CD3 binding domains

Abstract

The present disclosure relates to binding domains that bind CD3, and binding moieties comprising such binding domains. The present disclosure further relates to the use of such binding domains or binding moieties in the treatment of cancer. The present disclosure also relates to nucleic acid encoding such binding domains or binding moieties, and a vector and cell comprising such nucleic acid.

Description

[0001] Title: CD3 BINDING DOMAINS

[0002] TECHNICAL FIELD

[0003] The present disclosure relates to the field of antibodies. In particular it relates to the field of therapeutic antibodies for the treatment of disease, in particular for the treatment of cancer. More particularly it relates to novel CD3 binding domains and binding moieties comprising such CD 3 binding domains.

[0004] BACKGROUND

[0005] Monoclonal antibodies that bind to human CD3 were among the first antibodies developed for therapeutic use in humans. Monoclonal CD3 binding antibodies are typically used for their immune suppressive qualities, for instance in transplant rejection. Antibodies which are multispecific and comprise a binding domain targeting CD3 on T cells and for one or more surface target antigens on cancer cells, are capable of re-directing a T cell to a cancer cell, independent of T-cell receptor specificity, co-stimulation, or peptide antigen presentation.

[0006] Whereas there are T-cell engaging antibodies developed for treatment of various indications, these have a variety of liabilities including as it concerns efficacy and toxicity and there remains a need for novel CD3 binding moieties and therapeutic interventions that re-direct T cells to tumor cells comprising such CD3 binding moieties.

[0007] SUMMARY

[0008] One of the objects of the present disclosure is to provide new CD3 binding domains useful for the generation of pharmaceutical agents for the treatment of human disease, in particular for the generation of T-cell engagers for the treatment of cancer. This object is met by the provision of the CD3 binding domains described herein.

[0009] In certain embodiments, the present disclosure provides a polypeptide comprising an amino acid sequence as described herein, or a variant thereof, as well as methods for producing such variant. In certain embodiments, the present disclosure provides a CD3 binding domain comprising a polypeptide as described herein.

[0010] In certain embodiments, the present disclosure provides a CD3 binding domain, wherein the CD3 binding domain comprises a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences as described herein.

[0011] In certain embodiments, the present disclosure provides a binding moiety comprising a CD3 binding domain as described herein.

[0012] In certain embodiments, the present disclosure provides a binding moiety comprising a CD3 binding domain, wherein the CD3 binding domain comprises a heavy chain variable region comprising CDR1 , CDR2, and CDR3 sequences as described further herein.

[0013] In certain embodiments, the present disclosure provides a binding moiety that binds CD3, wherein the binding moiety competes with a binding moiety as described herein for binding to CD3.

[0014] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising an effective amount of a binding moiety as described herein.

[0015] In certain embodiments, the present disclosure provides a binding moiety as described herein, and a pharmaceutical composition as described herein, for use in therapy.

[0016] In certain embodiments, the present disclosure provides a binding moiety as described herein, and a pharmaceutical composition as described herein, for use in the treatment of cancer.

[0017] In certain embodiments, the present disclosure provides a method for treating a disease, comprising administering an effective amount of a binding moiety as described herein, or a pharmaceutical composition as described herein, to an individual in need thereof.

[0018] In certain embodiments, the present disclosure provides a method for treating cancer, comprising administering an effective amount of a binding moiety as described herein, or a pharmaceutical composition as described herein, to an individual in need thereof.

[0019] In certain embodiments, the present disclosure provides a nucleic acid sequence encoding the heavy chain variable region of a CD3 binding domain as described herein.

[0020] In certain embodiments, the present disclosure provides a vector comprising a nucleic acid sequence as described herein. In certain embodiments, the present disclosure provides a cell comprising a nucleic acid sequence encoding the heavy chain variable region of a CD3 binding domain as described herein.

[0021] In certain embodiments, the present disclosure provides a cell producing a CD3 binding domain or a binding moiety as described herein.

[0022] DETAILED DESCRIPTION

[0023] One of the objects of the present disclosure is to provide new CD3 binding domains useful for the generation of new pharmaceutical agents for the diagnosis and treatment of disease, in particular in humans and in particular for the diagnosis and treatment of cancer. This object is met by the provision of the CD3 binding domains described herein.

[0024] In certain embodiments, the present disclosure provides a polypeptide comprising the amino acid sequence as set forth in SEQ ID NO: 1 or 8, or a variant thereof. In certain embodiments, the polypeptide is an immunoglobulin heavy chain, or part thereof, that, when combined with a suitable light chain, or part thereof, binds to CD3. For example, the part of an immunoglobulin heavy chain can be a heavy chain variable region with a CHI region, or a heavy chain variable region. The part of a light chain can, for example, be a light chain variable region.

[0025] In certain embodiments, the present disclosure provides variants of the polypeptides as described herein. Such variants can be produced by making a modification in the amino acid sequence of SEQ ID NO: 1 or 8. A modification can be one or more modifications, such as for instance one or more amino acid substitutions, insertions, deletions, or a combination thereof. In certain embodiments, the modification is limited to at most one, two, three, four, five, six, seven, eight, nine, or ten modifications. In certain embodiments, the modification is limited to at most one, two, three, four or five amino acid substitutions. In certain embodiments, the modification is limited to one, two, or three amino acid substitutions.

[0026] In certain embodiments, the present disclosure provides a CD3 binding domain that comprises a polypeptide comprising the amino acid sequence as set forth in SEQ ID NO: 1 or 8.

[0027] In general, as described herein, antigen binding can be expressed in terms of specificity and affinity. The specificity determines which antigen or epitope thereof is specifically bound by the binding domain or binding moiety. The affinity is a measure for the strength of binding to a particular antigen or epitope. For the purpose of the present disclosure, a binding domain or binding moiety is considered to bind the antigen when it has an at least two times higher binding signal than the background signal in the same assay.

[0028] The term “CD3” (cluster of differentiation 3) refers to a protein complex, which is composed of a CD3y chain (UniProt (SwissProt) P09693), a CD35 chain (UniProt (SwissProt) P04234), CD3s chains (UniProt (SwissProt) P07766), and a CD3 zeta chain homodimer (UniProt (SwissProt) P20963). CD3s is known under various aliases some of which are: “CD3e Molecule, Epsilon (CD3-TCR Complex)”; “CD3e Antigen, Epsilon Polypeptide (TiT3 Complex)”; T-Cell Surface Antigen T3 / Leu-4 Epsilon Chain; T3E; T-Cell Antigen Receptor Complex, Epsilon Subunit Of T3; CD3e Antigen; CD3-Epsilon 3; IMD18; TCRE. Ids for CD3E Gene are HGNC: 1674; Entrez Gene: 916; Ensembl: ENSG00000198851; OMIM: 186830 and UmProtKB: P07766. These chains associate with the T-cell receptor (TCR) and the ^-chain to form a TCR complex that upon mitogenic signaling generates an activation signal in T lymphocytes. CD3 is expressed on T cells and NK T cells.

[0029] In certain embodiments, the CD3 binding domain further comprises a polypeptide comprising the amino acid sequence as set forth in SEQ ID NO: 15.

[0030] In certain embodiments, the present disclosure provides a CD3 binding domain comprising a heavy chain variable region comprising the heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2 ), and heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence selected from SEQ ID NOs: 1 and 8. In certain embodiments, each of the HCDRs may comprise at most three, two, or one amino acid variations. In certain embodiments, only one of the HCDR1 or HCDR2 may comprise at most three, two, or one amino acid variations. In certain embodiments, HCDR3 does not comprise any amino acid variations. In certain embodiments, the amino acid variation is a conservative amino acid substitution.

[0031] In general, as described herein, typically, a conservative amino acid substitution involves a variation of an amino acid with a homologous amino acid residue, which is a residue that shares similar characteristics or properties. Homologous amino acids are known in the art, as are routine methods for making amino acid substitutions in antibody binding domains without significantly impacting binding or function of the antibody, see for instance handbooks like Lehninger (Nelson, David L., and Michael M. Cox. 2017. Lehninger Principles of Biochemistry. 7th ed. New York, NY: W.H. Freeman) or Stryer (Berg, J., Tymoczko, J., Stryer, L. and Stryer, L., 2007. Biochemistry. New York: W.H. Freeman), incorporated herein in its entirety. In determining whether an amino acid can be replaced with a conserved amino acid, an assessment may typically be made of factors such as, but not limited to, (a) the structure of the polypeptide backbone in the area of the substitution, for example, a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, and / or (c) the bulk of the side chain(s). If a residue can be substituted with a residue which has common characteristics, such as a similar side chain or similar charge or hydrophobicity, then such a residue is preferred as a substitute. For example, the following groups can be determined: (1) non-polar: Ala (A), Gly (G), Vai (V), Leu (L), He (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gin (Q); (3) acidic: Asp (D), Glu (E); and (4) basic: Lys (K), Arg (R), His (H). Alternatively, the amino acids may be grouped as follows: (1) aromatic: Phe (F), Trp (W), Tyr (Y); (2) apolar: Leu (L), Vai (V), He (I), Ala (A), Met (M); (3) aliphatic: Ala (A), Vai (V), Leu (L), He (I); (4) acidic: Asp (D), Glu (E); (5) basic: His (H), Lys (K), Arg (R); and (6) polar: Gin (Q), Asn (N), Ser (S), Thr (T), Tyr (Y). Alternatively, amino acid residues may be divided into groups based on common side-chain properties: (1) hydrophobic: Met (M), Ala (A), Vai (V), Leu (L), He (I); (2) neutral hydrophilic: Cys (C), Ser (S), Thr (T), Asn (N), Gin (Q); (3) acidic: Asp (D), Glu (E); (4) basic: His (H), Lys (K), Arg R); (5) residues that influence chain orientation: Gly (G), Pro (P); and (6) aromatic: Trp (W), Tyr (Y), Phe (F).

[0032] The substitution of an amino acid residue with another present in the same group would be preferred. Accordingly, conservative amino acid substitution can involve exchanging a member of one of these classes for another member of that same class. Typically, the variation results in no, or substantially no, loss in binding specificity of the binding domain to its intended target.

[0033] Additional types of amino acid variations include variations resulting from somatic hypermutation or affinity maturation. Binding variants encompassed by the present disclosure include somatically hypermutated or affinity matured heavy chain variable regions, which are heavy chain variable regions derived from the same VH gene segments as the heavy chain variable regions described by sequence herein, the variants having amino acid variations, including non-conservative and / or conservative amino acid substitutions in one, two, or all three HCDRs. Routine methods for affinity maturing antibody binding domains are widely known in the art, see for instance Tabasinezhad M, et al. (Trends in therapeutic antibody affinity maturation: From in-vitro towards next-generation sequencing approaches. Immunol Lett. 2019 Aug;212: 106-113).

[0034] In certain embodiments, the present disclosure provides a CD3 binding domain that comprises a heavy chain variable region comprising: a) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively, according to IMGT, or heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequence as set forth in SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively, according to Kabat; b) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequence as set forth in SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively, according to IMGT, or heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequence as set forth in SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively, according to Kabat.

[0035] In certain embodiments, each of the HCDRs may comprise at most three, two, or one amino acid variations. In certain embodiments, only one of the HCDR1 or HCDR2 may comprise at most three, two, or one amino acid variations. In certain embodiments, HCDR3 does not comprise any amino acid variations. In certain embodiments, the amino acid variation is a conservative amino acid substitution.

[0036] In certain embodiments, the CD3 binding domain comprises a heavy chain variable region having the amino acid sequence as set forth in SEQ ID NO: 1 or 8, or having at least 80%, 85%, 90%, or 95% sequence identity thereto.

[0037] “Percent (%) identity” as referring to nucleic acid or amino acid sequences herein is defined as the percentage of residues in a candidate sequence that are identical with the residues in a selected sequence, after aligning the sequences for optimal comparison purposes. In order to optimize the alignment between the two sequences gaps may be introduced in any of the two sequences that are compared. Such alignment can be carried out over the full length of the sequences being compared. Alternatively, the alignment may be carried out over a shorter length, for example over about 20, about 50, about 100 or more nucleic acids / bases or amino acids. The alignment may also be carried out over individual CDR sequences. The sequence identity is the percentage of identical matches between the two sequences over the reported aligned region.

[0038] A comparison of sequences and determination of percentage of sequence identity between two sequences can be accomplished using a mathematical algorithm. The skilled person will be aware of the fact that several different computer programs are available to align two sequences and determine the identity between two sequences (Kruskal, J. B. (1983) An overview of sequence comparison In D. Sankoff and J. B. Kruskal, (ed.), Time warps, string edits and macromolecules: the theory and practice of sequence comparison, pp. 1 -44 Addison Wesley). The percent sequence identity between two amino acid sequences or nucleic acid sequences may be determined using the Needleman and Wunsch algorithm for the alignment of two sequences. (Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453). The Needleman- Wunsch algorithm has been implemented in the computer program Geneious Prime (Dotmatics). For the purpose of this disclosure, the Geneious Prime program and the Geneious alignment, Global alignment Needleman-Wunsch with Blosum62 cost matrix, are used to determine the percent identity of amino acid sequences. The parameters used are a gap-open penalty of 12 and a gap extension penalty of 3. For DNA sequences, the Geneious Prime program and the Geneious alignment, Global alignment Needleman-Wunsch with cost matrix Identity (1.0 / 0.0) are used. The parameters used are a gap-open penalty of 12 and a gap extension penalty of 3.

[0039] After alignment by the program Geneious Prime as described above the percentage of sequence identity between a query sequence and a sequence of this disclosure is calculated as follows: Number of corresponding positions in the alignment showing an identical amino acid or identical nucleotide in both sequences divided by the total length of the alignment after subtraction of the total number of gaps in the alignment.

[0040] In certain embodiments, a CD3 binding domain of the present disclosure also comprises CD3 binding domain variants, which, in addition to the variations in the HCDRs referred to above, comprise one or more variations in the framework regions. A variation can be any type of amino acid variation described herein, such as for instance a conservative amino acid substitution or non-conservative amino acid substitution resulting from somatic hypermutation or affinity maturation. In certain embodiments, a CD3 binding domain variant of the present disclosure comprises no variations in the CDR regions but comprises one or more variations in the framework regions. Such variants have at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity to the sequences disclosed herein, and are expected to retain CD3 binding specificity. Thus, in certain embodiments, a CD3 binding domain of the present disclosure comprises:

[0041] - a heavy chain variable region having at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1, which heavy chain variable region comprises a HCDR1 amino acid sequence as set forth in SEQ ID NO: 2; a HCDR2 amino acid sequence as set forth in SEQ ID NO: 3; and a HCDR3 amino acid sequence as set forth in SEQ ID NO: 4, according to IMGT, or a HCDR1 amino acid sequence as set forth in SEQ ID NO: 5; a HCDR2 amino acid sequence as set forth in SEQ ID NO: 6; and a HCDR3 amino acid sequence as set forth in SEQ ID NO: 7, according to Kabat; or

[0042] - a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 8, which heavy chain variable region comprises a HCDR1 amino acid sequence as set forth in SEQ ID NO: 9; a HCDR2 amino acid sequence as set forth in SEQ ID NO: 10; and a HCDR3 amino acid sequence as set forth in SEQ ID NO: 11, according to IMGT, or a HCDR1 amino acid sequence as set forth in SEQ ID NO: 12; a HCDR2 amino acid sequence as set forth in SEQ ID NO: 13; and a HCDR3 amino acid sequence as set forth in SEQ ID NO: 14, according to Kabat.

[0043] In certain embodiments, a CD3 binding domain of the present disclosure comprises a light chain or light chain variable region as described herein.

[0044] In certain embodiments, the CD3 binding domain of the present disclosure comprises one or more constant regions as described herein.

[0045] In certain embodiments, the present disclosure provides a method for producing a variant of a CD3 binding domain of the present disclosure, and a variant thus produced, wherein the method comprises:

[0046] - making a modification in the amino acid sequence as set forth in SEQ ID NO: 1 or 8;

[0047] - testing the modified CD3 binding domain for binding to human CD3; and

[0048] - selecting the modified CD3 binding domain if it binds to human CD3.

[0049] In certain embodiments, the present disclosure provides a binding moiety comprising a CD3 binding domain as described herein. In certain embodiments, a binding moiety is a monospecific binding moiety, in particular a bivalent monospecific antibody. A monospecific antibody according to the present disclosure is an antibody, in any antibody format, that comprises one or more binding domains with specificity for a single target or epitope. In certain embodiments, a monospecific binding moiety of the present disclosure may further comprise an Fc region or a part thereof. In certain embodiments, a monospecific binding moiety of the present disclosure is an IgGl antibody.

[0050] In certain embodiments, the binding moiety is a multispecific binding moiety, such as a bispecific or trispecific binding moiety.

[0051] In certain embodiments, a multispecific binding moiety is a multispecific antibody. In certain embodiments, a trispecific binding moiety is a trispecific antibody. In certain embodiments, a trispecific binding moiety is a trivalent trispecific antibody. A trispecific antibody according to the present disclosure is an antibody that has specificity for three different targets or epitopes. In certain embodiments, a bispecific binding moiety is a bispecific antibody. In certain embodiments, a bispecific binding moiety is a bivalent bispecific antibody. In certain embodiments, a bispecific binding moiety is a trivalent or tetravalent bispecific antibody. A bispecific antibody according to the present disclosure is an antibody that has specificity for two different targets or epitopes.

[0052] In certain embodiments, a multispecific antibody of the present disclosure may comprise an Fc region or a part thereof. In certain embodiments, a multispecific binding moiety of the present disclosure is an IgGl antibody.

[0053] In certain embodiments, the binding moiety of the present disclosure binds to human CD3.

[0054] In general, as described herein, a “binding moiety” refers to a proteinaceous molecule and includes for instance all antibody formats available in the art, such as for example a full length IgG antibody, immunoconjugates, diabodies, BiTEs, Fab fragments, scFv, tandem scFv, single domain antibody (like VHH and VH), minibodies, scFab, scFv-zipper, nanobodies, DART molecules, TandAb, Fab-scFv, F(ab)’2, F(ab)’2-scFv2, and intrabodies as well as any other formats known to a person of ordinary skill in the art.

[0055] In general, as described herein, a “Fab” typically means a binding domain comprising a heavy chain variable region, a light chain variable region, a CHI and a CL region. In general, as described herein, an “Fc region” typically comprises a hinge, CH2, and CH3 region. A suitable hinge, CH2, and CH3 regions include, but are not limited to, those as described herein. The Fc region mediates effector functions of an antibody, such as complementdependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC) and antibodydependent cell phagocytosis (ADCP). Depending on the therapeutic antibody or Fc fusion protein application, it may be desired to either reduce or increase the effector function.

[0056] In certain embodiments, a binding moiety of the present disclosure has Fc effector function. In certain embodiments, a binding moiety of the present disclosure has enhanced Fc effector function. In certain embodiments, a binding moiety of the present disclosure exhibits antibody-dependent cell-mediated cytotoxicity (ADCC). A binding moiety, such as an antibody, can be engineered to enhance the ADCC activity (for review, see Kubota T et al. Cancer Sci. 2009; 100(9): 1566-72). For instance, ADCC activity of an antibody can be improved when the antibody itself has a low ADCC activity, by slightly modifying the constant region of the antibody (Junttila TT. et al. Cancer Res. 2010;70(l l):4481-9). Changes are sometimes also made to improve storage or production or to remove C-terminal lysines (Kubota T et al. Cancer Sci. 2009; 100(9): 1566-72). Another way to improve ADCC activity of an antibody is by enzymatically interfering with the glycosylation pathway resulting in a reduced fucose (von Horsten HH. etal. Glycobiology. 2010;20(12):1607-18). Alternatively, or additionally, multiple other strategies can be used to achieve ADCC enhancement, for instance including glycoengineering (Kyowa Hakko / Biowa, GlycArt (Roche) and Eureka Therapeutics) and mutagenesis, all of which seek to improve Fc binding to low-affinity activating FcyRIIIa, and / or to reduce binding to the low affinity inhibitory FcyRIIb. In certain embodiments, a binding moiety of the present disclosure exhibits enhanced antibody-dependent cell-mediated cytotoxicity (ADCC). In certain embodiments, a binding moiety of the present disclosure is afucosylated.

[0057] In certain embodiments, the present disclosure provides a CD3 binding domain, wherein the CD3 binding domain competes with a binding moiety as described herein for binding to CD3.

[0058] In certain embodiments, the present disclosure provides a binding moiety that binds CD3, wherein the binding moiety competes with a binding moiety as described herein for binding to CD3. For the purpose of the present disclosure, “compete”, “competes”, or “competing” refers to an activity of a binding domain or binding moiety that displaces a binding domain or binding moiety as described herein from its target antigen, in a cross-blocking assay. Therefore, in certain embodiments, a binding domain that competes for binding with the binding domain as described herein, binds to human CD3 and displaces the binding domain as described herein, in a cross-blocking assay. In certain embodiments, a binding moiety that competes for binding with the binding moiety as described herein, binds to human CD3 and displaces the binding moiety as described herein, in a cross-blocking assay. In certain embodiments, a cross-blocking assay is a competitive ELISA. Methods of performing a competitive ELISA are known to a person of ordinary skill in the art.

[0059] In brief, in a competitive ELISA, antigen is coated on the wells of a microtiter plate and pre-incubated with or without the competing binding moiety. This is followed by addition of a biotin-labeled binding domain or binding moiety as described herein. The amount of labeled binding domain or binding moiety bound to the antigen in the wells is measured using avidinperoxidase conjugate and appropriate substrate. The amount of labeled biding domain or binding moiety that is bound to the antigen has an indirect correlation to the ability of the competing binding domain or binding moiety to compete for binding to the same antigen, i.e., the greater the affinity of the competing binding domain or binding moiety for the same antigen, the less labeled binding domain or binding moiety will be bound to the antigen-coated wells. A candidate competing binding domain or binding moiety is considered to compete for binding to the antigen, if the candidate binding domain or binding moiety can block binding of the binding domain or binding moiety of the present disclosure, to the target antigen, by at least 20%, or by at least 20-50%, or by at least 50%, as compared to the control performed in parallel in the absence of the candidate competing binding domain or binding moiety.

[0060] Light chain

[0061] In certain embodiments, a CD3 binding domain, or the CD3 binding domain of a binding moiety of the present disclosure comprises a light chain variable region comprising the light chain CDR1 (LCDR1), light chain CDR2 (LCDR2 ), and light chain CDR3 (LCDR3) of a light chain variable region having the amino acid sequence as set forth in SEQ ID NO: 15. In certain embodiments, each of the LCDRs may comprise at most three, two, or one amino acid variations. In certain embodiments, only one of the LCDR1 or LCDR2 may comprise at most three, two, or one amino acid variations. In certain embodiments, LCDR3 does not comprise any amino acid variations. In certain embodiments, the amino acid variation is a conservative amino acid substitution.

[0062] In certain embodiments, the light chain variable region comprises light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), having an amino acid sequence as set forth in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively, according to IMGT, or light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), having an amino acid sequence as set forth in SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 18, according to Kabat, respectively. In certain embodiments, each of the LCDRs may comprise at most three, two, or one amino acid variations. In certain embodiments, only one of the LCDR1 or LCDR2 may comprise at most three, two, or one amino acid variations. In certain embodiments, LCDR3 does not comprise any amino acid variations. In certain embodiments, the amino acid variation is a conservative amino acid substitution.

[0063] In certain embodiments, the light chain variable region has the amino acid sequence as set forth in SEQ ID NO: 15, or having at least 80%, 85%, 90%, or 95% sequence identity thereto.

[0064] In certain embodiments, a CD3 binding domain or binding moiety of the present disclosure also comprises variants, which, in addition to the variations in the heavy chain variable region and / or LCDRs referred to above, comprise one or more variations in the light chain variable framework regions. A variation can be any type of amino acid variation described herein, such as for instance a conservative amino acid substitution or non-conservative amino acid substitution resulting from somatic hypermutation or affinity maturation. In certain embodiments, a CD3 binding domain or binding moiety variant of the present disclosure comprises no variations in the LCDR regions but comprises one or more variations in the framework regions. Such variants have at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity to the sequences disclosed herein, and are expected to retain binding specificity. Thus, in certain embodiments, a CD3 binding domain or binding moiety of the present disclosure comprises: - a light chain variable region having at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 15, which light chain variable region comprises a LCDR1 amino acid sequence as set forth in SEQ ID NO: 16; a LCDR2 amino acid sequence as set forth in SEQ ID NO: 17; and a LCDR3 amino acid sequence as set forth in SEQ ID NO: 18, according to IMGT, or a LCDR1 amino acid sequence as set forth in SEQ ID NO: 19; a LCDR2 amino acid sequence as set forth in SEQ ID NO: 20; and a LCDR3 amino acid sequence as set forth in SEQ ID NO: 18, according to Kabat.

[0065] The CD3 binding domain and the CD3 binding domain of the binding moiety of the present disclosure have been generated with a common light chain, in particular with a common light chain referred to as VK1-39 / JK1. The CD3 binding domain and the CD3 binding domain of the binding moiety of the present disclosure can; however, comprise any suitable light chain, including but not limited to common light chains known in the art. Examples of common light chains known in the art include, but are not limited to: VK1-39 / JK5, comprising a light chain variable region comprising a light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), of a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 23. In certain embodiments, the light chain comprises a light chain variable region comprising a light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), of a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 23, wherein each of the LCDRs may comprise at most three, two, or one amino acid variations, for example substitutions. In certain embodiments, the light chain comprises a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 23, or having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto. In certain embodiments, the light chain comprises a light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) having an amino acid sequence as set forth in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 24, according to IMGT, respectively. In certain embodiments, the light chain comprises a light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) according to Kabat, which are as underlined in the amino acid sequence as set forth in SEQ ID NO: 23; VK3-15 / JK1, comprising a light chain variable region comprising a light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3(LCDR3), of a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 26. In certain embodiments, the light chain comprises a light chain variable region comprising a light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), of a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 26, wherein each of the LCDRs may comprise at most three, two, or one amino acid variations, for example substitutions. In certain embodiments, the light chain comprises a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 26, or having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto. In certain embodiments, the light chain comprises a light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) having an amino acid sequence as set forth in SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 29, respectively. In certain embodiments, the light chain comprises a light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) according to Kabat, which are as underlined in the amino acid sequence as set forth in SEQ ID NO: 26; VK3-20 / JK1, comprising a light chain variable region comprising a light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), of a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 31. In certain embodiments, the light chain comprises a light chain variable region comprising a light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), of a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 31, wherein each of the LCDRs may comprise at most three, two, or one amino acid variations, for example substitutions. In certain embodiments, the light chain comprises a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 31, or having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto. In certain embodiments, the light chain comprises a light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) having an amino acid sequence as set forth in SEQ ID NO: 32, SEQ ID NO: 28, and SEQ ID NO: 33, respectively. In certain embodiments, the light chain comprises a light chain CDR1 (LCDR1 ), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) according to Kabat, which are as underlined in the amino acid sequence as set forth in SEQ ID NO: 31; and VL3-21 / JL3, comprising a light chain variable region comprising a light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), of a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 35. In certain embodiments, the light chain comprises a light chain variable region comprising a light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), of a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 35, wherein each of the LCDRs may comprise at most three, two, or one amino acid variations, for example substitutions. In certain embodiments, the light chain comprises a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 35, or having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto. In certain embodiments, the light chain comprises a light chain CDR1 (LCDR1 ), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) having an amino acid sequence as set forth in SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38, respectively. In certain embodiments, the light chain comprises a light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) according to Kabat, which are as underlined in the amino acid sequence as set forth in SEQ ID NO: 35.

[0066] VK1-39 is short for Immunoglobulin Variable Kappa 1-39 Gene. The gene is also known as Immunoglobulin Kappa Variable 1-39; IGKV139; IGKV1-39; IgVKl-39. External Ids for the gene are HGNC: 5740; Entrez Gene: 28930; Ensembl: ENSG00000242371. An amino acid sequence for VK1-39 is given as SEQ ID NO: 22. This is the sequence of the V-region. The V- region can be combined with one of five J-regions. Suitable VJ-region sequences are indicated as VK1-39 / JK1 (SEQ ID NO: 15) and VK1-39 / JK5 (SEQ ID NO: 23); alternative names are IgVKl-39*01 / IGJKl*01 or IgVKl-39*01 / IGJK5*01 (nomenclature according to the IMGT database worldwide web at imgt.org). These names are exemplary and encompass allelic variants of the gene segments.

[0067] VK3-15 is short for Immunoglobulin Variable Kappa 3-15 Gene. The gene is also known as Immunoglobulin Kappa Variable 3-15; IGKV315; IGKV3-15; IgVK3-15. External Ids for the gene are HGNC: 5816; Entrez Gene: 28913; Ensembl: ENSG00000244437. An amino acid sequence for VK3-15 is given as SEQ ID NO: 25. This is the sequence of the V-region. The V- region can be combined with one of five J-regions. A suitable VJ-region sequence is indicated as VK3-15 / JK1 (SEQ ID NO: 26); alternative name is VK3-15*01 / IGJK1 *01 (nomenclature according to the IMGT database worldwide web at imgt.org). This name is exemplary and encompasses allelic variants of the gene segments.

[0068] VK3-20 is short for Immunoglobulin Variable Kappa 3-20 Gene. The gene is also known as Immunoglobulin Kappa Variable 3-20; IGKV320; IGKV3-20; IgVK3-20. External Ids for the gene are HGNC: 5817; Entrez Gene: 28912; Ensembl: ENSG00000239951. An amino acid sequence for VK3-20 is indicated as SEQ ID NO: 30. This is the sequence of the V-region. The V-region can be combined with one of five J-regions. A suitable VJ-region sequence is indicated as VK3-20 / JK1 (SEQ ID NO: 31); alternative name is IgVK3-20*01 / IGJKl*01 (nomenclature according to the IMGT database worldwide web at imgt.org). This name is exemplary and encompasses allelic variants of the gene segments.

[0069] VL3-21 is short for Immunoglobulin Variable Lambda 3-21 Gene. The gene is also known as Immunoglobulin Lambda Variable 3-21; IGLV321; IGLV3-21; IgVX3-21. External Ids for the gene are HGNC: 5905; Entrez Gene: 28796; Ensembl: ENSG00000211662.2. An amino acid sequence for VL3-21 is given as SEQ ID NO: 34. This is the sequence of the V- region. The V-region can be combined with one of five J-regions. A suitable VJ-region sequence is indicated as VL3-21 / JL3 (SEQ ID NO: 35); alternative name is IgVX3-21 / IGJX3 (nomenclature according to the IMGT database worldwide web at imgt.org). This name is exemplary and encompasses allelic variants of the gene segments.

[0070] Further, any light chain variable region of a CD3 antibody available in the art may be used, as may any other light chain variable region that can readily be obtained, such as from, for instance, an antibody display library by showing antigen binding activity when paired with an anti-CD3 heavy chain variable as described herein.

[0071] Constant regions

[0072] In certain embodiments, a CD3 binding domain of the present disclosure further comprises a CHI region. In certain embodiments, a CD3 binding domain of the present disclosure further comprises a CHI region, hinge, CH2 region, and CH3 region. A suitable CHI region includes, but is not limited to, the CHI region of which the amino acid sequence is set forth in SEQ ID NO: 40. A suitable hinge includes, but is not limited to, the hinge of which the amino acid sequence is set forth in SEQ ID NO: 39. Suitable CH2 and CH3 regions include, but are not limited to, the CH2 region of which the amino acid sequence is set forth in SEQ ID NO: 41 or 42 and the CH3 region of which the amino acid sequence is set forth in SEQ ID NO: 43, or 44, or 45. In certain embodiments, a CD3 binding domain of the present disclosure further comprises a CL region. A suitable CL region includes, but is not limited to, the CL region of which the amino acid sequence is set forth in SEQ ID NO: 21. Y1

[0073] A CL, CHI, hinge, CH2, and / or CH3 region may be modified according to methods known in the art in order to obtain favorable antibody characteristics, including for instance to promote heterodimerization of different heavy chains, to improve heavy-light chain pairing, and to enhance or reduce immune cell effector function. A CH3 region may comprise the terminal lysine residue, or lack the terminal lysine residue to improve manufacturability.

[0074] Constant regions of a binding moiety of the present disclosure may comprise one or more variations that modulate properties of the binding moiety other than its binding properties to the target antigens or epitopes. For instance, the constant regions may comprise one or more variations that favor heterodimerization of the CD3 heavy chains over homodimerization of two CD3 heavy chains. Suitable variations that favor heterodimerization of two different heavy chains are, but are not limited to, those described for instance in WO 2013 / 157953 or WO 2013 / 157954. Also, the constant regions may comprise one or more variations that reduce or improve effector function, preferably one or more variations that reduce effector function. Suitable variations that reduce effector function are, but are not limited to, for instance L235G and / or G236R (according to the European numbering system). Further, the constant regions may comprise one or more variations that facilitate separation of the binding moiety from a mixture wherein it is produced. Suitable variations that facilitate separation of the binding moiety from a mixture wherein it is produced are, but are not limited to, those described for instance in WO 2020 / 226502.

[0075] Nucleic acids, vectors, and cells

[0076] In certain embodiments, the present disclosure provides a nucleic acid comprising a nucleic acid sequence that encodes a polypeptide or a heavy chain variable region as described herein. In certain embodiments, a nucleic acid of the present disclosure further comprises a nucleic acid sequence encoding a CHI region. In certain embodiments, a nucleic acid of the present disclosure further comprises a nucleic acid sequence encoding a CHI region, a hinge, CH2 region, and CH3 region. In certain embodiments, a nucleic acid of the present disclosure may further comprise at least one nucleic acid sequence encoding a light chain variable region, or encoding a light chain variable region and a CL region. In certain embodiments, the light chain variable region can be a light chain variable region as described herein. In certain embodiments, the present disclosure provides a vector comprising a nucleic acid as described herein. In certain embodiments, a vector of the present disclosure comprises a nucleic acid sequence that encodes a polypeptide or heavy chain variable region as described herein and a CHI region. In certain embodiments, a vector of the present disclosure encodes a polypeptide or heavy chain variable regions as described herein, a CHI region, hinge, CH2 region, and CH3 region. In certain embodiments, a vector of the present disclosure further comprises a nucleic acid sequence that encodes a light chain variable (VL) region, or a light chain variable region and a light chain constant (CL) region. In certain embodiments, the light chain variable region is a light chain variable region of a light chain that is capable of pairing with multiple heavy chains having different epitope specificities.

[0077] In certain embodiments, the present disclosure provides a cell comprising a nucleic acid sequence encoding the heavy chain variable region of a CD3 binding domain as described herein. In certain embodiments, the present disclosure provides a cell comprising a nucleic acid sequence encoding the heavy chain variable region of a CD3 binding domain as described herein. In certain embodiments, the cell further comprises a nucleic acid sequence encoding a CHI region. In certain embodiment, the cell further comprises a nucleic acid sequence encoding a CHI region, a hinge, CH2 region, and CH3 region. In certain embodiments, a cell of the present disclosure further comprises at least one nucleic acid sequence encoding a light chain variable (VL) region, or a light chain variable region and a light chain constant (CL) region. In certain embodiments, the light chain variable region is a light chain variable region of a light chain that is capable of pairing with multiple heavy chains having different epitope specificities.

[0078] In certain embodiments, the present disclosure provides a cell producing a binding moiety as described herein. In certain embodiments, the cell is a recombinant cell comprising a vector as described herein.

[0079] Pharmaceutical compositions and methods of use

[0080] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising an effective amount of a binding moiety comprising a CD3 binding domain as described herein, and a pharmaceutically acceptable carrier. In certain embodiments, the binding moiety comprising a CD3 binding domain is an antibody, such as for instance an IgGl antibody. In certain embodiment, the present disclosure provides a binding moiety comprising a CD3 binding domain as described herein, or a pharmaceutical composition as described herein, for use in therapy. In certain embodiments, the binding moiety comprising a CD3 binding domain is an antibody, such as for instance an IgGl antibody. In certain embodiments, the binding moiety comprising a CD3 binding domain, or a pharmaceutical composition comprising the same, is for use in the treatment of cancer.

[0081] In certain embodiments, the present disclosure provides a method for treating a disease, comprising administering an effective amount of a binding moiety comprising a CD3 binding domain as described herein, or a pharmaceutical composition as described herein, to a subject in need thereof. In certain embodiments, the disease is cancer.

[0082] As used herein, the terms “individual”, "subject" and "patient" are used interchangeably and refer to a mammal such as a human, mouse, rat, hamster, guinea pig, rabbit, cat, dog, monkey, cow, horse, pig and the like, and in particular to a human subject having cancer.

[0083] The terms “treat,” “treating,” and “treatment,” as used herein, refer to any type of intervention or process performed on or administering an active agent or combination of active agents to a subject with the objective of curing or improving a disease or symptom thereof or which produces a positive therapeutic response. As used herein, "positive therapeutic response" refers to a treatment producing a beneficial effect, e.g. reversing, alleviating, ameliorating, inhibiting, or slowing down a symptom, complication, condition or biochemical indicia associated with a disease, as well as preventing the onset, progression, development, severity or recurrence of a symptom, complication, condition or biochemical indicia associated with a disease, such as, for example, amelioration of at least one symptom of a disease or disorder, e.g. cancer. A beneficial effect can take the form of an improvement over baseline, including an improvement over a measurement or observation made prior to initiation of therapy according to the method. For example, a beneficial effect can take the form of slowing, stabilizing, stopping or reversing the progression of a cancer in a subject at any clinical stage, as evidenced by a decrease or elimination of a clinical or diagnostic symptom of the disease, or of a marker of cancer. Effective treatment may, for example, decrease tumor size, decrease the presence of circulating tumor cells, reduce or prevent metastases of a tumor, slow or arrest tumor growth and / or prevent or delay tumor recurrence or relapse. The term “therapeutic amount" or “effective amount” refers to an amount of an agent or combination of agents that treats a disease, such as cancer. In some embodiments, a therapeutic amount is an amount sufficient to delay tumor development. In some embodiments, a therapeutic amount is an amount sufficient to prevent or delay tumor recurrence.

[0084] As used herein, an effective amount of the agent or composition is one that, for example, may: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, retard, slow to some extent and may stop cancer cell infiltration into peripheral organs; (iv) inhibit tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay occurrence and / or recurrence of tumor; and / or (vii) relieve to some extent one or more of the symptoms associated with the cancer.

[0085] An effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual to be treated, and the ability of the agent or combination of agents to elicit a desired response in the individual, which can be readily evaluated by the ordinarily skilled physician or other health care worker.

[0086] An effective amount can be administered to a subject in one or more administrations.

[0087] An effective amount can also include an amount that balances any toxic or detrimental effects of the agent or combination of agents and the beneficial effects.

[0088] The term “agent” refers to a therapeutically active substance, in the present case a binding domain or a binding moiety of the present disclosure, or a pharmaceutical composition of the present disclosure.

[0089] As used herein, "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded.

[0090] The articles “a” and “an” are used herein to refer to one or more of the grammatical object of the article. By way of example, “an element” means one or more elements.

[0091] A reference herein to a patent document or other matter is not to be taken as an admission that that document or matter was known or that the information it contains was part of the common general knowledge at the priority date of any of the claims.

[0092] All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety. CDRs and framework regions of antibodies have been described and defined in the art using a number of different systems, including for instance Kabat (see Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md., 1987 and 1991); Kabat et al., J. Biol. Chem.252:6609-6616 (1977)), IMGT (discussed in Giudicelli et al., Nucleic Acids Res. 25: 206-21 1 1997), Chothia (Chothia and Lesk J. Mol. Biol. 196: 901 -917, 1987; Chothia et al., Nature 342: 877-883, 1989; Al-Lazikam et al., J. Mol. Biol. 273: 927-948, 1997), North (North et al., “A New Clustering of Antibody CDR Loop Conformations”, Journal of Molecular Biology, 406, 228-256 (2011)), and the nomenclatures of Honnegher and Plukthun (Honnegher and Plukthun, J. Mol. Biol. 309: 657-670, 2001), MacCallum (MacCallum et al., J. Mol. Biol.262:732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008)), and Lefranc (Lefranc M.P. et al., Dev. Comp. Immunol., 27: 55-77 (2003)). In general, any numbering system can be used considering that an antibody exhibits its properties regardless of which numbering system is used to define the CDRs. When the amino acid sequence of a variable region is given, a skilled person can readily determine its CDRs based on different numbering systems. Thus, the present disclosure encompasses defining the CDRs in accordance with each numbering system available to a skilled person. In particular, the present disclosure encompasses defining the CDRs in accordance with the numbering systems of Kabat, IMGT, and Chothia. In certain embodiments, the CDRs are as defined in Table 10. In certain embodiments, the CDRs are as defined in Table 11. Amino acids in the constant regions are indicated according to the EU numbering system. Accession numbers are primarily given to provide a further method of identification of a target, the actual sequence of the protein bound may vary, for instance because of a mutation in the encoding gene such as those occurring in some cancers or the like. An antigen binding site of a binding domain or binding moiety of the disclosure can bind the antigen and a variety of variants thereof, such as those expressed by some antigen positive immune or tumor cells. HGNC stands for the HUGO Gene nomenclature committee. The number following the abbreviation is the accession number with which information on the gene and protein encoded by the gene can be retrieved from the HGNC database. Entrez Gene provides the accession number or gene ID with which information on the gene or protein encoded by the gene can be retrieved from the NCBI (National Center for Biotechnology Information) database. Ensembl provides the accession number with which information on the gene or protein encoded by the gene can be obtained from the Ensembl database. Ensembl is a joint project between EMBL-EBI and the Wellcome Trust Sanger Institute to develop a software system which produces and maintains automatic annotation on selected eukaryotic genomes.

[0093] When herein reference is made to a gene or a protein, the reference is preferably to the human form of the gene or protein. When herein reference is made to a gene or protein reference is made both to the natural gene or protein and to variant forms of the gene or protein as can be detected in tumors, cancers and the like, preferably as can be detected in human tumors, cancers and the like.

[0094] BRIEF DESCRIPTION OF THE DRAWINGS

[0095] The following naming conventions are used herein as follows. In the Figures, different CD3 binding domains are indicated with different letters, e.g. CD3 binding domain A is indicated as “A”, CD3 binding domain B is indicated as “B”, etc. Bivalent bispecific antibodies are indicated in the format BsAb-RSV-G-A, BsAb indicating that it is a bispecific antibody, RSV-Gthat one binding domain targets RSV-G, and A that the antibody comprises CD3 binding domain A. This similarly applies to bispecific antibodies targeting EGFR and CD3 or cMET and CD3, and to different CD3 binding domains, e.g. B, C, D, E, F, G, H, or I. Bispecific antibodies targeting cMET and CD3 comprising different cMET binding domains and the same CD3 binding domain are indicated in the format BsAb-cMETl-A, BsAb-cMET2-A, etc. Bivalent bispecific positive and negative control antibodies are indicated in the format RSV-GxCD3, EGFRxTT, EGFRxCD3, TTxCD3, where different CD3 binding domains are indicated with different letters, e.g. X, Y, or Z.

[0096] Figure 1 - Figures 1 A and IB show the binding activity of RSV-GxCD3 bispecific antibodies as determined by FACS. Binding activity is expressed as mean fluorescence intensity (MFI) as measured at different antibody concentrations.

[0097] Figure 2 - Figure 2A shows the off-rate of EGFRxCD3 bispecific antibodies for binding to human CD3 on HBP-ALL cells. Figure 2B shows the off-rate of EGFRxCD3 bispecific antibodies for binding to human CD3 on Jurkat E6 cells. The binding of the bispecific antibodies was calculated as the percentage (%) of binding left of the initial binding, as measured over time. Figure 3 - Figure 3 A shows the data of a reporter assay. The ability of EGFRxCD3 bispecific antibodies to induce T cell activation is expressed in relative light units (RLU) as measured at different antibody concentrations. Figure 3B shows the data of a cytotoxicity assay. The ability of EGFRxCD3 bispecific antibodies to induce T cell-meditated target cell killing is expressed as the percentage (%) of target cell lysis as measured at different antibody concentrations. The % target cell lysis is as compared to the sample where no IgG was added and calculated as follows: (percentage target cell lysis equals (=)

[0098] 100 minus (-) (RLU sample divided by (:) RLU no IgG) times (x) 100 percent). Figure 3C shows an overview of multiple CD3 binding domains plotted based on the average potency (in AUC) as measured in a cytotoxicity assay against the average off-rate (in AUC). The average off-rate is normalized to CD3 binding domain C. The size of the dots represents the affinity for binding to CD 3: the larger the dot the higher the affinity.

[0099] Figure 4 - Figures 4A and 4B show the data of a reporter assay. The ability of cMETxCD3 bispecific antibodies to induce T cell activation is expressed as the Stimulation Index as measured at different antibody concentrations.

[0100] EXAMPLES

[0101] In the Examples, which are used to illustrate the present disclosure but are not intended to limit the disclosure in any way, each binding domain of the bispecific antibodies comprises a light chain variable region variable region having an amino acid sequence as set forth in SEQ ID NO: 15 and a light chain constant region having an amino acid sequence as set forth in SEQ ID NO: 21. The bispecific antibodies preferably are IgGl antibodies comprising a CHI, hinge, CH2, and CH3. In the Examples, which are used to illustrate the present disclosure but are not intended to limit the disclosure in any way, bispecific antibodies were screened in IgGl format, wherein the RSV-G, EGFR, or cMET binding heavy chain comprises a CHI having an amino acid sequence as set forth in SEQ ID NO: 40, a CH2 having an amino acid sequence as set forth in SEQ ID NO: 42, and a CH3 having an amino acid sequence as set forth in SEQ ID NO: 44; and the CD3 binding heavy chain comprises a CHI having an amino acid sequence as set forth in SEQ ID NO: 40, a CH2 having an amino acid sequence as set forth in SEQ ID NO: 42, and a CH3 having an amino acid sequence as set forth in SEQ ID NO: 45. EXAMPLES

[0102] EXAMPLE 1 - Generation of CD3 binding domains

[0103] Binding domains, antibodies and heavy chain variable regions with binding specificity to human CD3 were obtained by immunizing transgenic mice comprising a common IGKV1-39 light chain (MeMo® mice) with TCR / CD3 containing lipoparticles.

[0104] The CD3 binding domain sequences disclosed herein, once characterized and sequenced through the techniques provided herein, can be subsequently obtained by any method known in the art.

[0105] The CD3 binding domains were combined with different second binding domains, including a mock target binding domain (RSV-G), EGFR binding domain, or cMET binding domain to produce bivalent bispecific IgG antibodies. The bispecific IgG antibodies were generated by transient co-transfection of two plasmid vectors: one encoding an IgG heavy chain with a CD3 binding heavy chain variable (VH) region and the other encoding an IgG heavy chain with a VH region targeting RSV-G, EGFR, or cMET. CH3 engineering technology as described in WO 2013 / 157954 and WO 2013 / 157953 was employed to ensure efficient heterodimerization and formation of bispecific antibodies. Both vectors further encode a common light chain comprising the IGKVl-39 / Jkl light chain variable region. Cell transfection, cell culture, and the harvesting and purification of antibodies was performed by methods known in the art. Further CH3 engineering technologies, for instance as described in WO 2021 / 235936, may be employed to ensure efficient dimerization and formation of bispecific antibodies.

[0106] EXAMPLE 2 - Binding characteristics of RSV-GxCD3 bispecific antibodies

[0107] ELISA

[0108] A panel of RSV-GxCD3 bispecific antibodies was assessed for the ability to bind human CD3 on HEK391 cells engineered to express human CD3 Bio-VLPs, using ELISA. The RSV- GxCD3 bispecific antibodies comprise the same RSV-G binding domain and different CD3 binding domains.

[0109] Plates were coated with HEK293 cells expressing human CD3 Bio-VLPs (Integral Molecular, Cat. nr. INT-2131B), or HEK293 “null” cells (Integral Molecular, Cat. nr. INT- 2128B) that do not express human CD3 Bio-VLPs, at 5 U / well in lx PBS, O / N, at 4°C. Plates were subsequently blocked with 4% skimmed milk (Marvel) in lx PBS. RSV-GxCD3 bispecific antibodies in 1% skimmed milk / lx PBS were added to the cells in 7-step 3-fold dilutions starting at 5000 pg / ml. Secondary antibody only was used as a negative assay control at the two highest concentrations only. Also, only the highest concentration of antibodies was tested on HEK293 “null” Bio-VLPs. Antibodies were incubated at 100 pl / well, at room temperature (RT) for 1 hour. Bound antibodies were detected using goat anti-human IgG (Fc) HRP conjugated (Bethyl Labs, Cat. nr. A80-104P) at 1:2000 in 1% skimmed milk / lx PBS, by incubation at 100 pl / well, at RT for 1 hour. Cells were washed 3X with IxPBS between steps and 8X with IxPBS prior to development. Development was performed by adding 100 pl of TMB solution (eBioscience, Cat. Nr. 00-4201-56) and the reaction was stopped with 100 pl of H2SO4 (Fisher Chemical, Cat. Nr. J / 8430 / 15). Plates were read at O.D. 450 nm.

[0110] The results for two CD3 binding domains are shown in Tables 1 and 2. Bispecific antibody BsAb-RSV-G-A, comprising a CD3 binding domain with a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 1, and bispecific antibody BsAb- RSV-G-B, comprising a CD3 binding domain with a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 8, were able to bind human CD3 but did not bind the cells not expressing human CD3.

[0111] AUC and EC50 values were determined using GraphPad prism software, applying a nonlinear regression fitting, asymmetric (five parameters) equation with robust fit. Bispecific antibody BsAb-RSV-G-A had an EC50 of 0.055 pg / ml and an area under the curve (AUC) of 19.200 in this assay. Bispecific antibody BsAb-RSV-G-B had an EC50 of 0.148 pg / ml and an area under the curve (AUC) of 15.700 in this assay.

[0112] Table 1. O.D. 450 nm values for binding to HEK293 huCD3 Bio-VLPs.

[0113] Table 2. O.D. 450 nm values for binding to HEK293 “null” Bio-VLPs.

[0114] FACS

[0115] The panel of RSV-GxCD3 bispecific antibodies was assessed for the ability to bind to human CD3 endogenously expressed on HPB-ALL cells, using FACS.

[0116] HPB-ALL cells were used at 0.15x106cells / well. Antibodies were added in 8-step semilog (3.16-fold) serial dilutions starting at 10 pg / ml and incubated at 50 pl / well, on ice, for 30 minutes, in FACS buffer (0.5% FBS / EDTA 1 : 1000 / lxPBS). A RSV-GxCD3 control antibody was included as positive assay control. Bound antibodies were detected using goat anti-human IgGPE (Invitrogen, Cat. nr. Hl 0104) at 1: 100 dilution.

[0117] The results are shown in Table 3 and Figure 1. Bispecific antibody BsAb-RSV-G-A, comprising a CD3 binding domain with a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 1, and bispecific antibody BsAb-RSV-G-B, comprising a CD3 binding domain with a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 8, were able to bind human CD3 endogenously expressed on HB-ALL cells. EC50 values were determined using GraphPad prism software, applying a nonlinear regression fitting, asymmetric (five parameters) equation with robust fit. AUC values were determined using GraphPad prism software.

[0118] Table 3. EC50 and AUC values for binding to HBP-ALL cells endogenously expressing human CD3.

[0119] Binding affinity

[0120] The panel of RSV-GxCD3 bispecific antibodies was assessed for their binding affinity to human CD3 using SPR.

[0121] The equipment used was Biacore T200. Chip / channels: CFJB468 and CFJB469 Series S LI chips (GE Healthcare, Cat. nr. 29-1049-93; Fc 1: HEK293 “null” Bio-VLPs; Fc 2: HEK293 huCD3 Bio-VLPs; and Fc 3: no Bio-VLPs (blank)). Running buffer: HBS-N (GE Healthcare, Cat nr. BR-1008-28). Controls: Blank: HBS-N. Analytes: bispecific and control antibodies at 5- step 2-fold serial dilutions starting at 300 nM in HBS-EP+ pH 7.4. Analytes injection, single cycle kinetics: 20 pl of five different concentrations (18.75-300 nM) of antibodies and RSV-G control antibody injected sequentially at 20 pl / min in Fc 1-3, contact time 60 seconds, followed by an off-rate of 60 seconds between samples, and a final off-rate of 300 seconds. The positive control antibody RSV-GxCD3 and the negative control antibody RSV-G were injected twice, in the beginning and at the end of the run. Regeneration: 4M NaCl injected at 30 pl / min in Fc 1-3, contact time 20 seconds, followed by a stabilization period of 90 sec.

[0122] The results are shown in Table 4. Bispecific antibody BsAb-RSV-G-A, comprising a CD3 binding domain with a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 1, has relative high affinity to HEK293 cells expressing human CD3 Bio- VLPs in this assay. Bispecific antibody BsAb-RSV-G-B, comprising a CD3 binding domain with a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 8, has medium affinity in this assay.

[0123] Table 4. Results ofRSV-GxCD3 bispecific antibodies affinity determination towards HEK293 huCD3 Bio-

[0124] VLPs.

[0125] EXAMPLE 3 - CD3 epitope binning

[0126] Several epitope binning studies were performed to group the CD3 binding domains based on competition for binding the same region or epitope of CD3, including using ELISA.

[0127] CD3 proteins AgOl (ACROBiosystems, CDD-H52W1), Ag02 (ACROBiosystems, CDG- H52W9) and Ag03 (ACROBiosystems, CDE- H5223) and Ag04 (produced at UPE), and TT protein Ag05 (AJ Vaccines, 2674) as negative control, were coated as indicated in Table 5.

[0128] Table 5. Overview of ELISA coating conditions.

[0129] The CD3 binding domains were tested in RSV-GxCD3 bispecific antibody format. The ELISA was performed using a 8-step 4-fold antibody dilution series starting at 10 pg / ml. Negative control antibody TT was also included. Epitope bins were made of analyzed antibodies that show similar binding behavior. Results are shown in Table 6. Both bispecific antibody RSV-G-A, comprising a CD3 binding domain with a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 1, and bispecific antibody RSV-G-B comprising a CD3 binding domain with a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 8 bind human CD3Ss and human CD3yS.

[0130] Table 6. Overview of the data from the CD3 epitope binning ELISA.

[0131] EXAMPLE 4 - Binding characteristics of EGFRxCD3 bispecific antibodies

[0132] FACS

[0133] A panel of EGFRxCD3 bispecific antibodies was assessed for their binding ability to CD3 expressing cells, EGFR expressing cells, and cells not expressing CD3 or EGFR.

[0134] Cells: CD3 expressing cells (Jurkat E6, HPB-ALL), EGFR expressing cells (293FF, CH0-K1 EGFR) and cells not expressing CD3 or EGFR (JRT3-T3.5) were used in the assay.

[0135] Antibodies: EGFRxCD3 bispecific antibodies, a positive control EGFR antibody, and a negative control TT antibody were used at a single concentration of 10 pg / ml in a FACS assay using Jurkat E6, 293 FF and JRT3-T3.5 cells. EGFRxCD3 bispecific antibodies, a positive control EGFR antibody, and a negative control TT antibody were titrated in serial 2-fold dilution starting from a concentration of 16 pg / ml in a FACS assay using Jurkat E6, HPB-ALL, and CHO-K1 EGFR cells. Antibodies were incubated at 50 pl / well, for 30 minutes at 2-8°C in dark in FACS buffer (PBS + 0.5% BSA + 2 mM EDTA).

[0136] Detection was performed with antibody Ab0026 (Invitrogen cat#H10104) at a concentration of 3 pg / ml, ratio 1: 100.

[0137] Read-out: The mean fluorescence intensity (MFI) of the stained cell population was plotted as a function of the (logarithm of the) primary antibody concentration used and from the obtained S-shaped curves, the relative affinity (i.e. the concentration of half-maximal signal) of the Fab arms for TCR / CD3 was determined. In addition, area under the curve (AUC) values were determined.

[0138] Results are shown in Tables 7 and 8. These tables show the results of bispecific antibodies comprising the same EGFR binding domain with different CD3 binding domains. The results indicate that bispecific antibody BsAb-EGFR-A, comprising a CD3 binding domain that comprises a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 1, has relative high binding activity to CD3 expressing cells as compared to other CD3 binding domains.

[0139] Table 7. Overview of AUC values measured by FACS on Jurkat E6, 293FF andJRT3-T3.5 cells.

[0140] Table 8. Overview of AUC values measured by FACS on Jurkat E6, HBP-ALL and CHO-K1 EGFR cells. Determination of off-rates

[0141] The panel of EGFRxCD3 bispecific antibodies was assessed for their off-rate in FACS. The FACS assay was set up using historical data to validate the assay conditions.

[0142] Cells: HPB-ALL cells and Jurkat-E6 cells were used in the assay.

[0143] Antibodies: Bispecific antibodies comprising the same EGFR binding arm and different CD3 binding arms were tested. Antibody EGFRxTT was included as negative assay control.

[0144] Assay: Exponentially growing HPB-ALL cells and Jurkat-E6 cells were harvested, spun down at 300 g for 3 minutes and washed once with 1 ml of ice-cold FACS blocking buffer (containing 0.1% (w / v) sodium azide). Cells were resuspended in 300 pl of 25 pg / ml of the bispecific antibodies, diluted in ice-cold FACS blocking buffer, and transferred to a 96-wells deep well plate and incubated for an hour on ice. After incubation, cells were spun down at 300 g for 3 minutes, washed twice with 0.5 ml of ice-cold FACS blocking buffer and resuspended in 0.5 ml of ice-cold FACS blocking buffer. Cells were spun down at 300 g for 3 minutes and resuspended in 0.45 ml of pre-warmed 37°C FACS blocking buffer to ensure that all samples were incubated at 37°C at / for the same time, keeping cells at 37°C with gentle shaking. At time points 30 seconds, 1 minute, 2 minutes, 4 minutes, 6 minutes, 8 minutes and 16 minutes, while samples are kept at 37°C with gentle shaking, 50 pl of the sample was immediately transferred to a pre-cooled 96-wells plate on ice. After incubation, cells were spun down at 300 g for 3 minutes and washed twice with ice-cold FACS blocking buffer. Cells were resuspended in 50 pl of PE- labeled anti-human IgG (Invitrogen H10104 lot: 1947097A -> 0.1 mg / ml) 3 pg / ml in FACS blocking buffer) and incubated for 30 minutes on ice in the dark. Cells were washed by adding 200 pl FACS blocking buffer (as kept on ice) directly to the plate. Cells were pelleted at 300 g for 3 minutes and washed once more. Cells were fixed and fluorescence was measured using the FACS Canto. At least 5000 events in the live gate were measured.

[0145] Read-out: Mean Fluorescence Intensity (MFI) was plotted as a function of time: the signal (MFI) obtained using staining at 4°C was set at 100% and all values were calculated as a percentage of this value.

[0146] Results are shown in Figure 2. Bispecific antibody BsAb-EGFR-A shows a relative high- off rate on HPB-ALL and Jurkat E6 cells in this assay. EXAMPLE 5 - Functional activity of EGFRxCD3 bispecific antibodies

[0147] Reporter assay

[0148] A panel of EGFRxCD3 bispecific antibodies was assessed for the ability to induce T cell activation using a reporter assay. The EGFRxCD3 bispecific antibodies comprise the same EGFR binding domain and different CD3 binding domains.

[0149] Cells: Jurkat-NFAT-RE-luc2 cells (Promega JI 33 A) were used as reporter cells and CHO-K1 stable expressing EGFR cells were used as target cells. Cells were used in an E:T ratio of 1 :1, at 50.000 reporter cells / well.

[0150] Antibodies: EGFRxCD3 bispecific antibodies and control TTxCD3 and EGFRxTT antibodies were used starting at 5000 ng / ml or 200 ng / ml, in a 10-step 5-fold serial dilution. Three control TTxCD3 antibodies were included covering low (TTxCD3-C), medium (TTxCD3- Y) and high (TTxCD3-Z) affinity binding to CD3. A combination of an anti-CD3 antibody (Ab358) and GaM-IgG (Ab351) was included as a positive assay control and antibody GaM-IgG (Ab351) was included as a negative assay control to confirm that the assay works.

[0151] Assay: Target cells were harvested, counted and suspended in assay medium to reach 8 x 106cells / ml. Reporter cells were thawed, gently mixed by pipetting 1 or 2 times and 1 ml was transferred to a 50 ml conical centrifuge tube. 4 ml of RT assay medium was added dropwise to the reporter cells. Reporter cells were counted and suspended in assay medium to reach 4 x 106cells / ml. CHO-EGFR+ target cells were mixed with the reporter cells, resulting in an in E:T ratio of 1 : 1. Antibodies and cells were incubated for 5 hours at 37 °C, 5% CO2.

[0152] Read-out: Reporter activity was quantified by luminescence measurement using the Envision Microplate Reader. Bio-Gio™ Luciferase Assay System (Promega G7941) was thawed protected from light. Assay plates were equilibrated for 10 minutes at room temperature. 50 pl of Bio-Gio™ per well was added and plates were incubate for 5 minutes at room temperature. Relative light unit (RLU) was measured in Envision reader.

[0153] The results are shown in Figure 3A. This data shows that combining an EGFR binding domain with a CD3 binding domain comprising a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 1 is capable of inducing T cell activation. The activity of the EGFRxCD3 bispecific antibodies is higher than that of the control CD3xTT antibodies, indicating that T cell activation is induced by binding to EGFR and CD3, and not merely by binding to CD3 on the T cells.

[0154] Cytotoxicity assay

[0155] The panel of EGFRxCD3 bispecific antibodies was assessed for the ability to induce T cell-mediated cytotoxicity. The EGFRxCD3 bispecific antibodies comprise the same EGFR binding domain and different CD3 binding domains. The experiment was performed in triplicate, including the use of a different T cell donor. The assay described here is a representative example.

[0156] Cells: BxPC3 cells were used as target cells and healthy donor-derived T cells (validated for being >90% viable ) as effector cells, at a E:T ratio of 5: 1 at 20.000 target cells / well.

[0157] Antibodies: EGFRxCD3 bispecific antibodies were tested in a 4-step 10-fold dilution range starting at 4000 ng / ml. Three TTxCD3 antibodies covering low (TTxCD3-C), medium (TTxCD3-Y) and high (TTxCD3-Z) affinity binding to CD3, and a EGFRxTT antibody, were included as controls.

[0158] Assay and readout:

[0159] Cell culture. BxPC3 target cells were cultured at least 10 days and without exceeding passage number 30 before cytotoxicity assays were performed. Cytotoxicity assays were performed on subculture days using cells that show 70-90% confluency.

[0160] Days 0 and 1. Target cells. Cells were harvested using TrypleSelect (Gibco, cat#12563- 029), followed by centrifugation (300 g, 5 min., room temperature), cell count and resuspension in assay media (RPMI1640 (Gibco, 21875-034) + 10% Human AB pooled serum (HS) (Sanquin) for plating at a density of 20,000 cells / well in clear assay plates at room temperature (Flat bottom, clear polystyrene; Greiner, 655180). Antibody dilutions. EGFRxCD3 bispecific antibodies were tested in a 4-step 10-fold dilution range starting at 4000 ng / ml prepared in assay medium. Three TTxCD3 antibodies covering low (TTxCD3-C), medium (TTxCD3-Y) and relative high (TTxCD3-Z) affinity binding to CD3, and a EGFRxTT antibody, were included as controls.

[0161] Tips were changed for each step of serial dilutions. Test antibody dilution plates were stored at room temperature before directly moving to the next step or on ice up to 30 min. The following items were added to the clear assay plates in the order stated below: a) 25 pl of specified test antibody solution b) 25 pl assay medium in wells without test antibodies (no IgG) c) 50 pl of target cells

[0162] 250 pl of PBS were added to the border wells and all empty wells. Cells were allowed to settle for 30 min. at room temperature before the next step. T cells. 1 ml of thawed T cells were transferred to a sterile 50 ml tube, and 9 ml wash medium (RPMI1640 + 10% HI-FBS) were added at room temperature very slowly, drop by drop, to the cells while gently swirling the tube. Following centrifugation (300 g, 5 minutes, room temperature), supernatants were removed and cell pellets were resuspended in the remaining layer of supernatant for a homogeneous cell suspension.

[0163] Following cell counts, cells were resuspended in assay medium at an appropriate density to obtain E:T of 5: 1 (by addition of 25 pl of T cells to the clear assays plates). Cells were allowed to settle and divide over entire well over 30 min. at room temperature, followed by incubation at 37°C, 5% CO2 for 48 hours in a metal incubator box.

[0164] Day 2. CellTiter-Glo® read-out to determine cell lysis of BxPC3 cells CellTiter-Glo® 2.0 (Promega G9242) and assay plates were equilibrated to room temperature before use. T cells and supernatants were collected from assay plates by pipetting up and down for 3 times and transferred into a V-bottom FACS plate and 75 pl of wash medium were added to wells of assay plate. T cell pellets were retrieved by spinning FACS plates at 300 g, 5 min room temperature. 75 pl of CellTiter-Glo® were added to the cells in the wells, mixed to induce lysis (5 minutes on an orbital shaker) and plates were incubated to stabilize luminescent signal (10 minutes at room temperature in the dark). For measurements, 140 pl of lysed cell suspension were transferred to white assay plates carefully to avoid bubble formation, luciferase signal was measured in Envision microplate reader and quantified in relative light units (RLU). Percentage of target cell lysis compared to no IgG control (relative target cell lysis) is calculated as follows:

[0165] Percentage target cell lysis= 100 - ((RLU sample) / (RLU no IgG)) *100%

[0166] Results are shown in Table 9 and Figure 3B. This data shows that combining an EGFR binding domain with a CD3 binding domain comprising a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 1 is capable of inducing T cell-mediated killing of target cells. The activity of the EGFRxCD3 bispecific antibodies is higher than that of the control CD3xTT antibodies, indicating that cytotoxicity is induced by binding to EGFR and CD3, and not merely by binding to CD3 on the T cells. Bispecific antibody BsAB-EGFR-A comprising a CD3 binding domain that comprises a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 1 is a potent antibody in this assay.

[0167] Table 9. AUC values of binding to HBP-ALL and Jurkat E6 cells and T cell-mediated killing of BxPC3 cells.

[0168] Figure 3C shows an overview of the average potency of the EGFR-CD3 antibodies in T- cell mediated killing of BxPC3 target cells over the three experiments plotted against the average off-rate normalized to BsAb-EGFR-C. BsAb-EGFR-A is one of the antibodies with the relative highest lysis activity and relative highest off-rate relative to other antibodies.

[0169] EXAMPLE 6 - Binding and functional characterization of cMETxCD3 bispecific antibodies

[0170] Binding

[0171] A panel of cMETxCD3 bispecific antibodies, all comprising a CD3 binding domain with a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 1, was assessed for the ability to bind cMET on BxPC3 cells and CD3 on HBP-ALL cells, using FACS. The panel showed a diversity in binding affinity to cMET and similar binding to CD3 (data not shown).

[0172] Reporter assay

[0173] The panel of cMETxCD3 bispecific antibodies was assessed for the ability to induce T cell activation, using a reporter assay.

[0174] Cells: Jurkat-NFAT-RE-luc2 cells (Promega JI 33 A) were used as reporter cells and BxPC3 (pancreatic), HCT-116 (colon), MDA-MB-231 (breast), A549 (lung), ES-2 (ovarian), JIMT-1 (breast), RKO (colon), and SK-BR-3 (breast) cancer cell lines were used as target cells. Cells were used at the day of subculture when they were at 70-80% confluency. Cell dissociation buffer was used to harvest the cells to prevent cleavage of surface antigen. Equal amounts of target cells (4 x 106cells / ml) were mixed with reporter cells (4 x 106cells / ml), resulting in an E:T ratio of 1: 1.

[0175] Antibodies: cMETxCD3 bispecific antibodies and positive assay control antibody EGFRxCD3-F were used in a 6-step 20-fold serial dilution starting at a concentration of 4 pg / ml, and negative control antibody (TTxCD3-A) was used in a 4-step 20-fold serial dilution starting at a concentration of 4 pg / ml.

[0176] Assay and readout:

[0177] Test antibody solutions and control antibody solutions were prepared in assay medium (RPMI1640 (Gibco, cat. no. 2187-034) + 10% HI-FBS (Sigma, cat. no. F7524)) in 96-well dilution plates (Cellstar, cat. no. 655180). 96-well (half area) white assay plates (Corning, cat. no. 3688) were filled with 25pl / well of test and control antibodies, and with 25 pl assay medium for the no-IgG wells. All empty wells of the assay plate were filled with 100 pl assay medium and 50 pl in the blank wells. Assay plates were stored at room temperature.

[0178] Target cells were centrifuged at 300 g for 5 minutes, at room temperature. Supernatant was removed, and cells were resuspended in assay medium at a density of 4xl06 / ml, and stored at room temperature.

[0179] Jurkat-NFAT-RE-luc2 cells were thawed using Thawstar and suspended in assay medium by very slowly adding the assay medium drop by drop to the cells while gently swirling the tube. Cells were centrifuged at 150 g, for 5 minutes, at room temperature, and cells were resuspended in assay medium at a density of 4xl06 / ml and stored at room temperature.

[0180] Equal volumes of target cells (4 x 106cells / ml) and Jurkat-NFAT-RE-luc2 reporter cells (4 x 106cells / ml) were mixed in an in E:T ratio of 1 : 1.

[0181] 25 pl / well of the target + reporter cell suspension were added to the assay plates for incubation at 37°C with 5% CO2 for 5 hours. The Steady-Gio® Luciferase Assay System (Promega E2510) and assay plates were equilibrated at room temperature, respectively, 4-5 hours and 10 minutes before use on the day of the assay.

[0182] Read out: RLU (Relative light unit) was measured using an Envision microplate reader; and the stimulation index, defined as the ratio of experimental activity to control activity (no IgG) was calculated.

[0183] The results are shown in Figure 4. This data shows that combining a cMET binding domain with a CD3 binding domain comprising a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 1 is capable of inducing T cell activation. The activity of the cMETxCD3 bispecific antibodies is higher than that of the CD3xTT antibody, indicating that T cell activation is induced by binding to cMET and CD3, and not merely by binding to CD3 on the T cells.

[0184] The data provided in the above Examples support that CD3 binding domains comprising a heavy chain variable region as described herein are useful as binding domains in T cell engager molecules.

[0185] Table 10. HCDR sequences of CD3 binding domain A, comprising a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 1, and CD3 binding domain B, comprising a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 8, according to IMGT and Kabat numbering systems. Both CDS binding domains comprise a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 15. The LCDRs according to IMGT are as set forth in SEQ ID NOs: 16-18. The LCDRs according to Kabat are as set forth in SEQ ID NOs: 19, 20, and 18.

[0186] Table 11. HCDR sequences of CDS binding domain A, comprising a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 1, and CDS binding domain B, comprising a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 8, according to the North numbering system. Both CDS binding domains comprise a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 15. The LCDRs according to North are as set forth in SEQ ID NOs: 52, 53, and 18.

[0187] SEQUENCES

[0188] SEQ ID NO: 1 - Heavy chain variable region

[0189] EVQLVQS GAEVKKPGS S VKVS CKAS GD AFKSKTFTIS WVRQ A PGQGLE WLGGI I P VFGTI TYAQKFQGRVTITADKSTNTAFMELSSLRSEDTAIYYCTRRGKWNPFDPWGPGTLVTVS S

[0190] SEQ ID NO: 2 - Heavy chain CDR1 according to IMGT

[0191] GDAFKSKTFT

[0192] SEQ ID NO: 3 - Heavy chain CDR2 according to IMGT

[0193] IIP VFGTI

[0194] SEQ ID NO: 4 - Heavy chain CDR3 according to IMGT

[0195] TRRGKWNPFDP

[0196] SEQ ID NO: 5 - Heavy chain CDR1 according to Kabat

[0197] SKTFTIS

[0198] SEQ ID NO: 6 - Heavy chain CDR2 according to Kabat

[0199] GIIPVFGTITYAQKFQG

[0200] SEQ ID NO: 7 - Heavy chain CDR3 according to Kabat

[0201] RGKWNPFDP

[0202] SEQ ID NO: 8 - Heavy chain variable region

[0203] EVQLLESGGGMVQPGGSLRLSCAASGFTFSYYDMTWVRQAPGKGLEWVSSISGSGGRT YYADSVKGRFTISRDNPKNTLYLHLNSLRAEDAAVYYCAKRGNWNPFDPWGQGTLVT vss

[0204] SEQ ID NO: 9 - Heavy chain CDR1 according to IMGT GFTFSYYD

[0205] SEQ ID NO: 10 - Heavy chain CDR2 according to IMGT

[0206] ISGSGGRT

[0207] SEQ ID NO: 11 - Heavy chain CDR3 according to IMGT

[0208] AKRGNWNPFDP

[0209] SEQ ID NO: 12 - Heavy chain CDR1 according to Kabat

[0210] YYDMT

[0211] SEQ ID NO: 13 - Heavy chain CDR2 according to Kabat

[0212] SISGSGGRTYYADSVKG

[0213] SEQ ID NO: 14 - Heavy chain CDR3 according to Kabat

[0214] RGNWNPFDP

[0215] SEQ ID NO: 15 - Light chain variable region

[0216] DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSR

[0217] FSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIK

[0218] SEQ ID NO: 16 - Light chain CDR1 according to IMGT

[0219] QSISSY

[0220] SEQ ID NO: 17 - Light chain CDR2 according to IMGT

[0221] AAS

[0222] SEQ ID NO: 18 - Light chain CDR3 according to IMGT, Kabat, North

[0223] QQSYSTPPT

[0224] SEQ ID NO: 19 - Light chain CDR1 according to Kabat RASQSISSYLN

[0225] SEQ ID NO: 20 - Light chain CDR2 according to Kabat

[0226] AASSLQS

[0227] SEQ ID NO: 21 - CL region

[0228] RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ

[0229] DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0230] SEQ ID NO: 22 - V region VK1-39

[0231] DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSR

[0232] FSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTP

[0233] SEQ ID NO: 23 - Light chain variable region

[0234] DIOMTOSPSSLSASVGDRVTITCRASOSISSYLNWYOOKPGKAPKLLIYAASSLQSGVPSR

[0235] FSGSGSGTDFTLTISSLOPEDFATYYCOQSYSTPPITFGOGTRLEIK

[0236] SEQ ID NO: 24 - Light chain CDR3 according to IMGT

[0237] QQSYSTPPIT

[0238] SEQ ID NO: 25 - V region VK3-15

[0239] EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPA

[0240] RFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWP

[0241] SEQ ID NO: 26 - Light chain variable region

[0242] EIVMTOSPATLSVSPGERATLSCRASOSVSSNLAWYOOKPGOAPRLLIYGASTRATGIPA

[0243] RFSGSGSGTEFTLTISSLOSEDFAVYYCOQYNNWPWTFGOGTKVEIK

[0244] SEQ ID NO: 27 - Light chain CDR1 according to IMGT

[0245] QSVSSN SEQ ID NO: 28 - Light chain CDR2 according to IMGT

[0246] GAS

[0247] SEQ ID NO: 29 - Light chain CDR3 according to IMGT

[0248] QQYNNWPWT

[0249] SEQ ID NO: 30 - V region VK3-20

[0250] EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPD

[0251] RFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSP

[0252] SEQ ID NO: 31 - Light chain variable region

[0253] EIVLTOSPGTLSLSPGERATLSCRASOSVSSSYLAWYOQKPGOAPRLLIYGASSRATGIPD

[0254] RFSGSGSGTDFTLTISRLEPEDFAVYYCOQYGSSPWTFGOGTKVEIK

[0255] SEQ ID NO: 32 - Light chain CDR1 according to IMGT

[0256] QSVSSSY

[0257] SEQ ID NO: 33 - Light chain CDR3 according to IMGT

[0258] QQYGSSPWT

[0259] SEQ ID NO: 34 - V region VL3-21

[0260] SYVLTQPPSVSVAPGETARITCGGDNIGRKSVYWYQQKSGQAPVLVIYYDSDRPSGIPER

[0261] FSGSNSGNTATLTISRVEAGDEADYYCQVWDGSSDH

[0262] SEQ ID NO: 35 - Light chain variable region

[0263] SYVLTOPPSVSVAPGETARITCGGDNIGRKSVYWYOOKSGOAPVLVIYYDSDRPSGIPER

[0264] FSGSNSGNTATLTISRVEAGDEADYYCQVWDGSSDHWVFGGGTKLTVL

[0265] SEQ ID NO: 36 - Light chain CDR1 according to IMGT

[0266] NIGRKS SEQ ID NO: 37 - Light chain CDR2 according to IMGT

[0267] YDS

[0268] SEQ ID NO: 38 - Light chain CDR3 according to IMGT

[0269] QVWDGSSDHWV

[0270] SEQ ID NO: 39 - hinge region

[0271] EPKSCDKTHTCPPCP

[0272] SEQ ID NO: 40 - CHI region

[0273] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS

[0274] GLYSLS S V VT VPS S SLGTQTYICNVNHKPSNTKVDKRV

[0275] SEQ ID NO: 41 - CH2 region

[0276] APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT

[0277] KPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK

[0278] SEQ ID NO: 42 - CH2-DM region

[0279] APELGRGPSVFLFPPI<PI<DTLMISRTPEVTCVVVDVSHEDPEVI<FNWYVDGVEVHNAI<T

[0280] KPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK

[0281] SEQ ID NO: 43 - CH3 region

[0282] GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD

[0283] SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0284] SEQ ID NO: 44 - CH3-DE region

[0285] GQPREPQVYTDPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTPPVLD

[0286] SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0287] SEQ ID NO: 45 - CH3-KK region GQPREPQVYTKPPSREEMTKNQVSLKCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD

[0288] SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0289] SEQ ID NO: 46 - Heavy chain CDR1 according to North KASGDAFKSKTFTIS

[0290] SEQ ID NO: 47 - Heavy chain CDR2 according to North GIIPVFGTIT

[0291] SEQ ID NO: 48 - Heavy chain CDR3 according to North TRRGKWNPFDP

[0292] SEQ ID NO: 49 - Heavy chain CDR1 according to North AASGFTFSYYDMT

[0293] SEQ ID NO: 50 - Heavy chain CDR2 according to North SISGSGGRTY

[0294] SEQ ID NO: 51 - Heavy chain CDR3 according to North AKRGNWNPFDP

[0295] SEQ ID NO: 52 - Light chain CDR1 according to North RASQSISSYLN

[0296] SEQ ID NO: 53 - Light chain CDR2 according to North YAASSLQS

Claims

CLAIMS1. A polypeptide comprising the amino acid sequence as set forth in SEQ ID NO: 1 or 8, or a variant thereof.

2. A method for producing a variant of a polypeptide of claim 1 , wherein the method comprises:- making a modification in the amino acid sequence as set forth in SEQ ID NO: 1 or 8.

3. A polypeptide obtainable by the method according to claim 2.

4. A CD3 binding domain comprising a polypeptide according to claim 1.

5. The CD3 binding domain according to claim 4, wherein the CD3 binding domain further comprises a polypeptide comprising the amino acid sequence as set forth in SEQ ID NO: 15.

6. A CD3 binding domain comprising a heavy chain variable region comprising the heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence selected from SEQ ID NO: 1 and 8, wherein each of the HCDRs may comprise at most three, two, or one amino acid variations.

7. The CD3 binding domain according to claim 6, wherein the CD3 binding domain comprises a heavy chain variable region comprising: a) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively, or heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequence as set forth in SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively; orb) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequence as set forth in SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively, or heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequence as set forth in SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively, wherein each of the HCDRs may comprise at most three, two, or one amino acid variations.

8. The CD3 binding domain according to claim 6 or 7, wherein the CD3 binding domain comprises a heavy chain variable region having the amino acid sequence as set forth in any one of SEQ ID NO: 1 or 8, or having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto.

9. The CD3 binding domain according to any one of claims 6-8, wherein the CD3 binding domain comprises a light chain variable region comprising the light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) of a light chain variable region having the amino acid sequence as set forth in SEQ ID NO: 15, wherein each of the LCDRs may comprise at most three, two, or one amino acid variations.

10. The CD3 binding domain according to any one of claims 6-9, wherein the CD3 binding domain comprises a light chain variable region comprising light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), having an amino acid sequence as set forth in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively, or light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), having an amino acid sequence as set forth in SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 18, respectively, wherein each of the LCDRs may comprise at most three, two, or one amino acid variations.

11. The CD3 binding domain according to any one of claims 6-10, wherein the CD3 binding domain comprises a light chain variable region having the amino acid sequence as setforth in SEQ ID NO: 15, or having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto.

12. A method for producing a variant of a CD3 binding domain of any one of claims 6-11, wherein the method comprises:- making a modification in the amino acid sequence as set forth in any one of SEQ ID NO: 1 or 8, and / or in the amino acid sequence as set forth in SEQ ID NO: 15;- testing the modified CD3 binding domain for binding to human wildtype and / or mutant CD 3; and- selecting the modified CD3 binding domain if it binds to human CD3.

13. A CD3 binding domain obtainable by the method according to claim 12.

14. A binding moiety comprising a CD3 binding domain according to any one of claims 4-11, or 13.

15. The binding moiety according to claim 14, wherein the binding moiety is a monospecific binding moiety, in particular a bivalent monospecific antibody.

16. The binding moiety according to claim 14, wherein the binding moiety is a multispecific binding moiety, in particular a bivalent bispecific antibody, a trivalent bispecific antibody, or a trivalent trispecific antibody.

17. A binding moiety that binds CD3, wherein the binding moiety competes with a binding moiety of any one of claims 14-16 for binding to CD3.

18. A pharmaceutical composition comprising an effective amount of the binding moiety according to any one of claims 14-17, and a pharmaceutically acceptable carrier.

19. The binding moiety according to any one of claims 14-17, or the pharmaceutical composition according to claim 18, for use in therapy.

20. The binding moiety according to any one of claims 14-17, or the pharmaceutical composition according to claim 18, for use in the treatment of cancer.

21. A method for treating a disease, comprising administering an effective amount of a binding moiety according to any one of claims 14-17, or the pharmaceutical composition according to claim 18, to a subject in need thereof.

22. A method for treating cancer, comprising administering an effective amount of a binding moiety according to any one of claims 14-16, or the pharmaceutical composition according to claim 18, to a subject in need thereof.

23. A nucleic acid sequence encoding a heavy chain variable region as defined in any one of claims 6-8.

24. A vector comprising a nucleic acid sequence as claimed in claim 23.

25. The vector according to claim 24, wherein the vector further comprises a nucleic acid sequence encoding a CHI region, in particular encoding a CHI region, hinge, CH2 region, and CH3 region.

26. The vector according to claim 24 or 25, wherein the vector further comprises at least one nucleic acid sequence encoding a light chain variable region, in particular encoding a light chain variable region and a CL region.

27. The vector according to claim 26, wherein the light chain variable region is a light chain variable region of a light chain that is capable of pairing with multiple heavy chains having different epitope specificities.

28. A cell comprising a nucleic acid sequence encoding the heavy chain variable region of a CD3 binding domain as defined in any one of claims 6-8.

29. The cell according to claim 28, wherein the cell further comprises a nucleic acid sequence encoding a CHI region, in particular a nucleic acid sequence encoding a CHI region, hinge, CH2 region, and CH3 region.

30. The cell according to claim 28 or 29, wherein the cell further comprises at least one nucleic acid sequence encoding a light chain variable region, in particular a light chain variable region as defined in any one of claims 9-11, in particular a light chain variable region as defined in any one of claims 9-11 and a CL region.

31. A cell producing a binding moiety as claimed in any one of claims 14-16.

32. The cell according to claim 31, wherein the cell is a recombinant cell comprising the vector as claimed in any one of claims 24-27.

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