met other than the first antigen-binding peptide, or binding to a tumor antigen other than c-Met, such as EGFR or CEA5. For example, said first antigen-binding peptide may be a Fab and said second antigen-binding peptide may be a scFv, or said first antigen-binding peptide may be a scFv and said second antigen-binding peptide may be a Fab.

In one embodiment, the bispecific molecule comprises a first antigen-binding peptide in the form of a Fab and a second antigen-binding peptide in the form of a scFv, or comprises a first antigen-binding peptide in the form of a scFv and a second antigen-binding peptide in the form of a Fab. In one embodiment, the first antigen-binding peptide, for example in the form of a Fab or scFv, is fused to an AG-SEED and the second antigen-binding peptide, for example in the form of a scFv of Fab, is fused to a GA-SEED, or said first antigen-binding peptide is fused to a GA-SEED and said second antigen-binding peptide is fused to an AG-SEED. The term “SEED”, as used herein, refers to strand-exchange engineered domain (SEED) CH3 heterodimers as disclosed in W02007/110205 A2. These heterodimeric SEED molecules are derivatives of human IgG and IgA CH3 domains and create complementary human SEED CH3 heterodimers that are composed of alternating segments of human IgA and IgG CH3 sequences. A pair of SEED CH3 domains preferably associates to form heterodimers in a 1:1 ratio when expressed in mammalian cells to form “SEEDbodies” (Sb). The term “GA-SEED” hereby indicates that the SEED molecule begins with an IgG sequence, followed by an IgA sequence, while “AG-SEED” refers to the fact that the SEED molecule begins with an IgA- derived sequence followed by an IgG-derived sequence.

In one embodiment, the bispecific molecule comprises a first antigen-binding peptide and a second antigen-binding peptide which are connected by heterodimerization, preferably using a knob-into-hole approach (for example a knob-into-hole approach as described in Merchant et al. 1998 or Xu et al 2015). In one embodiment, the bispecific molecule is provided in a knob- into-hole format. For example, the knob-into-hole format may be a format as described in Merchant et al. 1998 or Xu et al 2015. For example, the bispecific molecule comprising a first antigen-binding peptide and a second antigen-binding peptide connected by a knob-into-hole approach may comprise any sequence of SEQ ID NO: 39-44, preferably any or all sequences of SEQ ID NO: 39-41 or SEQ ID NO: 42-44. In one embodiment, the bispecific molecule comprises or consists of sequences of SEQ ID NO: 39-41 or the bispecific molecule comprises or consists of sequences of SEQ ID NO: 42-44.

The term “first antigen-binding peptide”, as used herein, preferably relates to the antigenbinding peptide of the invention, for example an antigen-binding fragment of an anti-c-Met antibody, such as a Fab, Fab', a F(ab')2, a scFv, di-scFv, or a VH domain. The term “second antigen-binding peptide”, as used herein, preferably relates to a further antigen-binding peptide of the bispecific molecule, for example an antigen-binding fragment of an antibody, such as a Fab, Fab', a F(ab')2, a scFv, di-scFv, or a VH domain. The second antigen-binding peptide may bind to the same antigen as the first antigen-binding peptide or may bind to a different antigen. For example, said second antigen-binding peptide may bind to a cell surface receptor, particularly to a cell surface receptor overexpressed on cells of a tumor, such as a solid tumor. In a preferred embodiment, the second antigen-binding peptide binds to a tumor antigen. The second antigen-binding peptide may bind to the same tumor antigen as the first antigen-binding peptide, particularly c-Met, or may bind to a different tumor antigen as the first antigen-binding peptide, such as EGFR or CEA5. In one embodiment, the first antigenbinding peptide binds to c-Met, and the second antigen-binding peptide binds to c-Met, particularly binds to an epitope of c-Met other than the first antigen-binding peptide, or binds to a tumor antigen other than c-Met, such as EGFR or CEA5.

As used herein, a “tumor antigen” is, in its broadest sense, an antigen that allows recruitment of a molecule, such as an antigen-binding peptide or a bispecific molecule according to the present invention, to the site of a tumor. Upon recruitment of said molecule, a therapeutic action or diagnostic action (e.g. labelling of the tumor site) can be achieved. The tumor antigen may either be an antigen that is present on the surface of the tumor cells or an antigen associated with the tumor microenvironment. In one embodiment, said tumor antigen is an antigen that is present on the surface of a tumor cell. In one embodiment, the term “tumor antigen” indicates an antigen that is present at the cell surface of a tumor cell and allows for distinction of the tumor cell over other cell types.

In one embodiment, said tumor antigen is part of a molecule (e.g. a protein) that is expressed by a tumor cell and accessible from the extracellular environment. A tumor antigen may differ (i.e. qualitatively differ) from its counterpart in corresponding non-tumor cells (e.g., where the molecule is a protein by one or more amino acid residues). Alternatively, the tumor antigen may be identical to its counterpart in corresponding non-tumor cells, but present on the surface of the tumor cells at a higher level than on the surface of corresponding non-tumor cells. For example, the tumor antigen may be present only on the surface of the tumor cells, but not on the surface of non-tumor cells, or the tumor antigen may be present on the surface of tumor cells at a higher level (e.g. at least 5-fold higher, preferably at least 100-fold higher) than on the surface of non-tumor cells. In an embodiment, the tumor antigen is present on the surface of tumor cells at a level that is at least 1000-fold higher than on the surface of non- tumor cells. In one embodiment, the tumor antigen is present only on the surface of the tumor cells, but not on the surface of non-tumor cells. In one embodiment, the tumor antigen is present on the surface of tumor cells at a higher level (e.g. at least 5-fold higher, preferably at least 100-fold higher) than on the surface of non-tumor cells. In one embodiment, the tumor antigen is present on the surface of tumor cells at a level that is at least 1000-fold higher than on the surface of non-tumor cells.

The term “tumor”, as used herein, refers to an abnormal cell mass formed by neoplastic cell growth. A tumor can be benign or malignant. Preferably, in the present disclosure the term “tumor” refers to a malignant tumor. The tumor can for example be, but is not limited to, a tumor present in a lung, breast, ovary, kidney, gastrointestinal tract, thyroid, pancreas, or head and neck. In one embodiment, the tumor is a c-MET expressing tumor. As used herein, the term “cancer” refers to a malignant neoplasm, preferably to a c-MET expressing cancer. Cancer can include a hematological cancer or a solid tumor. For example, the cancer can be a lung cancer (e.g., non-small cell lung cancer; NSCLC), breast cancer, ovarian cancer, kidney cancer, gastrointestinal cancer, particularly colon and/or stomach cancer, thyroid cancer, pancreas cancer, or head and neck cancer. The cancer is preferably a solid malignant tumor. In one embodiment, the cancer is a lung cancer, breast cancer, ovarian cancer, kidney cancer, gastrointestinal cancer, thyroid cancer, pancreas cancer, or head and neck cancer.

Non-limiting examples of tumor antigens are c-MET, EGFR, and CEA5 (Carcinoembryonic antigen 5). The bispecific molecule may, for example, bind to c-Met, or to c-Met and EGFR or CEA5. Particularly, the first antigen-binding peptide of the bispecific molecule may bind to c- Met, and the second antigen-binding peptide of the bispecific molecule may bind to c-MET, EGFR, or CEA5. An example of a previous bispecific anti-EGFR x anti-c-MET antibody is Amivantamab (Syed YY. Amivantamab: First Approval. Drugs. 2021 Jul;8i(n):i349-1353.). In one embodiment, the bispecific molecule is a biparatopic molecule, wherein the first and the second antigen-binding peptide of the bispecific molecule each bind to c-MET.

Insofar as the designations of antigens indicated in the present disclosure are gene designations, these designations refer to the protein(s) encoded by said gene. Sources for information on cell surface expression and methods to identify and verify tumor antigens are known to a skilled person and described in the literature (see e.g. Bornstein, AAPS J. (2015), vol. 17(3), p. 525-534; Bander, Methods Mol Biol (2013), vol. 1045, p. 29-40; Antibody-Drug Conjugates: Fundamentals, Drug Development, and Clinical Outcomes to Target Cancer", 1st edition (2016), editors Olivier and Hurvitz, publisher John Wiley & Sons, Inc. (U.S.); Vigneron et al., Cancer Immun. (2013), vol. 13, p. 15; Hong et al., BMC Syst Biol. (2018), vol. 12 (Suppl 2), p. 17; de Souza et al., Cancer Immun. (2012), vol. 12, p. 15; Immune Epitope Database and Analysis Resource (https://www.iedb.org); Cancer Cell Line Encyclopedia (https://portals.broadinstitute.org/ccle); OASIS Database (http://oasis-genomics.org/)).

The term “biparatopic molecule”, as used herein, relates to a molecule that binds to two different epitopes on the same antigen, particularly that binds to two non-overlapping epitopes on the same antigen. In one embodiment, the bispecific molecule is a biparatopic molecule, wherein the first antigen-binding peptide binds to c-Met and the second antigen-binding peptide binds to c-Met; particularly the second antigen-binding peptide binds to an epitope of c-Met other than the epitope of c-Met to which the first antigen-binding peptide binds. In a preferred embodiment, the biparatopic molecule is a biparatopic antibody or antigen-binding fragment thereof. In one embodiment, each of the first and the second antigen-binding peptides are a Fab or scFv, or one of the first and the second antigen-binding peptides is a Fab and the other is a scFv.

In one embodiment, said bispecific molecule comprises an antigen-binding peptide of the present invention; preferably an antigen binding peptide having a VH domain comprising or consisting of an amino acid sequence selected from SEQ ID NO: 20-31, preferably selected from SEQ ID NO: 20-22, more preferably selected from SEQ ID NO: 20-21, and, optionally, a VL domain comprising or consisting of an amino acid sequence of SEQ ID NO: 35; and further comprises an antigen-binding peptide binding to a tumor antigen, preferably an antigen-binding peptide binding to c-Met, more preferably an antigen binding peptide having a VH domain comprising or consisting of an amino acid sequence of SEQ ID NO: 36 and a VL domain comprising or consisting of an amino acid sequence of SEQ ID NO: 37.

In one embodiment, said bispecific molecule comprises a first antigen-binding peptide having a VH domain comprising or consisting of an amino acid sequence selected from SEQ ID NO: 20-31, preferably selected from SEQ ID NO: 20-22, more preferably selected from SEQ ID NO: 20-21, and a VL domain comprising or consisting of an amino acid sequence of SEQ ID NO: 35; wherein said first antigen-binding peptide is a Fab or scFv; and further comprises an antigen-binding peptide binding to a tumor antigen, preferably an antigen-binding peptide binding to c-Met, more preferably an antigen binding peptide having a VH domain comprising or consisting of an amino acid sequence of SEQ ID NO: 36 and a VL domain comprising or consisting of an amino acid sequence of SEQ ID NO: 37; wherein said second antigen-binding peptide is a Fab or scFv; wherein, preferably, if said first antigenbinding peptide is a Fab, said second antigen-binding peptide is a scFv, and if said first antigenbinding peptide is a scFv, said second antigen-binding peptide is a Fab.

In one embodiment, said bispecific molecule comprises a first antigen-binding peptide having a VH domain comprising or consisting of an amino acid sequence selected from SEQ ID NO: 20-21, and a VL domain comprising or consisting of an amino acid sequence of SEQ ID NO: 35; wherein said first antigen-binding peptide is a Fab; and further comprises an antigen-binding peptide binding to a tumor antigen, preferably an antigen-binding peptide binding to c-Met, more preferably an antigen binding peptide having a VH domain comprising or consisting of an amino acid sequence of SEQ ID NO: 36 and a VL domain comprising or consisting of an amino acid sequence of SEQ ID NO: 37; wherein said second antigen-binding peptide is a scFv.

In one embodiment, said bispecific molecule comprises a first antigen-binding peptide having a VH domain comprising or consisting of an amino acid sequence selected from SEQ ID NO: 20-21, preferably an amino acid sequence of SEQ ID NO: 21, and a VL domain comprising or consisting of an amino acid sequence of SEQ ID NO: 35; wherein said first antigen-binding peptide is a scFv; and further comprises an antigen-binding peptide binding to a tumor antigen, preferably an antigen-binding peptide binding to c-Met, more preferably an antigen binding peptide having a VH domain comprising or consisting of an amino acid sequence of SEQ ID NO: 36 and a VL domain comprising or consisting of an amino acid sequence of SEQ ID NO: 37; wherein said second antigen-binding peptide is a Fab.

In one embodiment, the bispecific molecule, preferably bispecific antibody or antigen-binding fragment thereof, is coupled to a diagnostic and/ or therapeutic agent, preferably a therapeutic agent, more preferably a cytotoxic agent, such as exatecan.

In one embodiment, the bispecific molecule comprises or consists of one or more amino acid sequences selected from SEQ ID NO: 39-44, preferably comprises or consists of one or more, preferably all, sequences of SEQ ID NO: 39-41 or of SEQ ID NO: 42-44. In one embodiment, the bispecific molecule comprises or consists of the sequences of SEQ ID NO: 39-41 or the bispecific molecule comprises or consists of the sequences of SEQ ID NO: 42-44.

In one embodiment, the bispecific molecule is a heterodimeric immunoglobulin molecule, preferably comprising

(i) a first and/or second Fab or scFv fragment which specifically bind(s) to human c-MET, and

(ii) an antibody hinge region, an antibody CH2 domain and an antibody CH3 domain comprising a hybrid protein-protein interaction interface domain, wherein said interaction interface domain is formed by amino acid segments of the CH3 domain of a first member and amino acid segments of the CH3 domain of a second member, wherein said protein-protein interface domain of the first chain is interacting with the protein-protein-interface of the second chain by homodimerization of the corresponding amino acid segments of the same member of the immunoglobulin superfamily within said interaction domains; wherein the first Fab or scFv fragment comprises an amino acid sequence selected from SEQ ID NO: 20-31. In one embodiment, the bispecific molecule of the invention is of the knob-into- hole format or of the SEED format, preferably of the knob-into-hole format. In one embodiment, the bispecific molecule of the invention is an antibody of the knob-into-hole format or an antibody of the SEED format, preferably an antibody of the knob-into-hole format.

In one embodiment, the bispecific molecule is afucosylated, preferably is an afucosylated bispecific antibody or antigen-binding fragment thereof. The term “antigen-binding peptide-drug conjugate”, as used herein, relates to an antigenbinding peptide linked to a payload, particularly an antigen-binding peptide linked to a diagnostic and/or therapeutic agent. In a preferred embodiment, the antigen-binding peptide- drug conjugate is an antibody-drug conjugate. The term “DAR”, as used herein, relates to the drug to antibody ratio and indicates the level of loading of the payload on the ADC. The antigen-binding peptide-drug conjugate may comprise a linker which links the payload and the antigen-binding peptide; such as a linker based on chemical motifs including disulfides, hydrazones, peptides, or thioethers.

In one embodiment, said antigen-binding peptide-drug conjugate is an antibody-drug conjugate, comprising an antibody comprising or consisting of an antigen-binding peptide of the present invention, or an antibody comprising or consisting of a bispecific molecule of the present invention; and further comprising a diagnostic and/ or therapeutic agent, preferably a therapeutic agent, more preferably a cytotoxic agent, such as exatecan; wherein, optionally, the antibody-drug conjugate comprises a linker.

The term “diagnostic and/or therapeutic agent”, as used herein, may refer to a diagnostic agent, to a therapeutic agent, and/or to an agent which is diagnostic and therapeutic, particularly a diagnostic and therapeutic agent. In a preferred embodiment, the diagnostic and/or therapeutic agent is a therapeutic agent, more preferably a cytotoxic agent, such as exatecan.

The term “diagnostic agent”, as used herein, preferably relates to an agent which can be used to detect the antigen-binding peptide specifically bound to c-MET, preferably human c-MET and/or c-MET variants thereof. For example, the diagnostic agent may comprise a radioactive isotope, fluorescent probe, fluorophore, chemiluminescent agent, or a detectable label, such as an enzyme, enzyme substrate, enzyme cofactor, enzyme inhibitor, dye, metal ion, biotin, or streptavidin, particularly which allow the detection of the antigen-binding peptide bound to c- MET. The term “coupled”, as used herein, preferably refers to the diagnostic and/or therapeutic agent being non-covalently attached, such as by ionic and/or hydrophobic interaction, and/or being covalently attached to said antigen-binding peptide and/or to said bispecific molecule. Examples of a detectable label include alkaline phosphatase, horseradish peroxidase, beta-galactosidase, Tobacco Etch Virus nuclear-inclusion-a endopeptidase ("TEV protease"). Exemplary fluorophores include 1,8-ANS, 4-methylumbelliferone, 7-amino-4- methylcoumarin, 7-hydroxy-4-methylcoumarin, Acridine, Alexa Fluor 350™, Alexa Fluor 405™, AMCA, AMCA-X, ATTO Rho6G, ATTO Rhoil, ATTO Rhoi2, ATTO Rhoi3, ATTO Rhoi4, ATTO Rhoioi, Pacific Blue, Alexa Fluor 430™, Alexa Fluor 480™, Alexa Fluor 488™, BODIPY 492/515, Alexa Fluor 532™, Alexa Fluor 546™, Alexa Fluor 555™, Alexa Fluor 594™, BODIPY 505/515, Cy2, cyQUANT GR, FITC, Fluo-3, Fluo-4, GFP (EGFP), mHoneydew, Oregon Green™ 488, Oregon Green™ 514, EYFP, DsRed, DsRed2, dTomato, Cy3-5, Phycoerythrin (PE), Rhodamine Red, mTangerine, mStrawberry, mOrange, mBanana, Tetramethylrhodamine (TRITC), R-Phycoerythrin, ROX, DyLight 594, Calcium Crimson, Alexa Fluor 594™, Alexa Fluor 610™, Texas Red, mCherry, mKate, Alexa Fluor 660™, Alexa Fluor 680™ allophycocyanin, DRAQ-5, carboxynaphthofluorescein, C7, DyLight 750, Cellvue NIR780, DM-NERF, Eosin, Erythrosin, Fluorescein, FAM, Hydroxycoumarin, IRDyes (IRD40, IRD 700, IRD 800), JOE, Lissamine rhodamine B, Marina Blue, Methoxy coumarin, Naphtho fluorescein, PyMPO, 5-carboxy-4',5'-dichloro-2',7'-dimethoxy fluorescein, 5- carboxy-2', 4', 5', 7' -tetrachlorofluorescein, 5-carboxyfluorescein, 5-carboxyrhodamine, 6- carboxyrhodamine, 6-carboxytetramethyl amino, Cascade Blue, Cy2, Cy3, Cy5,6-FAM, dansyl chloride, HEX, 6-JOE, NBD (7-nitrobenz-2-oxa-i,3-diazole), Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, phthalic acid, terephthalic acid, isophthalic acid, cresyl fast violet, cresyl blue violet, brilliant cresyl blue, para- aminobenzoic acid, erythrosine, phthalocyanines, azomethines, cyanines, xanthines, succinylfluoresceins, rare earth metal cryptates, europium trisbipyridine diamine, a europium cryptate or chelate, diamine, di cyanins, La Jolla blue dye, and quantum dots. Examples of radioactive isotopes include 47Ca, 14C, 137CS, i57Cr, 57C0, 60C0, 67CU, 6yGa, 1231, 1251, 1291, 131I, 32P, 758c, 8sSr, 35S, 20iTh, or 3H.

The term “therapeutic agent”, as used herein, refers to any compound useful for therapeutic purposes. For example, a therapeutic agent may be any compound that is administered to a patient for the treatment of a malignancy, such as cancer. Therapeutic agents include but are not limited to drugs and drug-like molecules, proteins, peptides, antibodies, antibody fragments, aptamers, and small molecules. Protein therapeutic agents include, for example, peptides, enzymes, structural proteins, receptors and other cellular or circulating proteins as well as fragments and derivatives thereof, the aberrant expression of which gives rise to one or more disorders. In one embodiment, the therapeutic agent is selected from chemotherapeutic agents, cytostatic agents, and cytotoxic agents. For example, cytostatic agents include alkylating agents, antimetabolites, antibiotics, mitotic inhibitors, hormones, and hormone antagonists. Alkylating agents may, for example, include Busulfan (Myleran), Carboplatin (Paraplatin), Chlorambucil, Cisplatin, Cyclophosphamide (Cytoxan), Dacarbazine (DTIC- Dome), Estramustine Phosphate, Ifosphamide, Mechlorethamine (Nitrogen Mustard), Melphalan (Phenylalanine Mustard), Procarbazine, Thiotepa, Uracil Mustard, antimetabolites may e.g. include Cladribine, Cytarabine (Cytosine Arabinoside), Floxuridine (FUDR, 5- Fluorodeoxyuridine), Fludarabine, 5-Fluorouracil (5FU), Gemcitabine, Hydroxyurea, 6- Mercaptopurine (6MP), Methotrexate (Amethopterin), 6-Thioguanine, Pentostatin, Pibobroman, Tegafur, Trimetrexate, Glucuronate, antibiotics may e.g. include Aclarubicin, Bleomycin, Dactinomycin (Actinomycin D), Daunorubicin, Doxorubicin (Adriamycin), Epirubicin, Idarubicin, Mitomycin C, Mitoxantrone, Plicamycin (Mithramycin), or mitotic inhibitors may e.g. include Etoposide (VP-16, VePesid), Teniposide (VM-26, Vumon), Vinblastine, Vincristine, Vindesine, hormones, or hormone antagonists which may e.g. be used include Buserelin, Conjugate Equine Estrogen (Premarin), Cortisone, Chlorotriansene (Tace), Dexamethasone (Decadron), Diethylstilbestrol (DES), Ethinyl Estradiol (Estinyl), Fluoxymesterone (Halotestin), Flutamide, Goserelin Acetate (Zoladex), Hydroxyprogesterone Caproate (Delalutin), Leuprolide, Medroxyprogesterone Acetate (Provera), Megestrol Acetate (Megace), Prednisone, Tamoxifen (Nolvadex), Testolactone (Teslac), Testosterone. For example, the diagnostic and/or therapeutic agent maybe coupled to a first domain and/or a second domain of the antigen-binding peptide of the invention, for example to a variable light chain or a variable heavy chain. For example, the diagnostic and/or therapeutic agent maybe coupled to a first antigen-binding peptide and/or a second antigen-binding peptide of the bispecific molecule of the invention, for example to a variable light chain or a variable heavy chain of the first antigen-binding peptide or of the second antigen-binding peptide.

For example, the diagnostic and/or therapeutic agent may be covalently attached to said antigen-binding peptide or said bispecific molecule by selective chemical modification of cysteine or histidine residues in the antigen-binding peptide or the bispecific molecule. For example, if present, hinge-region disulfides of the antigen-binding peptide or the bispecific molecule can be selectively reduced to make free sulfhydryls available for targeted labeling. For example, site-specific reduction with mercaptoethylamine (MEA) or site-specific conjugation using thiol-reactive linkers maybe used for coupling the diagnostic and/or therapeutic agent to said antigen-binding peptide or said bispecific molecule. Furthermore, enzyme-mediated bioconjugation, such as by using sortase A (srtA) or transglutaminase (TGase), may be used for coupling the diagnostic and/or therapeutic agent to said antigen-binding peptide or said bispecific molecule.

In one embodiment, the diagnostic and/or therapeutic agent, particularly the therapeutic agent, is a cytotoxic agent. Cytotoxic agents may include cytotoxic agents which disrupt microtubule assembly and cytotoxic agents which target the DNA structure. Typically, diagnostic and/or therapeutic agents, such as cytotoxic agents, are coupled to said antigenbinding peptide or said bispecific molecule via a linker. The term “linker”, as used herein, preferably relates to an amino acid sequence, such as a not naturally occurring amino acid sequence, that connects two molecules, such as an antigenbinding peptide and a diagnostic and/or therapeutic agent. For example, the linker maybe a cleavable or non-cleavable linker. For example, cleavable linkers may be cleaved by proteases, acids, or by reduction of a disulfide bond. Cleavable linkers include, for example, valinecitrulline linkers, hydrazone linkers, and disulfide linkers. Non-cleavable linkers include, for example, maleimidocaproyl linkers to MMAF (mc-MMAF), N-maleimido methyl cyclohexane- i-carboxylate (MCC) linkers, or mercapto-acetamidocaproyl linkers. In one embodiment, the linker is a hydrophobic linker. In a preferred embodiment, the linker is a thiol-reactive linker.

Pharmaceutically acceptable excipients, for example as comprised by a composition of the invention, may be selected from carriers, diluents, fillers, binders, lubricants, disintegrants, glidants, colorants, pigments, taste masking agents, sweeteners, flavorants, plasticizers, and any acceptable auxiliary substances such as absorption enhancers, penetration enhancers, surfactants, co-surfactants, and specialized oils. Suitable pharmaceutically acceptable excipient(s) may, for example, be selected based on the dosage form, the intended mode of administration, the intended release rate, and manufacturing reliability. Examples of common types of pharmaceutically acceptable excipient(s) include, for example, polymers, waxes, calcium phosphates, and sugars.

The term “nucleic acid”, as used herein, relates to a nucleotide sequence, such as ribonucleic acid or deoxyribonucleic acid. In one embodiment, said nucleic acid encodes an antigenbinding peptide of the present invention and/ or a bispecific molecule of the present invention. In one embodiment, said nucleic acid encodes for a sequence which is at least 50 %, preferably at least 85 % identical to a nucleic acid sequence encoding an antigen-binding peptide of the present invention and/or a bispecific molecule of the present invention. In one embodiment, said nucleic acid at least 50 %, preferably at least 85 % identical to a nucleic acid sequence encoding an antigen-binding peptide of the present invention and/or a bispecific molecule of the present invention results in the same peptide product as a too % identical sequence, due to the redundancy of the genetic code.

The term “isolated nucleic acid”, as used herein, preferably relates to nucleic acids which are separated from constituents, such as cellular constituents, which the nucleic acids are normally associated with in nature; for example, the isolated nucleic acid is at least 80%, 90%, 95% pure by weight, i.e. devoid of contaminating constituents. For example, the isolated nucleic acid may be a DNA molecule that is separated from sequences with which it is immediately contiguous (in the 5' and 3' directions) in the naturally occurring genome of the organism from which it was derived. For example, the isolated nucleic acid may be a DNA molecule inserted into an expression vector, such as a plasmid or a viral vector, or integrated into the genomic DNA of a prokaryote or eukaryote. Accordingly, the present invention also provides expression vectors which comprise the nucleic acid of the invention.

The nucleic acid of the invention may be used in the manufacture of the antigen-binding peptide of the invention, the bispecific molecule of the invention, and/ or the antigen-binding peptide-drug conjugate of the invention, particularly by means of expression in a cell culture. Exemplary expression vectors which may be used for expressing the antigen-binding peptide of the invention and/or the bispecific molecule of the invention comprise pCMV, pcDNA, P4X3, P4X4, P4X5, P4X6, PVL1392, PVL1393, PACYC177, PRS420, pBABEpuro, pWPXL, and pXP-derived vectors.

The present invention also relates to a recombinant cell comprising the isolated nucleic acid of the invention, for example the isolated nucleic acid provided in an expression vector, and its use in the manufacture of the antigen-binding peptide of the invention, the bispecific molecule of the invention, and/or the antigen-binding peptide-drug conjugate of the invention. The recombinant cell maybe, for example, a yeast cell, insect cell, or mammalian cell. For example, the recombinant cell maybe an insect cell selected from Sf9, Sf2i, S2, His, and BTI-TN-5B1-4 cells; or a yeast cell selected from Saccharomyces cerevisiae, Hansenula polymorpha, Schizosaccharomyces pombe, Schwartniomyces occidentalis, Kluyveromyceslactis, Yarrowia lipolytica and Pichia pastoris; or a mammalian cell selected from HEK293, HEK293T, HEK293E, HEK 293F, NSo, per.C6, MCF-7, HeLa, Cos-1, Cos-7, PC-12, 3T3, Vero, vero-76, PC3, U87, SAOS-2, LNCAP, DU145, A431, A549, B35, H1299, HUVEC, Jurkat, MDA-MB-231, MDA-MB-468, MDA-MB-435, Caco-2, CHO, CH0-K1, CHO-B11, CHO-DG44, BHK, AGEi.HN, Namalwa, WI-38, MRC-5, HepG2, L-929, RAB-9, SIRC, RK13, 11B11, 1D3, 2.4G2, A-10, B-35, C-6, F4/80, IEC-18, L2, MH1C1, NRK, NRK-49F, NRK-52E, RMC, CV-1, BT, MDBK, CPAE, MDCK.1, MDCK.2, and D-17.

The antigen-binding peptide of the invention, the bispecific molecule of the invention, and/ or the antigen-binding peptide-drug conjugate of the invention maybe used for the manufacture of a medicament for the treatment of cancer. For example, the antigen-binding peptide of the invention, the bispecific molecule of the invention, and/or the antigen-binding peptide-drug conjugate of the invention may be formulated into a pharmaceutical composition for administration to a patient in need thereof, such as a patient suffering from or being at risk of acquiring a cancer.

The present invention also relates to a method of preventing or treating a cancer which comprises administering to a patient in need thereof a therapeutically effective amount of the antigen-binding peptide of the invention, the bispecific molecule of the invention, the antigenbinding peptide-drug conjugate of the invention, and/or the composition, preferably pharmaceutical composition, of the invention. For example, the method of preventing or treating a cancer may comprise administering to a patient in need thereof, particularly a patient suffering from or being at risk of acquiring a cancer, from about 0.001 mg/kg to about 50 mg/kg, or from about 0.005 mg/kg to about 45 mg/kg, or from about 0.01 mg/kg to about 40 mg/kg, or from about 0.05 mg/kg to about 35 mg/kg, or from about 0.1 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 2.5 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 12.5 mg/kg, 15 mg/kg, 17.5 mg/kg, 20 mg/kg, 22.5 mg/kg, 25 mg/kg to about 26 mg/kg, 27 mg/kg, 28 mg/kg, 29 mg/kg, 30 mg/kg, 32.5 mg/kg, 35 mg/kg, 37.5 mg/kg, 40 mg/kg, 42.5 mg/kg, 45 mg/kg of the antigen-binding peptide of the invention, the bispecific molecule of the invention, the antigen-binding peptide-drug conjugate of the invention, and/or the composition, preferably pharmaceutical composition, of the invention. The term “mg/kg”, as used herein, preferably refers to mg of the molecule or composition to be administered/kg body weight.

In one embodiment, the term “patient” relates to a human or an animal, preferably a human. The patient may suffer from or being at risk of acquiring cancer. In one embodiment, the patient suffers from or is at risk of acquiring a cancer that expresses high levels of c-MET or of a c-MET variant. The term “high levels”, as used herein in the context of levels of c-MET or c- MET variants, refers to c-MET expression levels that are at least 2x, 5X, tox, 15X, 20x, 25X, or 5Ox higher than in a control (e.g. tissue obtained from a healthy individual or a cell line that does not express c-MET, e.g. as assessed by qPCR, Western blotting, immunohistochemistry). For example, c-Met expression in a cancer of a patient may be assessed in tumor tissue which, for example, may be obtained by needle aspiration or surgical biopsy. c-Met expression may, for example, be assessed by means of immunohistochemistry or by analyzing circulating tumor DNA (ctDNA) in the patient's blood. Preferably, obtaining the sample of a patient does not form part of the present invention.

The antigen-binding peptide of the invention, the bispecific molecule of the invention, the antigen-binding peptide-drug conjugate of the invention, and/or the composition, preferably pharmaceutical composition, of the invention may, for example, be used for diagnostic purposes to detect c-MET expression in a patient sample. The term “sample”, as used herein, may refer to tissue samples or cell samples obtained from tumor tissue of a patient or to a control, such as a cell line or a control tissue from a healthy donor, e.g. a human subject not inflicted with cancer. The sample may comprise cells of a cancer cell line, such as KP-4, U87MG, A549, NCI-H441, MKN-45, or EBC-i, e.g. obtainable from ATCC. Detecting c-MET expression in a sample may comprise contacting the sample with the antigenbinding peptide of the invention, the bispecific molecule of the invention, the antigen-binding peptide-drug conjugate of the invention, and/or the composition of the invention under conditions that allow specific binding to c-MET and subsequently detecting the antigenbinding peptide of the invention, the bispecific molecule of the invention, the antigen-binding peptide-drug conjugate of the invention, and/or the composition of the invention, preferably by means of detecting a radioactive isotope, fluorescent probe, fluorophore, chemiluminescent agent, or a detectable label of said diagnostic and/ or therapeutic agent.

The inventors have found that previous antigen-binding peptides, e.g. the previous antigenbinding peptide CS06 as disclosed in Sellmann et al. 2016, exhibits poor conjugatability, particularly a production of ADCs from said previous antigen-binding peptide is hindered. Advantageously, the antigen-binding peptides of the invention exhibit high thermal stability, strongly reduced hydrophobicity, retained affinities supporting the desired functionality and enhanced conjugatability, particularly compared to previous antigen-binding peptides, such as CS06 as disclosed in Sellmann et al. 2016. Moreover, the bispecific antigen-binding peptides and ADCs of the invention are highly active on c-MET expressing tumor cell lines.

The terms “of the [present] invention”, “in accordance with the invention”, “according to the invention” and the like, as used herein are intended to refer to all aspects and embodiments of the invention described and/or claimed herein. The term “present invention” may be used herein with a broad meaning in the sense of “present disclosure”. As used herein, the term “comprising” is to be construed as encompassing both “including” and “consisting of’, both meanings being specifically intended, and hence individually disclosed embodiments in accordance with the present invention. The term “comprising”, when used in connection with an amino acid sequence of a SEQ ID NO:, herein, is typically meant to allow for the presence of additional residues adjacent to such sequence, which additional residues are not listed in the respective SEQ ID NO:. The term “comprising” is, however, also meant to include the possibility that such sequence does not include any additional residues (not listed in the respective SEQ ID NO:), in which case, such term “comprising” is used in the sense of and to be understood as “consisting of’. As an example, a region which “comprises” an amino acid sequence of SEQ ID NO:xyz, includes the sequence that is listed in SEQ ID:xyz, but may also include additional adjacent residues that are not listed in SEQ ID NO:xyz; such terminology does, however, also include the possibility that the region merely “consists of’ the amino acid sequence of SEQ ID NO:xyz, in which case no additional adjacent residues are meant to be encompassed by and present in said region. Where used herein, “and/or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, “A and/ or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein. In the context of the present invention, the terms “about” and “approximately” denote an interval of accuracy that the person skilled in the art will understand to still ensure the technical effect of the feature in question. The term typically indicates deviation from the indicated numerical value by ±20%, ±15%, ±10%, and for example ±5%. As will be appreciated by the person of ordinaiy skill, the specific such deviation for a numerical value for a given technical effect will depend on the nature of the technical effect. For example, a natural or biological technical effect may generally have a larger such deviation than one for a man-made or engineering technical effect. Where an indefinite or definite article is used when referring to a singular noun, e.g. "a", "an" or "the", this includes a plural of that noun unless something else is specifically stated. The term “about”, as used herein in the context of numbers or data, intends to include deviations (± ) which usually are to be considered in the respective technical field.

SEQUENCES

BRIEF DESCRIPTION OF THE FIGURES

The present invention is now further described by reference to the following figures. All methods mentioned in the figure descriptions below were carried out as described in detail in the examples.

Figure 1 shows exemplary biparatopic anti-c-MET antibodies favoring inter- over intra-target binding, (a) Scheme of inter- versus intra-molecular binding of a biparatopic antibody. Simultaneously addressing two epitopes on the same c-MET monomer yields high apparent affinity, but no further effects, and hence is not desired. Combinations of paratopes mediating binding to different c-MET monomers ideally results in enhanced cross-linking, internalization, target degradation and, if ADCs are applied, enhanced payload release, (b) In silico docking against c-MET (PDB code 2UZY) suggests differential Fab arm positions for selected reference antibodies and CS06 & B10V5. Onartuzumab epitope derived from available X-ray structure with PDB code 4K3J. (c) Fab arms 1 and 2 of biparatopic reference antibody REGN5093 show orientations supporting inter-molecular c-MET engagement. Visualization obtained via docking with restraints from experimental data taken from DaSilva et al. Clin Cancer Res 2020;26:1408-19. (d) Proposed inter-c-MET cross-linking property of a B10V5 x CS06 biparatopic antibody.

Figure 2 shows exemplary optimized CS06 sequences and library design, (a) Predicted aggregation hot spots in CS06 Fv and respective amino acids in positions marked red. (b) Positional variability in CS06 HCDR3 as observed during affinity maturation, indicating essential versus alterable amino acids, (c) Library design varying hydrophobic residues defined from information shown in (a) and (b). (d) Sequential sequence engineering towards lower hydrophobicity. Single HCDR2 or HCDR3 point mutations with retained affinity but lowered hydrophobicity in 3.X series were combined into 2- to 4-point mutant sequences in 5.X variants, (e) In comparison to parental sequences (top panel) and focused engineering, library approaches yielded a comparatively broad diversity within the resulting cloned library (second logo plot) and distinct sequence motifs upon one CHO display sorting round (third plot) or four consecutive rounds of affinity sorting on the surface of yeast (three bottom logo plots).

Figure 3 shows yeast surface and mammalian display for hydrophobicity engineering, (a) Yeast surface display sort for display and retained antigen binding plus resulting hit panel for in vitro analyses, (b) Differential display of rationally designed parental (VH1.0) and optimized (VH5.1) CS06 indicating applicability of screening for manufacturability properties such as hydrophobicity by mammalian display, (c) CHO display sort for manufacturability (as indicated by high display) and retained target binding plus resulting most enriched output sequences, (d) Diversity of H-CDR2-CDR3 sequences illustrated using UMAP dimensionality reduction. Figure 4 shows an improved conjugatability and high potency of engineered biparatopic ADCs, (a) Scheme of a final biparatopic ADC. (b) Key data on biparatopic ADC conjugation, (c) In vitro potency of biparatopic ADCs on c-MET expressing human tumor cell lines NCI-H441, HCC-827 and EBC-i in comparison to re-produced DAR6 REGN5093 reference. Doseresponse curves of REGN5093 and biparatopic ADCs from independent experiments had been merged. Representative graphs with error bars indicating mean ± SD of technical triplicates.

Figure 5 shows an overview on key properties of final biparatopic lead candidates. Constructs marked bold were selected for subsequent antibody drug conjugation. HIC retention time reference for clinical stage antibody was cetuximab with 5.8 min. n.d., not determined.

Figure 6 shows the in vitro binding and physico-chemical qualification data for selected optimized CS06 IgGi variants. Variants designed but not listed here were not produced. VH6.18 & VH6.21 are lead structures. HIC retention time reference for clinical stage antibody was cetuximab with 19.6 min for samples 1-33 (a, b) and 5.8 for samples 34-36 (c). n.d., not determined; NA, not applicable.

Figure 7 shows the geomean IC₅₀ [M] (n=3-4) of biparatopic c-MET ADCs and comparable REGN in-house ADC on cMET expressing cell lines.

Figure 8 shows full gating information for Yeast and CHO diplay sorts, (a) YSD sorting round 1 for CS06 display (anti-lambda-PE) and c-MET binding detected by anti-His-APC. (b) Sort round 2. (c) Pre-gating for sort round 3, shown in (d) and re-analysis of sorted population (e). (f) Pre-gating for CHO display CS06 library sorting shown in (g) and Figure 3c plus reference stainings second step control in sort (h), second step control stain in separate well (i) and sort stained in well (j).

Figure 9 shows a one-armed versus biparatopic cellular binding comparison. Biparatopic antibodies shown in blue (□), while respective corresponding mono-valent variants are shown in red (•) for CS06 and grey (A) for B10V5 scFv (comparative B10V5 Fab data not available).

Figure 10 shows exemplary mutations of exemplary antigen-binding peptides of the invention compared to reference sequence VH1.0. The shown exemplary antigen-binding peptides of the invention have significantly lowered hydrophobicity and acceptable affinity. Furthermore, the shown exemplary antigen-binding peptides of the invention have enhanced thermal stability. In the following, reference is made to the examples, which are given to illustrate, not to limit the present invention.

EXAMPLES

Example 1: Materials and Methods

Molecular modeling of mono-specific and multi-specific antibody structures

Structural models of variable domains and full-length antibodies were created using the antibody modeler tool in the molecular modeling software package moe. scFv constructs and biparatopic constructs were built by adding linkers via moe's protein builder, followed by a conformational search of the linker via moe's linker modeler. Finally, the modeled constructs were subjected to energy minimization. Visualization of 3D structures was done with PyMOL.

Antibody-antigen docking

The crystal structure of v-MET was taken from PDB code 2UZY. Docking hypotheses of the variable antibody regions shown in Figure lb were generated using PIPER (Kozakov et al., 2006) via Schrodingers BioLuminate.

In silico developability assessment

The in silico developability profiles (shown in Figure 6) were computed using an internal pipeline termed “Sequence Assessment Using Multiple Optimization Parameters (SUMO)” (Evers et al., 2023). This approach automatically generates antibody models based on the provided sequences of the variable regions, identifies the human-likeness by sequence comparison to the most similar human germline sequence, determines structure-based surface-exposed chemical liability motifs (unpaired cysteines, methionines, asparagine deamidation motifs and aspartate deamidation sites) as well as sites susceptible to post- translational modification (N-linked glycosylation). Moreover, a small set of orthogonal computed physico-chemical descriptors including the isoelectric point (pl) of the variable domain, Schrodingers AggScore (Sankar et al., 2018) as predictor for hydrophobicity and aggregation tendency calculated for the complete variable domain as well as the complementarity-determining regions (CDRs) only and the calculated positive patch energy of the CDRs were determined. These scores were complemented with a green to yellow to red color coding, indicating scores within one standard deviation from the mean over a benchmarking dataset of multiple biotherapeutics approved for human application as green, scores above one standard deviation as yellow and those above two standard deviations as red. For the AggScore values, these cutoffs were slightly adjusted based on correlation analyses to experimental hydrophobic interaction chromatography (HIC) data. Combinatorial variant library design and generation

DNA libraries were generated via synthesis with S. cerevisiae codon usage and mutations incorporated into the parental CS06 VH sequence as shown in Figure lb (Twist Biosciences): two positions in H-CDR2 and 7 positions in H-CDR3 were exchanged against hydrophilic amino acids. The library was constrained to contain 5% single-point and 10% double-point mutations in H-CDR1 and 15% double-point as well as 70% triple-point mutations in H-CDR3. In addition, specific amino acid motifs, relating to glycosylation motifs, asparagine deamidation, aspartate deamidation, lysine glycation, integrin binding, CDnc/CDi8 binding, fragmentation and hydrophobicity were explicitly excluded.

For yeast surface display (YSD) Fab library generation, randomized CS06 VH gene strings were amplified using CS06 FR1 and FRq-specific primers carrying yeast gap repair overhangs (CSo6_Twist_fwd: TGTTTTTCAATATTTTCTGTTATTGCTAGCGTTTTAGCAGG-

Gcaagtccaattagttcaa and CSo6_Twist_rev: AGAAGATGGAGCCAATGGAAAAA- CAGATGGACCTTTTGTAGAAGCagaagagacagtgac). CS06 VL gene was synthesized as a gene string at GeneArt and was also amplified using specific primers to introduce gap repair overhangs (CSo6_VL_fwd: GCCAGCATTGCTGCTAAAGAAGAAGGGG-

TACAACTCGATAAAAGAcaattggtcttgactcaatc and CSo6_VL_rev: GGATGGCG- GGAACAGAGTGACCGAAGGGGCGGCCTTCGGCTGACCagaacggtcaattttgtac). Next, VH library was cloned into YSD vector using S. cerevisiae strain EBY100 MATa (URA3-52 trpi leu2Di his3D2OO pep4::HIS3 prbiDi.6R cant GAL (pIU2ii:URA3)) and the VL chain was cloned using BJ5464 cells (MATa URA3-52 trpi leu2Dihis3D2OO pep4::HIS3 prbiDi.6R cani GAL) via gap repair cloning according to the optimized protocol of Benatuil et al. (Benatuil et al., 2010). EBY100 cells comprising the randomized heavy chain diversity were mated with BJ5464 cells comprising the CS06 VL chain to produce diploid yeast cells capable of Fab display (Weaver-Feldhaus et al., 2004).

CS06 display CHO library was cloned in a similar fashion as described above to realize direct fusion of the randomized CS06 heavy chain to GGGGS-linked transmembrane domain of PDGFR. The library was subsequently generated as reported in Gaa et al. (Gaa et al., 2023).

Library sorting

YSD sorting was performed as described in references (Elter et al., 2021; Schroter et al., 2018). Briefly, CS06 Fab display was induced by incubation of diploid yeast library cells in SG-Trp- Leu medium + 10% (w/v) polyethylene glycol 8000 for 48 h at 20°C, 120 rpm agitation. For sorting rounds 1 and 2, yeast cells were incubated with 1 pM (rh)c-MET-ECD-His6 (inhouse) in PBS, while for sorting rounds 3 and 4 the antigen concentration was reduced to 8 nM and 0.5 nM c-MET-ECD-His6, respectively. Surface display of correctly assembled Fab molecules was detected using 25 pg/ ml goat-F(ab’)₂ anti-human lambda-PE antibody (Southern Biotech) and specific c-MET-ECD-His6 antigen binding was detected using 5 pg/ml SureLight APC anti-6xHis tag antibody (Abeam) in PBS. Cell sorting was performed using BD FACSAria Fusion flow cytometer. Following FACS, sorted cells were transferred to 50 ml SD-Trp-Leu cultivation medium and were expanded at 3O°C, 120 rpm agitation for 48-72 h.

CHO library sorting for high display and target binding was conducted applying a Sony SH800S flow cytometer after staining with too nM c-MET-ECD-His6 in PBS and detected using 15 pg/ml anti-Penta-His Alexa Fluor 647 conjugate (Qiagen) in PBS following manufacturer’s recommendations. Sorting was conducted applying a too pm nozzle in sort mode "purity” with gates adjusted to include approximately 2% events, as shown in Figure 3c and Figure 8f-j .

Sequence evaluation and comparative analyses

Sanger sequencing was used for sequence analyses of YSD output diversities, and the resulting forward + reverse consensus sequences were analyzed with Geneious Prime software version 2022.1.1.

For CS06 mammalian display sort output evaluation, bulk cell output cDNA was generated as previously described (Gaa et al., 2023). The CS06 VH sequences were amplified using fusion primers targeting the flanking vector sequence. The VH amplicons were purified with AMPure (Beckman Coulter) and amplified with index primers for Illumina sequencing. The final sequencing library was purified using a Pippin Prep (Sage Science). The VH domains were sequenced on a MiSeq using the v3 600 cycle kit according to the manufacturer’s protocol. FASTQ files were uploaded to Geneious Biologies (https: //www.geneious.com/biopharma) for analysis. Reads were overlapped, filtered for length, and the VH sequences were annotated using the IMGT/V-QUEST reference library (https://www.imgt.org/vquest/refseqh.html). Normalized counts for HCDR2 and HCDR3 were used to identify amino acid variants of high prevalence.

The in silico developability profile was computed using an internal pipeline termed “Sequence Assessment Using Multiple Optimization Parameters (SUMO)” (Evers et al., 2023). In brief, automatically generated VH models allow evaluation of “humanlikeness” by sequence comparison to the most similar human germline and determine structure-based surface- exposed chemical liability motifs as well as N-linked glycosylation. In addition, orthogonal physicochemical descriptors such as the isoelectric point (pl) of the variable domain, Schrodingers AggScore as predictor for hydrophobicity and aggregation tendency and the calculated positive patch energy of the CDRs are determined. These scores were complemented by color coding, indicating scores within one standard deviation from the mean over a benchmarking dataset as green, scores above one standard deviation as yellow and above two standard deviations as red. For comparative visualization of sequence panels derived from three different approaches, the respective sequences pools were projected into a two- dimensional space using UMAP (Becht et al., 2019). Engineered antibody qualification

Recombinant transient antibody expression and purification as well as thermal stability determination were conducted as described by Yanakieva et al. (Yanakieva et al., 2022). Hydrophobic interaction chromatography analysis was conducted via two methods with vaiying gradients. For all processes, a Butyl-NPR (2.5 pm 4.6 mm x too mm) column (TOSOH Bioscience 42168) was applied at 25°C and protein samples formulated in PBS pH 7.4 were mixed with ammonium sulfate at 1 M end concentration prior to analyses. A long gradient method was run at a flow rate of 0.75 ml/min using a linear gradient of 50 mM sodium phosphate + 1.5 M ammonium sulfate pH 7.0 to 50 mM sodium phosphate + 5% isopropanol pH 7.0 in 33 min. A short gradient method was applied with a flow rate of 0.5 ml/min using a linear gradient of 1.2 M ammonium sulfate, lx PBS, pH 6.47, 170.1 mS/cm to 50% methanol, o.ix PBS, pH 8.39, 0.998 mS/cm in 15 min. Typically, 20 pg of protein sample were loaded onto the column. Absorbance was monitored at 214 nm using a multi-wavelength detector (Agilent). ChemStation software (Agilent) was used to integrate the peak areas.

Affinity determination was conducted by Biolayer interferometry as previously described (Sellmann 2016). EC50 values were determined via flow cytometry (iQue 3 screener, Sartorius) in 1:2 titration series from 500 nM to 0.01 nM on c-MET expressing human lung carcinoma EBC-i cells (Riken Bioresource Center Cel Bank JCRB0920 031496, cultured under recommended conditions) and AF-488 AffiniPure Fab Fragment Goat-anti-Human IgG, Fey fragment specific detection antibody (Jackson Immuno Research). Analyses were conducted with GraphPad Prism 9.1.2 software (GraphPad Software LLC).

ADC generation

ADCs were generated via interchain reduction and subsequent reaction with maleimide containing drug-linkers (DL). MAbs were thawed at 20°C and mAb concentration was adjusted to 5 mg/ml using conjugation buffer (50 mM Histidine pH 6.5, 100 mM NaCl). Subsequently, interchain disulfides were reduced incubating the mAb solution with 10 molar equivalents (relative to the mAb) of tris(2-chloroethyl) phosphate (TCEP) at 20°C for 2 h. Afterwards, the solution was incubated with 12 molar equivalents (relative to the mAb) of DL for 1 h at 20°C to conjugate the DL to the antibody. The reaction was stopped by addition of 12 molar equivalents (relative to the mAb) of N-acetyl cysteine (NAC) for 20 min at 20°C. ADCs were separated from unconjugated DL via size exclusion chromatography (SEC) using a HiLoad Superdex 200 Increase column in combination with an Akta LC system (Cytiva) and conjugation buffer as running buffer. Fractionated samples containing the ADC material were pooled and concentrated using Amicon Ultra 15 50K centrifugal (Millipore) followed by a final buffer exchange into 10 mM Histidine, 40 mM NaCl, 6% Trehalose, 0.05% TWEEN, pH 5.5 using HiTrap Desalting columns in combination with an Akta LC system (Cytiva). Final ADC material was filtered through a 0.22 |um sterile filter unit (Millipore) and shock frozen in liquid nitrogen until further use.

In vitro potency assessment

In vitro potency assessment of the ADCs was determined on c-MET expressing human lung cancer cell lines HCC-827 (lung adenocarcinoma, ATCC® CRL-2868™, originally obtained from the American Type Culture Collection (ATCC), Manassas, VA, USA), NCI-H441 (lung adenocarcinoma, papillary, ATCC® HTB-174™) and EBCi (squamous cell carcinoma, Health Science Research Resources Bank, now National Institutes of Biomedical Innovation, Health and Nutrition, Japanese Cancer Research Bank, JCRB0920 031496) using the CellTiter Gio® Luminescent Cell Viability assay (#07575, Promega Corporation, Madison, WI, USA). The cell lines HCC-827 and NCI-H441 were cultured in RPMI1640 medium with GlutaMAX ™ supplement (#61870-010, Gibco™, purchased from Thermo Fisher Scientific, Waltham, MA, USA), 1 mM sodium pyruvate (#113670-070, Gibco ™, Thermo Fisher Scientific), 2.5 g/L Glucose (#08769, Sigma Aldrich, St. Louis, MO, USA or #A24949-OI, Gibco™, Thermo Fisher Scientific) and 10% FBS (#80615, Sigma Aldrich). The cell line EBC-1 was cultured in MEM Eagle Medium (#M2279, Sigma Aldrich) including 2 mM Glutamine (#35050-061, Gibco ™ GlutaMAX™, Thermo Fisher Scientific) and 10% FBS (#80615, Sigma Aldrich). The day before treatment, 2500 cells/well (NCI-H441 or EBCi) or 1250 cells/well (HCC827) were plated in sterile 96-well flat bottom microplates (90 pl volume each, #165303, Thermo Fisher Scientific) and incubated at 5% CO2, 37°C. Background wells were supplied with respective culture medium. After overnight incubation, a lox starting concentration of the ADCs and a 1:4 serial dilution was prepared with RPMI1640 medium with GlutaMAX™ supplement, 1 mM sodium pyruvate and 10% FBS. A total of 10 pl was added to the respective wells in technical triplicates. Control wells were treated with a respective amount of RPMI1640 medium. Following 6 days of incubation, 100 pl CellTiter Gio ® reagent was added to each well and plates were incubated for 2 min with shaking at 300 rpm and for additional 20 min at room temperature, protected from light. Afterwards, luminescence was measured on a Varioskan Flash plate reader (Thermo Fisher Scientific). Relative light units (RLU) were processed by subtracting the background and by normalization the data to untreated control cells. The processed data was used to describe the dose-response by %effect vs. concentration [M] with the equation log(inhibitor) vs. response-variable slope (four parameters) (GraphPad Prism version 8.2.0 for Windows, GraphPad software, La Jolla, California, USA). Graphs were displayed with error bars indicating the standard deviation (SD) of technical triplicates. For determining IC₅₀ values, data were processed by using Genedata Screener (Genedata). Experiments were performed several times and Geometric mean values of determined IC₅₀s were indicated as geomean IC₅₀ [M]. Example 2: Results

Docking for educated choice of optimal paratope combination

The inventors previously reported identification of fully human antibodies with pico-molar affinities to distinct epitopes on SEMA and IPT1 domains of c-MET ECD, B10V5 & CS06 (Sellmann et al., 2016). Comparative docking studies of the B10V5 and CS06 variable domains against c-MET ECD (PDB code 2UZY) suggested paratope orientations likely supporting inter- rather than intra-molecular c-MET ECD binding (Figure lb). Originally identified by scFv Phage Display, both binding moieties could be adapted to both Fab and scFv format (Sellmann et al., 2016) to build Fab x scFv heterodimeric Fc biparatopic antibodies. Initial biparatopic strand-exchanged engineered domain (SEED) (Davis et al., 2010) and DuoBody (Labrijn et al., 2013) designs yielded high apparent affinities (Figure 5) and strong internalization capacities indicating applicability for MET degradation or biparatopic ADC approaches.

Biparatopic antibody generation and optimization

Sequence optimization towards framework-based germline humanization of B10V5 was successfully achieved in one design cycle applying SUMO, a procedure for in silico sequence assessment and optimization using multiple optimization parameters (Evers et al., 2023). Humanization of CS06 could be accomplished too, but the parental as well as the designed variants showed high HIC retention times (Figure 2d) that were attributed to pronounced solvent-exposed hydrophobic patches in HCDR2 and HCDR3 (Figure 2a). In addition, these properties resulted in poor to no conjugatability (Figure 4). Sequence optimized variants are herein referred to as B10V5 and CS06, and CS06 VH variants discussed below were optimized for suitable hydrophobicity profiles. Three comparative approaches were performed to reduce hydrophobicity while retaining sufficient affinity (Figure 2): (a) iterative design of CS06 HCDR2 and HCDR3 sequence variants; (b) a library approach varying hydrophobic CDR residues screened by yeast surface display (YSD); (c) sorting the same library for display and target binding via mammalian display.

Hydrophobicity engineering

Hydrophobicity reduction was performed in two iterative design cycles after structural antibody modeling and structure-based in silico prediction of aggregation hot spots: in the first design cycle, solvent-exposed hydrophobic residues in HCDR2 or HCDR3 were replaced by specific polar residues, resulting in 18 single-point mutations (termed VH3.1 - VH3.18 in Figure 2d) of the parental CS06 sequence. Those variants that revealed reduced hydrophobicity and favorable binding affinities after synthesis and experimental characterization were combined into a total of seven 2-, 3- or 4-point mutations in the second design cycle (VH5.1 - VH5.7 in Figure 2d). Encouragingly, the optimized candidates showed strongly reduced hydrophobicity and still comparable binding affinity compared the parental CS06 sequence (VHi.o). The 4-point mutational variant VH5.1 showed the lowest retention time in HIC and, encouragingly, only an affinity loss of < factor 2 compared to VH1.0 and was therefore nominated as new frontrunner for further studies.

Hydrophilicity screening library design and Yeast Surface Display sorting

Enabling the search within a larger combinatorial screening space, knowledge obtained from affinity-tolerating hydrophobicity-reducing single-point mutations was applied to design a focused combinatorial library exchanging hydrophobic HCDR2 and HCDR3 key residues to pre-defined portions against any polar or soluble amino acid (Figure 3c). Resulting amino acid distribution was assessed by Sanger sequencing of 96 single clones obtained after multiple rounds of FACS and in high accordance with desired proportions (Figure 2c and 3d). YSD sorting was conducted in four rounds for display & retained target-specific binding. Resulting clones were sequenced (Figure 2e) and re-produced as IgGi for evaluation of target-specific binding and physicochemical properties, including hydrophobicity (Figure 6). Advantageously, all engineered variants showed higher purities, higher thermal stabilities and, most relevant, strongly reduced HIC retentions times similar or below a reference antibody (cetuximab). Affinities were reduced to varying extent, but due to avidity, reduction in cellular binding (as measured by EC50) was limited (Figure 5). In silico developability assessment suggested favorable overall profiles for most variants (Figure 6).

Mammalian display sorting for manufacturability

As interim results had indicated a strongly reduced hydrophobicity profile for the preliminary engineered variant CS06 VH5.1, the display level on the surface of CHO cells was assessed in comparison to parental VH1.0 sequence. Indeed, display levels differed by 3 to 4-fold (Figure 3b), indicating the feasibility to sort for both enhanced physicochemical properties as well as target affinity via a mammalian display approach. Library cloning was employing the same diversity as before, and sorting was conducted in a similar fashion to YSD, with a gating strategy aiming at high displaying clones with retained target binding. Evaluation was conducted by NGS and analysis of most prevalent clones showing suitable in silico developability properties.

Comparative sequence and antibody evaluation

We compared the output of three approaches in several dimensions: First, amino acid exchanges occurred in similar positions and quantities in all three resulting panels and correlated with previous data (Figure 3 versus Figure 4). Secondly, amino acids variability was higher from library output then rational design, reflecting the significantly higher combinatorial space (Figure 3a+c), and higher from NGS analyses than Sanger sequencing due to the different nature of these sequencing technologies. Third, there was overlap of resulting sequence variants between all three approaches (d). Comparative analyses of resulting properties between antibodies derived from the different optimization approaches revealed enhanced thermal stability and lowered hydrophobicity in variants derived from all approaches, particularly from CHO display and YSD. 14 of 30 variants identified by YSD and Sanger sequencing showed affinities within 10-fold difference to the parental clone and most similar cellular binding potency due to avidity (Figure 6). Hits from CHO display sorts were evaluated by NGS and chosen for high prevalence and suitable in silico developability properties. Of 12 clones, 3 still bound c-MET with affinity loss higher than 10-fold (Figure 6). Advantageously, CS06 variants such as VH6.18 and VH6.21 showed greatly enhanced hydrophobicity profile, suitable physicochemical properties, and retained cellular binding as IgGi. Based on enhanced hydrophobicity profile, suitable physicochemical properties, retained cellular binding as IgGi and absence of in silico developability flags, CS06 variants VH6.18 and VH6.21 were chosen for assembly of final lead candidates in combination with sequence optimized B10V5. Biparatopic antibodies were produced as N-terminal fusions of Fab and scFv to a knob-into-hole heterodimeric Fc portion (Schaefer et al., 2011). Reference construct one- armed B10V5 was historically available as SEED construct (Davis et al., 2010). Advantageously, while yields and purities were acceptable, thermal stabilities were enhanced and HIC retention times were strongly reduced in all CS06 VH6.18 & VH6.21 variants when compared to the parental VH1.0 reference. Affinities of one-armed engineered variants were reduced to the three-digit nanomolar range, with CS06 VH6.18 and VH6.21 Fab constructs retaining cellular binding EC50S in the range of 30 - 50 nM. When rendered biparatopic however, apparent affinities to recombinant target protein were too high to measure due to very slow off-rates and cellular binding EC50S were in the picomolar range. Advantageously, CS06 Fab x B10V5 scFv constructs yielded higher binding potency and maximal binding in flow cytometry studies than monovalent references (Figure 9). In summary, suitable properties for the three lead combinations B10V5 scFv x CS06 VH6.18 Fab, B10V5 scFv x CS06 VH6.21 Fab, B10V5 Fab x CS06 VH6.21 scFv (further referred to as 6.i8_Fab; 6.2i_Fab; 6.2i_scFv) suggested assessment of improved capability for payload conjugation in comparison to parental bispecific B10V5 scFvx CS06 VH1.0 Fab (termed 1.0 Fab).

Antibody drug conjugation and potency evaluation

To evaluate the impact of manufacturability optimization on ADC production efficiency and yields, conjugation trials with the parental i.o_Fab and the engineered variants 6.i8_Fab; 6.2i_Fab and 6.2i_scFv were performed. First, analytical test reactions were set up to assess conjugation efficiency and monomeric purity of the conjugates. To attach the thiol-reactive drug-linker, interchain disulfide bridges were reduced with TCEP and afterwards incubated with the drug-linker for 2 h. Subsequently, monomeric purity was analyzed via analytical size exclusion chromatography (SEC) and drug to antibody ratio (DAR) via polymeric reversed- phase HPLC (PLRP). While conjugation efficiency appeared to be most efficient for i.o_Fab and 6.i8_Fab (both DAR=5.8, Fig XY) it was slightly less efficient for 6.2i_scFv (DAR=5-3) and 6.2i_Fab (DAR=5.1). In contrast, the monomeric purity of the i.o_Fab conjugate with only 76.7% was rather low and contained a high degree of low molecular weight species (LMW, 23.3%) indicating a low stability of this conjugate. Advantageously, the engineered constructs showed a much higher purity with 6.i8_Fab: 90.0%, 6.2i_scFv: 91.3% and 6.2i_Fab: 92.6%. In a next step, larger reactions were carried out including preparative SEC purification and concentration to assess the behavior of the respective conjugates throughout different downstream processing (DSP) steps. Analytical SEC and PLRP analyses yielded similar observations as before of high degree of LMWs in i.o_Fab preparative SEC. Although the LMWs could be separated, subsequent ultrafiltration was not successful as all material was adsorbed to the filter membrane omitting recoveiy of significant sample amounts. Such effects can be critical during manufacturing since many DSP steps for ADCs typically include ultrafiltration steps. Although the conditions for conjugation were not optimized, advantageously, improved yields were obtained for engineered variants with best final yield for 6.i8_Fab with 36%, followed by 6.2i_scFv with 23% and 6.2i_Fab: 16 %.

Advantageously, while several approaches failed to produce an ADC from parental CS06 VH1.0 containing biparatopic references, all three engineered biparatopic ADCs could be conjugated and produced with reasonable yields, indicating CS06 hydrophobicity engineering had a positive impact on ADC producibility. To demonstrate that the hydrophobicity engineering did not alter ADC functionality, in vitro potency on several c-MET expressing tumor cell lines was evaluated and shown to be in the subnanomolar to one-digit nanomolar range, similar to an internal REGN5093 DAR6 re-produced ADC (Figure 4c and Figure 7).

Example 3: Discussion

Next-generation designs can overcome past obstacles of therapeutic anti-c-MET biologies by supporting differentiated modes of action. Biparatopic antibodies are a promising approach that can lead to enhanced cellular binding, increased internalization by target cross-linking, and degradation, resulting in signal transduction inhibition and higher potency cytotoxicity when applied as ADCs. Several approaches have been described for degradation enhancing mono-specific biologies, bispecific antibodies aiming at crosslinking of c-MET with another tumor associated antigen (Syed, 2021) or further approaches for antibody-induced target degradation. The approach described here represents, to our knowledge, the first comparative protein engineering for manufacturability and applicability to ADC conjugation and yields deep insights into the capabilities of rational versus library approaches, as well as finally enhanced conjugation properties and a potent biparatopic anti-c-MET ADC. A comparison of CHO versus yeast surface display was based on recent reports on manufacturability screening on the surface of mammalian or yeast cells. Differential display of optimized to parental CS06 variants proves to our knowledge for the first time the ability to screen for developability properties beyond aggregation propensities such as a suitable hydrophobicity profile. Different numbers of sorting rounds and post-sort sequence identification methodologies do not allow a full comparison of yeast versus CHO display outputs. Advantageously, both approaches yielded optimized variants with high thermal stabilities and hydrophilicities, indicating successful enrichment for “manufacturability”. Both sort outputs indicate conversion overtime to parental tyrosine residues in essential positions (ill; 112,2; 112,1; 112) and distinct suitable amino acids for example H/R in position 111,1 or H/D in position 111,2. Whereas sequence information was available for rounds 2 - 4 of YSD enabling variant selection from yet diverse round 2/3 output while neglecting fully converged round 4 samples, only one round of CHO display sorting was conducted and available for hit identification. Resulting success rates for retained target binding were higher for Yeast-derived clones. Although choice of most prevalent variants in sort output NGS datasets can yield suitable hits, CHO hit rates could be further enhanced by application of deep learning focusing on pre- versus post-sort enriched sequences and iterative sorting in 2 to 3 rounds. Advantageously, full in vitro and in silico qualification of resulting CS06 variants indicated strongly enhanced physicochemical properties (Figure 6). Combination of a B10V5 and CS06 binding moiety into a heterodimeric biparatopic antibody restored and exaggerated apparent affinities and were suitable for subsequent ADC generation. ADCs with lower c-MET affinities could potentially yield advantages of lower normal tissue on-target engagement resulting in an enhanced therapeutic index.

While several approaches failed to produce an ADC from parental CS06 VH1.0 containing biparatopic references, all three engineered biparatopic ADCs could be conjugated and produced with reasonable yields, indicating CS06 hydrophobicity engineering had an advantageous impact on ADC producibility. Engineering and Exatecan conjugation finally resulted in highly active biparatopic ADCs showing similar in vitro potency on c-MET expressing cell lines as a re-produced REGN5093 ADC containing the same drug-linker and comparable DAR. In conclusion, the inventors herein provide highly potent and manufacturable antigen-binding peptides, advantageously even highly potent and manufacturable ADCs such as biparatopic ADCs. REFERENCES

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The features of the present invention disclosed in the specification, the claims, and/or in the accompanying figures may, both separately and in any combination thereof, be material for realizing the invention in various forms thereof.

Claims

1. An antigen-binding peptide binding to c-Met, comprising a first domain, preferably a heavy chain variable domain, comprising a first complementarity-determining region comprising or consisting of an amino acid sequence GGTFSSNA (SEQ ID NO: 1), a second complementarity-determining region comprising or consisting of an amino acid sequence IIPXIX₂GTA (SEQ ID NO: 2), and a third complementarity-determining region comprising or consisting of an amino acid sequence ARDQRGX₃X₄X₅YYYX₆GMDV (SEQ ID NO: 3); wherein

Xi is an amino acid selected from I, E, and S,

X₂ is an amino acid selected from F, H, and Q,

X₃ is an amino acid selected from Y, S, H, and E,

X₄ is an amino acid selected from Y, R, H, Q, and S,

X₅ is an amino acid selected from Y, D, and H, and

2. The antigen-binding peptide according to claim 1, wherein said first domain comprises or consists of an amino acid sequence which is at least 90% identical, preferably at least 95 % identical, more preferably at least 98% identical, even more preferably at least 99% identical, optionally identical, to an amino acid sequence

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSNAISWVRQAPGQGLEWMGGIIPX.X, GTAIYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDQRGX₃X₄X₅YYYX₆G MDVWGQGTTVTVSS (SEQ ID NO: 4); wherein

Xi is an amino acid selected from I, E, and S,

X₂ is an amino acid selected from F, H, and Q,

X₃ is an amino acid selected from Y, S, H, and E,

X₄ is an amino acid selected from Y, R, H, Q, and S,

X₅ is an amino acid selected from Y, D, and H, and

3. The antigen-binding peptide according to claim 1 or 2, wherein X₄ is not Y and/ or X₅ is not Y, wherein, preferably,

Xi is I and X₂ is H, or

Xi is I and X₂ is Q, or

Xi is E and X₂ is F, or

Xi is S and X₂ is F; wherein, more preferably,

Xi is I and X₂ is H, or

Xi is I and X₂ is Q; wherein, even more preferably,

Xi is I and X₂ is Q.

4. The antigen-binding peptide according to any one of the foregoing claims, wherein

X₅ is an amino acid selected from D and H, wherein, preferably,

Xi is I,

X₂ is H or Q,

X₃ isY, S, or H,

X₄ is R or H,

X₅ is D or H, and

X_(l is Y or S; wherein, more preferably,

Xi is I,

X₂ is H or Q,

X₃ isY, S, or H,

X₄ is H and X-, is D, or X₄ is R and X-, is H, and

X₆ isY or S; wherein, even more preferably,

Xi is I, X₂ is H, X₄ is H, and X₅ is D, or

Xi is I, X₂ is Q, X₄ is H, and X₅ is D, or

Xi is I, X₂ is Q, X₄ is R, and X₅ is H.

5. The antigen-binding peptide according to any one of the foregoing claims, wherein said first domain comprises a first complementarity-determining region comprising or consisting of an amino acid sequence GGTFSSNA (SEQ ID NO: 1); a second complementarity-determining region comprising or consisting of an amino acid sequence selected from

IIPIQGTA (SEQ ID NO: 5),

IIPIHGTA (SEQ ID NO: 6),

IIPEFGTA (SEQ ID NO: 7), and

IIPSFGTA (SEQ ID NO: 8); and a third complementarity-determining region comprising or consisting of an amino acid sequence selected from ARDQRGSHDYYYYGMDV (SEQ ID NO: 9),

ARDQRGYRHYYYSGMDV (SEQ ID NO: 10),

ARDQRGHHDYYYYGMDV (SEQ ID NO: 11),

ARDQRGYHDYYYYGMDV (SEQ ID NO: 12),

ARDQRGSYDYYYYGMDV (SEQ ID NO: 13),

ARDQRGYQDYYYYGMDV (SEQ ID NO: 14),

ARDQRGYRDYYYYGMDV (SEQ ID NO: 15),

ARDQRGESDYYYYGMDV (SEQ ID NO: 16),

ARDQRGYHHYYYYGMDV (SEQ ID NO: 17),

ARDQRGYRHYYYYGMDV (SEQ ID NO: 18), and

6. The antigen-binding peptide according to any one of the foregoing claims, wherein said first domain comprises or consists of an amino acid sequence which is at least 90% identical, preferably at least 95 % identical, more preferably at least 98% identical, even more preferably at least 99% identical, optionally identical, to an amino acid sequence selected from the group consisting of

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSNAISWVRQAPGQGLEWMGGIIPIQGT AIYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDQRGSHDYYYYGMDVW GQGTTVTVSS (SEQ ID NO: 20),

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSNAISWVRQAPGQGLEWMGGIIPIQGT AIYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDQRGYRHYYYSGMDVW GQGTTVTVSS (SEQ ID NO: 21),

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSNAISWVRQAPGQGLEWMGGIIPIHGT AIYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDQRGHHDYYYYGMDV WGQGTTVTVSS (SEQ ID NO: 22),

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSNAISWVRQAPGQGLEWMGGIIPIHGT AIYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDQRGYRHYYYYGMDVW GQGTTVTVSS (SEQ ID NO: 23), QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSNAISWVRQAPGQGLEWMGGIIPIQGT AIYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDQRGYHHYYYSGMDVW GQGTTVTVSS (SEQ ID NO: 24),

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSNAISWVRQAPGQGLEWMGGIIPIHGT AIYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDQRGYHDYYYYGMDVW GQGTTVTVSS (SEQ ID NO: 25),

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSNAISWVRQAPGQGLEWMGGIIPEFGT

AIYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDQRGSYDYYYYGMDVW GQGTTVTVSS (SEQ ID NO: 26),

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSNAISWVRQAPGQGLEWMGGIIPSFGT

AIYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDQRGESDYYYYGMDVW GQGTTVTVSS (SEQ ID NO: 27),

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSNAISWVRQAPGQGLEWMGGIIPIHGT

AIYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDQRGYQDYYYYGMDVW GQGTTVTVSS (SEQ ID NO: 28),

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSNAISWVRQAPGQGLEWMGGIIPIQGT

AIYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDQRGYRDYYYYGMDVW GQGTTVTVSS (SEQ ID NO: 29),

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSNAISWVRQAPGQGLEWMGGIIPIHGT AIYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDQRGYHHYYYYGMDV WGQGTTVTVSS (SEQ ID NO: 30), and

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSNAISWVRQAPGQGLEWMGGIIPIHGT AIYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDQRGYRDYYYYGMDVW GQGTTVTVSS (SEQ ID NO: 31); preferably selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22; more preferably selected from SEQ ID NO: 20 and SEQ ID NO: 21.

7. The antigen-binding peptide according to any one of the foregoing claims, wherein said antigen-binding peptide comprises a second domain, preferably a light chain variable domain, comprising a first complementarity-determining region comprising or consisting of an amino acid sequence NIRNVG (SEQ ID NO: 32), a second complementarity-determining region comprising or consisting of an amino acid sequence DDD (SEQ ID NO: 33), and a third complementarity-determining region comprising or consisting of an amino acid sequence QVWDSATDQRV (SEQ ID NO: 34); wherein, optionally, said second domain comprises or consists of an amino acid sequence which is at least 90% identical, preferably at least 95 % identical, more preferably at least 98% identical, even more preferably at least 99% identical, optionally identical, to an amino acid sequence SYVLTQPPSVSVAPGKTARITCGGNNIRNVGVHWYQQKPGQAPVLWYDDDDRPSGI PERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSATDQRVFGGGTKLTVL

(SEQ ID NO: 35).

8. The antigen-binding peptide according to any one of the foregoing claims, wherein said antigen-binding peptide is an antibody or an antigen-binding fragment thereof; wherein, preferably, said antigen-binding peptide is selected from an antibody, a Fab, Fab', a F(ab')2, a scFv, di-scFv, a VH domain, a single-domain antibody (sdAb), a diabody, a triabody, and a tetrabody; wherein, more preferably, said antigen-binding peptide is selected from an antibody, a Fab, and a scFv.

9. The antigen-binding peptide according to any one of the foregoing claims, wherein said antigen-binding peptide binds to human c-Met ECD with a KD of 10 nM or less, preferably 8.5 nM or less, as determined using biolayer interferometry; and/or wherein said antigen-binding peptide has a half maximal effective concentration (EC₅o) for binding to c-MET expressing cells of 2 nM or less, preferably 1.6 nM or less, more preferably 1.2 nM or less, as determined using flow cytometry; and/or wherein said antigen-binding peptide has a hydrophobic interaction chromatography retention time of 20 min or less, preferably 19.6 min or less, even more preferably 19 min or less, as determined using hydrophobic interaction chromatography; wherein, optionally, said antigen-binding peptide has a ratio of KDantigen-binding peptide to KDwildtype < 10, afld/OT a ratio of EC50antigen-binding peptide tO EC50wildtype — 1, preferably < 0.8, and/or a hydrophobic interaction chromatography retention time of < 19.6 min.

10. A bispecific molecule, preferably a bispecific antibody or antigen-binding fragment thereof, comprising or consisting of i) a first antigen-binding peptide, wherein said first antigen-binding peptide is an antigen-binding peptide as defined in any one of the foregoing claims; and ii) a second antigen-binding peptide; wherein, preferably, said second antigen-binding peptide binds to a tumor antigen; wherein, more preferably, said second antigen-binding peptide binds to c-Met.

11. The bispecific molecule according to claim 10, wherein said bispecific molecule is a biparatopic molecule, preferably a biparatopic antibody or antigen-binding fragment thereof; wherein, optionally, said second antigen-binding peptide comprises or consists of a heavy chain variable domain comprising or consisting of an amino acid sequence which is at least 90% identical, preferably at least 95 % identical, more preferably at least 98% identical, even more preferably at least 99% identical, optionally identical, to an amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGS TYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDRRITHTYWGQGTLVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS

DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

(SEQ ID NO: 36), and/or a light chain variable domain comprising or consisting of an amino acid sequence which is at least 90% identical, preferably at least 95 % identical, more preferably at least 98% identical, even more preferably at least 99% identical, optionally identical, to an amino acid sequence SYVLTQPPSVSVAPGQTARITCGGDSLGSKIVHWYQQKPGQAPVLVVYDDAARPSGIP ERFSGSNSGNTATLTISRVEAGDEADYYCQVYDYHSDVEVFGGGTKLTVLGQPKAAP SVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNK YAASSYLSLTPEQWKSHKSYSCQVTHEGSTVEKTVAPTECS

(SEQ ID NO: 37).

12. An antigen-binding peptide-drug conjugate, preferably an antibody-drug conjugate, wherein said antigen-binding peptide-drug conjugate comprises an antigen-binding peptide according to any one of claims 1-9 or a bispecific molecule according to any one of claims 10-11; and further comprises a diagnostic and/or therapeutic agent, preferably a therapeutic agent, more preferably a cytotoxic agent, such as exatecan; wherein, optionally, the antigen-binding peptide-drug conjugate comprises a linker.

13. A composition, preferably a pharmaceutical composition, comprising an antigenbinding peptide according to any one of claims 1-9, a bispecific molecule according to any one of claims 10-11, or an antigen-binding peptide-drug conjugate according to claim 12, and a pharmaceutically acceptable excipient.

14. An antigen-binding peptide according to any one of claims 1-9, a bispecific molecule according to any one of claims 10-11, an antigen-binding peptide-drug conjugate according to claim 12, or a composition according to claim 13, for use in a method of preventing or treating a cancer.

15. An isolated nucleic acid encoding an antigen-binding peptide according to any one of claims 1-9 or a bispecific molecule according to any one of claims 10-11.

16. A recombinant cell comprising the isolated nucleic acid according to claim 15.