CD3-binding polypeptide constructs

Abstract

The present invention relates to a polypeptide or polypeptide construct comprising a binding domain that binds to an extracellular epitope of the human CD3 epsilon chain, comprising or consisting of a VH region and a VL region, wherein i) the VH region has a CDR-H1 sequence of X1YAX2N (wherein X1 is K, V, S, G, R, T, or I; and X2 is M or I); RIRSKYNNYATYYADX1VKX2 (wherein X1 is S or Q; and X2 is D, G, K, S, or E); and a CDR-H3 sequence of HX1NFGNSYX2SX3X4AY (wherein X1 is G, R, or A; X2 is I, L, V, or T; X3 is Y, W, or F; and X4 is W, F, or Y); ii) the VL region comprises a CDR-H3 sequence of X1SSTGAVTX2X3X4YX5N (wherein X1 is G, R, or A; X2 is S or T; and X3 is W, F, or Y). X4 is G or S; X4 is N or Y; and X5 is P or A); CDR-L2 sequences of X1TX2X3X4X5X6 (wherein X1 is G or A; X2 is K, D, or N; X3 is F, M, or K; X4 is L or R; X5 is A, P, or V; and X6 is P or S); and X1LWYSNX2WV (wherein X1 is V, A, or T; and and X2 is R or L); iii) one or more of the CDR sequences of the VH region of i) and / or the VL region of ii) comprise one or a combination of amino acid substitutions selected from X24V and X24F in CDR-H1; D15 and X116A in CDR-H2; H1, X12E, F4, and N6 in CDR-H3; and X11L and W3 in CDR-L3. The present invention also relates to polynucleotides encoding the polypeptides or polypeptide constructs of the present invention, vectors comprising said polynucleotides, and host cells transformed or transfected with said polynucleotides or vectors. Furthermore, the present invention also provides processes for producing said polypeptides or polypeptide constructs and pharmaceutical compositions comprising said polypeptides or polypeptide constructs of the present invention. Furthermore, the present invention relates to medical uses of said polypeptides or polypeptide constructs and kits comprising said polypeptides or polypeptide constructs.

Description

[Technical Field]

[0001] The present invention relates to a polypeptide or polypeptide construct comprising a binding domain that binds to an extracellular epitope of the human CD3 epsilon chain, comprising or consisting of a VH region and a VL region, wherein i) the VH region has a CDR-H1 sequence of X1YAX2N (wherein X1 is K, V, S, G, R, T, or I; and X2 is M or I); RIRSKYNNYATYYADX1VKX2 (wherein X1 is S or Q; and X2 is D, G, K, S, or E); and a CDR-H3 sequence of HX1NFGNSYX2SX3X4AY (wherein X1 is G, R, or A; X2 is I, L, V, or T; X3 is Y, W, or F; and X4 is W, F, or Y); ii) the VL region comprises a CDR-H3 sequence of X1SSTGAVTX2X3X4YX5N (wherein X1 is G, R, or A; X2 is S or T; and X3 is W, F, or Y). X4 is G or S; X4 is N or Y; and X5 is P or A); CDR-L2 sequences of X1TX2X3X4X5X6 (wherein X1 is G or A; X2 is K, D, or N; X3 is F, M, or K; X4 is L or R; X5 is A, P, or V; and X6 is P or S); and X1LWYSNX2WV (wherein X1 is V, A, or T; and and X2 is R or L); iii) one or more of the CDR sequences of the VH region of i) and / or the VL region of ii) comprise one or a combination of amino acid substitutions selected from X24V and X24F in CDR-H1; D15 and X116A in CDR-H2; H1, X12E, F4, and N6 in CDR-H3; and X11L and W3 in CDR-L3. The present invention also relates to polynucleotides encoding the polypeptides or polypeptide constructs of the present invention, vectors comprising said polynucleotides, and host cells transformed or transfected with said polynucleotides or vectors. Furthermore, the present invention also provides processes for producing said polypeptides or polypeptide constructs and pharmaceutical compositions comprising said polypeptides or polypeptide constructs of the present invention. Furthermore, the present invention relates to medical uses of said polypeptides or polypeptide constructs and kits comprising said polypeptides or polypeptide constructs. [Background technology]

[0002] T cell engagers (TCEs) exploit the ability of T cells to recognize foreign peptides on mutated or infected cells via the T cell receptor. T cell recognition is mediated by clonotypic alpha-beta and gamma-delta T cell receptors (TcRs), which interact with peptide-loaded molecules of peptide-MHC (pMHC) with low affinity. The antigen-specific chain of the TcR lacks a signaling domain and instead binds to the conserved multisubunit signaling apparatus CD3. The mechanism by which TcR ligation is directly transmitted to the signaling apparatus is of fundamental interest in T cell biology. Briefly, it is the CD3 subunit that converts cell surface antigen binding into an intracellular phosphorylation signaling cascade. These phosphorylation events result in the activation of transcription factors such as NFAT and NFkB, which lead to the increased expression of cytokines and effector proteins such as granzymes and perforin. It is this feature that TCEs exploit by simultaneously binding target antigens on tumor cells and CD3 on T cells to form an artificial immune synapse, ultimately leading to the destruction of cells expressing the target antigen.

[0003] From a clinical perspective, TCEs not only need to exhibit efficient cell-killing activity, but also other desirable properties, such as a manageable side effect profile. However, these aspects are not the only considerations for the development of TCEs. Another area important for drug development is the pre-patient stage, i.e., properties of concern for the TCE outside the patient's body, such as shelf life, scalability of production, solubility, stability, and ease of formulation. In other words, a key feature from a pharmaceutical perspective is the developability of a given TCE, or portions thereof, to address specific needs, such as in drug manufacturing or administration. Bringing together requirements from clinical and pharmacological perspectives is critical for the advancement of TCE platforms in the context of commercialization. Given the biological complexity of having a TCE that induces immune synapse formation, engineering a TCE with respect to other properties, such as shelf life, scalability of production, solubility, stability, and ease of formulation, while maintaining its effectiveness in mediating target cell killing, can be challenging.

[0004] The thermal stability of TCE is an important feature both inside and outside the patient's body. Higher thermal stability of TCE reduces the formation of high molecular weight aggregates, which in turn provides TCE that is active for a long time in the body but is more stable during storage. Furthermore, the reduction of high molecular weight aggregates also leads to a more favorable immunogenic profile of TCE upon administration, thereby reducing the risk of side effects. Summary of the Invention [Problem to be solved by the invention]

[0005] Traditionally, the VH and VL domains that constitute the anti-target and anti-CD3 binding domains of TCEs have been the focus of efforts to improve stability. This is particularly true for the Fvs that are part of TCEs. Hydrophobic interactions between VH and VL are thought to strongly determine Fv stability. In many Fvs, the interaction may not be strong enough to ensure stability for in vitro and / or in vivo applications. This has led to the coupling of the V domains of Fvs with linkers, resulting in single-chain Fvs (scFvs). While linkers have been shown to be effective in holding the V domains (variable domains) together, they are not always effective in keeping scFvs active or sufficiently active for a given application, and thus further stability improvements are needed. One approach to enhancing stability is to increase the interaction between the two V domains, thereby increasing the stability of biologically active Fvs that remain target-binding monomers. While approaches to improving the stability of scFvs have been described in the art, there remains a need in the art to improve the stability of Fvs that are part of TCEs. [Means for solving the problem]

[0006] The present invention relates to a polypeptide or polypeptide construct comprising a binding domain that binds to an extracellular epitope of the human CD3 epsilon chain, the binding domain comprising or consisting of a VH region and a VL region, i) the VH region is a CDR-H1 sequence of X1YAX2N (wherein X1 is K, V, S, G, R, T, or I; and X2 is M or I); CDR-H2 sequence of RIRSKYNNYATYYADX1VKX2 (wherein X1 is S or Q; and X2 is D, G, K, S, or E); and CDR-H3 sequence of HX1NFGNSYX2SX3X4AY (wherein X1 is G, R, or A; X2 is I, L, V, or T; X3 is Y, W, or F; and X4 is W, F, or Y). Includes; ii) the VL region is a CDR-L1 sequence of X1SSTGAVTX2X3X4YX5N (wherein X1 is G, R, or A; X2 is S or T; X3 is G or S; X4 is N or Y; and X5 is P or A); a CDR-L2 sequence of X1TX2X3X4X5X6 (wherein X1 is G or A; X2 is K, D, or N; X3 is F, M, or K; X4 is L or R; X5 is A, P, or V; and X6 is P or S); and CDR-L3 sequence of X1LWYSNX2WV (wherein X1 is V, A, or T; and X2 is R or L) Includes; iii) one or more CDR sequences of the VH region of i) and / or the VL region of ii) CDR-H1 X24V and X24F; D15 (preferably E) and X116A of CDR-H2; H1 (preferably A or N), X12E, F4 (preferably I), and N6 (preferably S or T) of CDR-H3; and X11L and W3 (preferably Y) of CDR-L3 The present invention relates to a polypeptide or polypeptide construct comprising one or more amino acids selected from:

[0007] The term "polypeptide construct" (alternatively referred to herein as "compound") refers to an antigen-binding (or epitope-binding) molecule that contains the paratope-containing domain itself. In the context of the present invention, a polypeptide construct is understood as an organic polymer that does not occur in nature and that contains at least one continuous, unbranched amino acid chain that has been engineered. An example of a polypeptide construct that is a single polypeptide is a BiTE® molecule, which contains a core structure that contains at least one functional target-binding domain together with at least one complete, functional CD3-binding domain on a single polypeptide chain; these domains are directly linked by a flexible peptide ("linker") without any intervening domains, unlike, for example, Xmab, which contains a target-binding factor and a CD3-binding factor on different polypeptide chains. In the context of the present invention, such polypeptide constructs that contain multiple amino acid chains are also envisioned. While the term "polypeptide" is preferably used in connection with single-chain forms of the compounds of the present invention, "polypeptide construct" may also be more appropriate to describe polypeptides that contain multiple polypeptide chains, for example, two, three, or four polypeptide chains. Furthermore, the term "polypeptide construct" is also suitable for describing compounds of the present invention that contain one or more non-amino acid-based components, such as human serum albumin (HSA). The amino acid chain of a polypeptide typically contains at least 50 amino acids, preferably at least 100, 200, 300, 400, or 500 amino acids. In the context of the present invention, it is also envisaged that the amino acid chain of the polymer is linked to an entity that is not composed of amino acids.

[0008] The polypeptide comprises structural and / or functional features based on the structure and / or function of an antibody, e.g., a full-length immunoglobulin molecule. Thus, the polypeptide construct specifically and preferably selectively or immunospecifically binds to its target or antigen, more precisely to an epitope of said target or target antigen, and / or comprises a heavy chain variable region (VH) and / or a light chain variable region (VL) naturally found in an antibody or domains derived from said antibody. Thus, the construct may alternatively be considered to comprise a paratope-like structure and an epitope-binding structure as found in a naturally occurring antibody or fragment thereof. The polypeptide construct according to the present invention comprises the minimal structural requirements of an antibody capable of immunospecific target binding, i.e., a paratope that immunospecifically or immunoselectively recognizes an epitope on a target antigen, unless otherwise specified. This minimum requirement can be defined, for example, by the presence of at least three light chain CDRs (i.e., CDR1, CDR2, and CDR3 of the VL region, also referred to as CDR-L1, CDRL2, and CDR-L3) and / or three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3 of the VH region, also referred to as CDR-H1, CDR-H2, and CDR-H3), preferably all six CDRs. Thus, a polypeptide construct may be characterized by the presence of three or six CDRs in the binding domain, and the skilled artisan will know where (and in what order) these CDRs are located within the paratopic binding structure. In the context of a CD3-binding domain of a polypeptide or polypeptide construct, said paratopic binding structure is identified as a binding domain characterized by the presence of a VH region and a VL region comprising the CDRs. Thus, a polypeptide / polypeptide construct according to the invention comprises at least one paratopic binding structure which is a binding domain that selectively, immunospecifically and / or immunoselectively binds to an extracellular epitope of the human CD3ε (also referred to herein as CD3ε or "CD3 epsilon") chain comprising a VH region and a VL region with CDRs. Thus, a polypeptide / polypeptide construct according to the invention comprises a paratope that immunospecifically and / or immunoselectively binds to human CD3ε.The term "CDR" and its plural "CDRs" refer to the complementarity-determining regions, three of which constitute the binding properties of the light chain variable region (CDR-L1, CDR-L2, and CDR-L3) and three of which constitute the binding properties of the heavy chain variable region (CDR-H1, CDR-H2, and CDR-H3). CDRs contain most of the residues of an antibody (or construct or binding domain) responsible for specific interactions with the antigen and therefore contribute to the functional activity of an antibody molecule; CDRs are the primary determinants of antigen specificity. The exact definition of CDR boundaries and lengths depends on different classification and numbering systems. Thus, CDRs may be referred to by any other boundary definition, including Kabat, Chothia, contact, or any numbering system described herein. Despite the different boundaries, each of these systems has some overlap in what constitutes the so-called "hypervariable regions" within the variable sequences. Therefore, CDR definitions according to these systems may differ in length and boundary regions with respect to the adjacent framework regions. For example, Kabat (an approach based on interspecies sequence variability), Chothia (an approach based on crystallographic studies of antigen-antibody complexes), and / or MacCallum (Kabat et al., supra; Chothia et al., J. Mol. Biol., 1987, 196:901-917; and MacCallum et al., J. Mol. Biol., 1996, 262:732). Yet another standard for characterizing antigen-binding sites is the AbM definition used by Oxford Molecular's AbM antibody modeling software. See, for example, Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Antibody Engineering Lab Manual (Eds.: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg). Two residue identification techniques can be combined to define hybrid CDRs, as long as they define overlapping but non-identical regions. However, numbering according to the so-called Kabat system is preferred.The term "antigen-binding structure" as used herein refers to any polypeptide / polypeptide construct comprising an antigen-binding structure or any molecule having binding activity for a specific target antigen. The antigen-binding structure or antigen-binding molecule is not limited to those derived from a living organism and may be, for example, a polypeptide derived from an artificially designed sequence. These antigen-binding structures or antigen-binding molecules may be any natural polypeptide, synthetic polypeptide, recombinant polypeptide, etc. Because the antigen-binding structure according to the present invention specifically binds to a portion of an antigen, i.e., specifically binds to an epitope of CD3ε, the antigen (epitope)-binding structure may also be broadly defined herein as a "paratope structure." Therefore, the polypeptide / polypeptide construct according to the present invention may also be defined as a domain comprising a paratope that preferably immunospecifically or immunoselectively binds to a target antigen / target epitope; in certain embodiments comprising at least one additional paratope, it preferably immunospecifically or immunoselectively binds to an additional, different, or the same target antigen / target epitope. Thus, whenever the specification refers to a domain of a construct or molecule of the invention, the construct comprises at least one paratopic structure (or paratope) that binds to human CD3ε, as specified herein, in particular by any one of the appended claims. In certain embodiments, said construct comprises at least one further paratope that also binds to human CD3ε or a different target antigen as defined herein.

[0009] The term "antibody" as used in accordance with the present invention includes full-length antibodies, including camelid antibodies and other immunoglobulins produced by biotechnological or protein engineering methods or processes. These full-length antibodies may be, for example, monoclonal antibodies, recombinant antibodies, chimeric antibodies, deimmunized antibodies, humanized antibodies and human antibodies, as well as antibodies from other species such as mouse, hamster, rabbit, rat, goat or non-human primate.

[0010] The "polypeptides / polypeptide constructs" of the present invention may also comprise the structure of naturally occurring full-length immunoglobulins. For example, an antibody construct may comprise (at least) two full-length antibody heavy chains and two full-length antibody light chains. However, given that the polypeptides / polypeptide constructs according to the present invention preferably comprise a linker connecting the VH and VL regions of the CD3-binding domain, preferably resulting in an scFv, and / or in other embodiments at least one additional binding domain comprising a paratope, they do not occur in nature and their function differs significantly from naturally occurring products. For this reason, the polypeptides or polypeptide constructs of the present invention are preferably artificial "composite" molecules comprising scFvs and / or, in some embodiments, distinct paratopes / binding domains with different specificities and / or selectivities.

[0011] As mentioned above, the polypeptides of the present invention may comprise more than one polypeptide chain; i.e., polypeptides comprising two or more polypeptide chains, particularly polypeptides that form three-dimensional protein-like structures capable of immunospecific binding to CD3ε, are also contemplated by the present invention. Thus, the definition of the term "polypeptide construct" includes molecules consisting of only one polypeptide chain as well as molecules consisting of two, three, four, or more polypeptide chains, which may be identical (homodimers, homotrimers, or homooligomers) or different (heterodimers, heterotrimers, or heterooligomers). Examples of the above-identified antibodies and their fragments, variants, derivatives, and constructs derived therefrom are described, inter alia, in Harlow and Lane, Antibodies: A Laboratory Manual, CSHL Press (1988); Kontermann and Duebel, Antibody Engineering, Springer, 2nd ed. 2010; and Little, Recombinant Antibodies for Immunotherapy, Cambridge University Press 2009.

[0012] "Polypeptides / polypeptide constructs" of the invention may also comprise fragments of full-length antibodies, such as VH, VHH, VL, (s)dAb, Fv, light chain (VL-CL), Fd (VH-CH1), heavy chain, Fab, Fab', F(ab')2 or "rIgG" (a "half antibody" consisting of a heavy and light chain), which are defined as comprising the VH and VL regions, although not all of the above mentioned fragments are applicable to the CD3ε binding domain, but are applicable to embodiments relating to at least one further binding domain. Polypeptides / polypeptide constructs according to the invention may also comprise modified fragments of antibodies, also called antibody variants or antibody derivatives. Examples include scFv, di-scFv or bis(s)-scFv, scFv-Fc, scFv-zipper, scFab, Fab2, Fab3, diabodies, single chain diabodies, tandem diabodies (Tandab), tandem di-scFv, tandem tri-scFv, structures such as: (VH-VL-CH3)2, (scFv-CH3)2, ((scFv)2-CH3+CH3), ((scFv)2-CH3) or (scFv-CH3-scFv)2, multibodies such as triabodies or tetrabodies, and VHH, VH or VL, optionally and preferably "minibodies" embodied by structures that are single domain antibodies, such as nanobodies or single variable domain antibodies, comprising just one variable region that specifically binds to an antigen or target independently of other variable regions or domains, but are defined as comprising a VH and a VL region, and therefore not all of the above fragments are applicable to the CD3ε binding domain, but are applicable to embodiments involving at least one additional binding domain. Further possible formats of polypeptides / polypeptide constructs according to the present invention are crossbodies, maxibodies, hetero-Fc constructs, mono-Fc constructs and scFc constructs. Examples of these formats are described herein below.

[0013] Furthermore, the definition of the term "polypeptide construct" includes bivalent and polyvalent / multivalent polypeptides / polypeptide constructs as well as bispecific and multispecific / multispecific polypeptides / polypeptide constructs that selectively and preferably specifically bind to two, three or more antigen structures (epitopes) via different binding domains. For example, if a polypeptide construct has two binding domains for one target (CD3 epsilon) and one binding domain for another target, such as those described herein below, or vice versa, this polypeptide construct may have more valency than specificity; in either case, this polypeptide construct is trivalent and bispecific. Generally, the term "bispecific" includes the meaning that the polypeptide construct binds to at least two different antigens, such as said CD3ε and a further target, such as those specified herein below.

[0014] The terms "paratope," "antigen-binding domain," "epitope-binding domain," "binding domain," or "domain binding to" in the context of the present invention characterize a domain of a construct that selectively and preferably specifically or immunospecifically binds to / interacts with / recognizes an epitope on a target or on an antigen (here CD3). The terms "binding domain," or "domain binding to," or "domain" in the context of the present invention characterize a domain of a construct that immunospecifically binds to / interacts with / recognizes an epitope on a target or on an antigen. The structure and function of the CD3ε-binding domain (also referred to as the first binding domain in the case of a polypeptide / polypeptide construct comprising further, and consequently second, third, etc., binding domains), and preferably also the structure and / or function of any further binding domains (e.g., binding to cell surface antigens, e.g., tumor antigens, etc.), are based on the structure and / or function of an antibody, e.g., a full-length immunoglobulin polypeptide. Thus, a "binding domain" or "domain that binds to" may comprise the minimum structural requirements of an antibody that enable immunospecific target binding. While the structural requirements of a CD3ε-binding domain are specified as including a VH and a VL region with three corresponding CDRs per region, the minimum structural requirements for any additional binding domain may be defined, for example, by the presence of at least three light chain CDRs (i.e., CDR1, CDR2, and CDR3 of the VL region) and / or three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3 of the VH region), preferably all six CDRs. A "domain that binds to" (or "binding domain") typically comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), but need not comprise both and may comprise only a VH or a VL. Fd fragments, for example, often retain some antigen-binding function of an intact antigen-binding domain.The terms "paratope," "antigen-binding structure," and "epitope-binding structure," as used herein, also refer to a portion of an antibody (or molecule according to the invention) comprising a region that specifically binds to and is complementary to all or a portion of an antigen or a portion thereof; i.e., the antibody can bind only to a specific portion of the antigen. The specific portion is called an "epitope." The antigen-binding domain can be provided by one or more antibody variable domains. Preferably, the antigen-binding domain contains an antibody variable region comprising both an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). Such preferred antigen-binding domains include, for example, "single-chain Fv (scFv)," "single-chain antibody," "Fv," "single-chain Fv2 (scFv2)," "Fab," and "F(ab')2." The CD3ε-binding domain is preferably in the form of an scFv.

[0015] Examples of formats of a "domain that binds to" or "domain containing a paratope" (or "binding domain", "antigen-binding structure", "epitope-binding structure") include, but are not limited to, a full-length antibody, a fragment of a full-length antibody (e.g., VH, VHH, VL), a (s)dAb, an Fv, a light chain (VL-CL), an Fd (VH-CH1), a heavy chain, Fab, Fab', F(ab')2, or a "r" fragment, unless otherwise defined. IgG" ("half antibodies"), antibody variants or antibody derivatives such as scFv, di-scFv or bi(s)-scFv, scFv-Fc, scFv-zipper, scFab, Fab2, Fab3, diabodies, single chain diabodies, tandem diabodies (Tandab's), tandem di-scFv, tandem tri-scFv, "minibodies" (selected from formats such as (VH-VL-CH3)2, (scFv-CH3)2, ((scFv)2-CH3+CH3), ((scFv)2-CH3) or (scFv-CH3-scFv)2), multibodies (e.g. triabodies or tetrabodies) and single domain antibodies (e.g. nanobodies or single variable domain antibodies comprising only one variable region which may be VHH, VH or VL). Further examples of formats of "domains that bind to" (or "binding domains") include: (1) antibody fragments or antibody variants (e.g., Fab) comprising a VL, VH, CL, and CH1; (2) antibody fragments or antibody variants (e.g., F(ab')2) comprising two linked Fab fragments; (3) antibody fragments or antibody variants (e.g., Fd) comprising a VH and CH1; (4) antibody fragments or antibody variants (e.g., light chains) comprising a VL and CL; (5) antibody fragments or antibody variants (e.g., Fv) comprising a VL and VH; (5) dAb fragments having a VH domain (Ward et al., (1989) Nature 341:544-546); (6) antibody variants comprising at least three isolated CDRs of the heavy and / or light chain; and (7) single-chain Fvs (scFvs).Examples of embodiments of constructs or binding domains according to the invention are described, for example, in WO 00 / 006605, WO 2005 / 040220, WO 2008 / 119567, WO 2010 / 037838, WO 2013 / 026837, WO 2013 / 026833, US 2014 / 0308285, US 2014 / 0302037, WO 2014 / 144722, WO 2014 / 151910 and WO 2015 / 048272. In the context of the present invention, a paratope is understood as an antigenic site which is part of a polypeptide as described herein and which recognizes and binds to an antigen. A paratope is typically a small region of at least about five amino acids. As understood herein, a paratope typically comprises a portion of the heavy chain (VH) and light chain (VL) sequences derived from an antibody. Each binding domain of the polypeptide of the present invention has a paratope comprising a set of six complementarity-determining regions (CDR loops), three of which are contained within the VH and VL sequences derived from an antibody.

[0016] It is envisaged for the compounds, in particular the constructs of the invention, that a) the construct is a single-chain polypeptide or a single-chain construct, b) the CD3ε binding domain is in the format of an scFv, c) any further, e.g., second binding domain and / or third domain is in the format of an scFv, d) the first and said further, e.g., said second and / or third domain are linked via a linker, preferably a peptide linker, more preferably a glycine / serine or glycine / glutamine linker, and / or e) the construct comprises a domain that confers an extended serum half-life, e.g., an Fc-based domain or human serum albumin (HSA). In the latter case, the term "polypeptide construct" is a preferred embodiment that makes clear that it comprises more than a single peptide chain. A preferred serum half-life extending Fc-based domain (also referred to as an "HLE" domain) comprises two polypeptide monomers, each comprising a hinge, a CH2 domain, and a CH3 domain, fused together via a peptide linker (see, e.g., SEQ ID NOs: 18 and 19); the format, from N-terminus to C-terminus, is hinge-CH2-CH3-linker-hinge-CH2-CH3.

[0017] The construct of the present invention is preferably an "in vitro-generated construct" and / or a "recombinant construct." In the context of the present invention, the term "in vitro-generated" refers to a construct according to the above definition in which all or part of the binding domain or variable region (e.g., at least one CDR) has been generated by non-immune cell selection on a protein chip, such as in vitro phage display, or any other method that allows testing of candidate amino acid sequences for their antigen-binding ability. Thus, the term preferably excludes sequences generated solely by genome rearrangement in an animal's immune cells. It is envisioned that the first and / or second domain of the construct can be produced or obtained by phage display or library screening methods, rather than by grafting CDR sequences from a pre-existing (monoclonal) antibody onto a scaffold. A "recombinant construct" is (among other things) a construct generated or manufactured using recombinant DNA technology or genetic engineering.

[0018] The constructs of the present invention are contemplated to be monoclonal. As used herein, a polypeptide or construct designated "monoclonal" (mAb) is obtained from a population of substantially homogeneous antibodies / constructs, i.e., the individual antibodies / constructs within the population are identical (particularly with respect to their amino acid sequence) except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in minor amounts. Monoclonal antibodies / constructs are highly specific, directed against a single epitope within an antigen, in contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (or epitopes). In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by hybridoma culture and are therefore uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody / construct as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method.

[0019] For the preparation of monoclonal antibodies, any technique that provides antibodies produced by continuous cell line cultures can be used. For example, the monoclonal antibodies used can be made by the hybridoma method first described by Koehler et al., Nature, 256:495 (1975), or can be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). Additional examples of techniques for producing human monoclonal antibodies include the trioma technique, the human B-cell hybridoma technique (Kozbor, Immunology Today 4 (1983), 72), and the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985), 77-96).

[0020] Hybridomas can then be screened using standard methods, such as enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (BIACORE™) analysis, to identify one or more hybridomas that produce antibodies that selectively and preferably specifically or immunospecifically bind to a particular antigen. Any form of related antigen can be used as an immunogen, for example, recombinant antigen, its naturally occurring form, any variant or fragment, and antigenic peptides thereof. Surface plasmon resonance, as employed in the BIAcore™ system, can be used to increase the efficiency of binding of phage antibodies / constructs to epitopes of target antigens (Schier, Human Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13).

[0021] Another exemplary method for generating constructs or binding domains includes screening protein expression libraries, such as phage display or ribosome display libraries. Phage display is described, for example, in Ladner et al., U.S. Pat. No. 5,223,409; Smith (1985) Science 228:1315-1317; Clackson et al., Nature, 352:624-628 (1991); and Marks et al., J. Mol. Biol., 222:581-597 (1991).

[0022] In addition to using display libraries, relevant antigens can be used to immunize non-human animals, such as rodents (e.g., mice, hamsters, rabbits, or rats). In one embodiment, the non-human animal comprises at least a portion of a human immunoglobulin gene. For example, mouse strains deficient in mouse antibody production can be engineered with large fragments of the human Ig (immunoglobulin) locus. Hybridoma technology can be used to produce and select antigen-specific monoclonal antibodies derived from genes with the desired specificity. See, e.g., Xenomouse™ mice, Green et al. (1994) Nature Genetics 7:13-21, U.S. Patent Application Publication No. 2003-0070185, WO 96 / 34096, and WO 96 / 33735.

[0023] Monoclonal antibodies can also be obtained from non-human animals and then modified, e.g., humanized, deimmunized, chimerized, etc., using recombinant DNA techniques known in the art. Examples of modified constructs or binding domains include humanized variants of non-human antibodies / constructs, "affinity matured" constructs or binding domains (see, e.g., Hawkins et al. J. Mol. Biol. 254, 889-896 (1992) and Lowman et al., Biochemistry 30, 10832-10837 (1991)), and antibody variants or mutants with altered effector function (see, e.g., U.S. Pat. No. 5,648,260; Kontermann and Duebel (2010), supra; and Little (2009), supra).

[0024] In immunology, affinity maturation is the process by which B cells produce antibodies with increasing affinity for antigens during an immune response. Repeated exposure to the same antigen causes the host to produce antibodies with successively higher affinities. Similar to natural prototypes, in vitro affinity maturation is based on the principles of mutation and selection. In vitro affinity maturation has been successfully used to optimize antibodies, antibody fragments, antibody variants, constructs, or binding domains. Random mutations within CDRs are introduced using radiation, chemical mutagens, or error-prone PCR. Additionally, genetic diversity can be increased by chain shuffling. Two or three rounds of mutation and selection using display methods such as phage display typically yield antibodies, antibody fragments, antibody variants, constructs, or binding domains with affinities in the low nanomolar range.

[0025] A preferred type of amino acid substitution mutation of the constructs or binding domains of the invention involves substituting one or more residues within the hypervariable regions of a parent antibody structure (e.g., a humanized or human antibody structure). Generally, the resulting variants selected for further development have improved biological properties relative to the parent antibody structure from which they were generated. A convenient method for generating such substitution variants involves affinity maturation using phage display. Briefly, several sites (e.g., 6-7 sites) in the hypervariable region are mutated to generate all possible amino acid substitutions at each site. The variants thus generated are displayed in a monovalent manner from filamentous phage particles as fusions with the M13 gene III product packaged within each particle. Phage-displayed variants are then screened for their biological activity (e.g., binding affinity) as disclosed herein. Alanine scanning mutagenesis can also be performed to identify candidate hypervariable region sites (candidates for modification) that contribute significantly to antigen binding. Alternatively, or in addition, analysis of a crystal structure of the complex between the antigen and the construct or binding domain can be beneficial in identifying contact points between the binding domain and its specific antigen. Such contact and adjacent residues are candidates for substitution by the techniques detailed herein. Once such variants are generated, the panel of variants can be subjected to screening as described herein, and antibodies, antigen-binding fragments thereof, constructs, or binding domains with superior properties in one or more relevant assays can be selected for further development.

[0026] The constructs and binding domains of the present invention particularly include "chimeric" versions in which a portion of the heavy and / or light chain is identical to or homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chain is identical to or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as to fragments or variants of such antibodies, so long as the desired biological activity is exhibited (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric constructs or binding domains of interest herein include "primatized" constructs comprising variable domain antigen-binding sequences derived from a non-human primate (e.g., Old World monkey, ape, etc.) and human constant region sequences. Various techniques for producing chimeric antibodies or constructs have been described. See, e.g., Morrison et al., Proc. Natl. Acad. ScL USA 81:6851, 1985; Takeda et al., Nature 314:452, 1985; Cabilly et al., U.S. Pat. No. 4,816,567; Boss et al., U.S. Pat. No. 4,816,397; Tanaguchi et al., EP 0171496, EP 0173494, and GB 2177096.

[0027] Antibodies, polypeptide constructs, antibody fragments, antibody variants, or binding domains can also be modified by specific deletion of human T-cell epitopes (a method called "deimmunization"), for example, using the methods disclosed in WO 98 / 52976 or WO 00 / 34317. Briefly, the heavy and light chain variable regions of the antibody, construct, or binding domain can be analyzed for peptides that bind to MHC class II. These peptides represent potential T-cell epitopes (e.g., as defined in WO 98 / 52976 and WO 00 / 34317). To detect potential T-cell epitopes, a computer modeling method called "peptide threading," as described in WO 98 / 52976 and WO 00 / 34317, can be applied; in addition, databases of human MHC class II-binding peptides can be searched for motifs present in VH and VL sequences. These motifs bind to any of the 18 major MHC class II DR allotypes and therefore constitute potential T cell epitopes. Potential T cell epitopes detected can be eliminated by substituting a small number of amino acid residues within the variable domain or variable region, or preferably by single amino acid substitutions. Conservative substitutions are typically made. Often, but not exclusively, amino acids common to positions in human germline antibody sequences can be used. Human germline sequences are disclosed, for example, in Tomlinson, et al. (1992) J. Mol. Biol. 227:776-798; Cook, GP, et al. (1995) Immunol. Today Vol. 16(5):237-242; and Tomlinson et al. (1995) EMBO J. 14:14:4628-4638. The V BASE directory (www2.mrc-lmb.cam.ac.uk / vbase / list2.php) provides a comprehensive catalog of human immunoglobulin variable region sequences (compiled by Tomlinson, LA et al., MRC Centre for Protein Engineering, Cambridge, UK).These sequences can be used as a source of human sequences, for example, for the framework regions and CDRs. For example, the consensus human framework regions described in U.S. Patent No. 6,300,064 can be used.

[0028] "Humanized" antibodies, their variants or fragments, constructs, and binding domains are based primarily on human-sequence immunoglobulins, which contain minimal sequence derived from non-human immunoglobulin. In most cases, humanized antibodies, their variants or fragments, constructs, and binding domains are based on a human immunoglobulin (recipient antibody) in which residues from a hypervariable region or CDR are replaced by residues from a hypervariable region or CDR of a non-human species (donor antibody), such as a rodent (e.g., mouse, hamster, rat, or rabbit), possessing the desired specificity, affinity, capacity, and / or biological activity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, as used herein, "humanized" antibodies, their variants or fragments, constructs, and binding domains can also comprise residues that are not found in either the recipient antibody or the donor antibody. These modifications are made to further refine and optimize antibody performance. Humanized antibodies, variants or fragments thereof, constructs, and binding domains can also comprise at least a portion of an immunoglobulin constant region (e.g., Fc), typically that of a human immunoglobulin. For further details, see Nature, 321:522-525 (1986); Reichmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992).

[0029] Humanized antibodies, their variants or fragments, constructs, and binding domains can be created by replacing sequences of the (Fv) variable region not directly involved in antigen binding with equivalent sequences from a human (Fv) variable region. Exemplary methods for producing such molecules are provided by Morrison (1985) Science 229:1202-1207; Oi et al. (1986) BioTechniques 4:214, and U.S. Pat. Nos. 5,585,089, 5,693,761, 5,693,762, 5,859,205, and 6,407,213. These methods involve isolating, manipulating, and expressing nucleic acid sequences encoding all or part of immunoglobulin (Fv) variable regions from at least one of the heavy or light chains. Such nucleic acids can be obtained from hybridomas and other sources producing antibodies against a predetermined target, as described above. The recombinant DNA encoding the humanized antibody, variant or fragment thereof, construct, or binding domain can then be cloned into an appropriate expression vector.

[0030] Humanized antibodies, their variants or fragments, constructs, and binding domains can also be produced using transgenic animals (e.g., mice) that express human heavy and light chain genes but lack the ability to express endogenous mouse immunoglobulin heavy and light chain genes. Winter describes an exemplary CDR-grafting method that can be used to prepare the humanized molecules described herein (U.S. Pat. No. 5,225,539). All CDRs of a given human sequence can be replaced with at least a portion of a non-human CDR, or only a portion of the CDRs can be replaced with non-human CDRs. It is sufficient to replace only the number of CDRs necessary for the humanized molecule to bind to a given antigen.

[0031] Humanized antibodies, variants or fragments thereof, constructs, or binding domains may be optimized by introducing conservative substitutions, consensus sequence substitutions, germline substitutions, and / or back mutations. Such altered immunoglobulin molecules can be generated by any of several techniques known in the art (e.g., Teng et al., Proc. Natl. Acad. Sci. USA, 80:7308-7312, 1983; Kozbor et al., Immunology Today, 4:7279, 1983; Olsson et al., Meth. Enzymol., 92:3-16, 1982, and EP 239400).

[0032] The human anti-mouse antibody (HAMA) response has led the industry to prepare chimeric or other humanized antibodies / constructs. However, it is expected that some human anti-chimeric antibody (HACA) response will be observed, particularly with chronic or high-dose use of antibodies or constructs. Therefore, it would be desirable to provide constructs that include a human binding domain for the target to eliminate the concerns and / or impact of HAMA or HACA responses.

[0033] Thus, according to one embodiment, the polypeptide construct comprises at least one additional binding domain, said binding domain being "human." The terms "human antibody," "human construct," and "human binding domain" include antibodies, constructs, and binding domains, respectively, having antibody-derived regions, such as variable and constant regions or domains, that substantially correspond to human germline immunoglobulin sequences known in the art, including, for example, those described by Kabat et al. (1991) (supra). Human constructs or binding domains of the invention may comprise amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced in vitro by random or site-directed mutagenesis or in vivo by somatic mutation), for example in the CDRs, particularly CDR3. Human constructs or binding domains may have at least one, two, three, four, five, or more positions replaced with amino acid residues not encoded by human germline immunoglobulin sequences. The definition of human antibodies, constructs and binding domains as used herein also contemplates fully human antibodies, constructs and binding domains that contain only artificially and / or genetically unmodified human sequences of antibodies, such as those that may be derived using techniques or systems such as Xenomouse™ mice.

[0034] Polypeptides / polypeptide constructs comprising at least one human binding domain avoid some of the problems associated with antibodies or constructs with non-human variable and / or constant regions, such as those of rodents (e.g., mouse, rat, hamster, or rabbit). The presence of such rodent-derived proteins can lead to rapid clearance of the antibody or construct or can result in an immune response against the antibody or construct by the patient. To avoid the use of rodent-derived constructs, human antibody function can be introduced into rodents to produce fully human antibodies, creating humanized or fully human constructs.

[0035] The ability to clone and reconstruct megabase-sized human loci in YACs and introduce them into the mouse germline provides a powerful approach for elucidating the functional elements of very large or coarsely mapped loci and for generating useful models of human disease. Furthermore, the use of such techniques to replace mouse loci with their human equivalents can provide unique insights into the expression and regulation of human gene products during development, their communication with other systems, and their involvement in disease induction and progression.

[0036] An important practical application of such a strategy is the "humanization" of the mouse humoral immune system. The introduction of human immunoglobulin (Ig) loci into mice in which the endogenous Ig genes have been inactivated provides an opportunity to study the mechanisms underlying the programmed expression and assembly of antibodies and their role in B-cell development. Furthermore, such a strategy would provide an ideal source for the generation of fully human monoclonal antibodies (mAbs), a significant milestone in realizing the potential of antibody therapy in human diseases. Fully human antibodies or constructs derived therefrom are expected to minimize the immunogenicity and allergic reactions inherent in mouse or mouse-derived mAbs, thereby increasing the efficacy and safety of the administered antibodies / constructs. The use of fully human antibodies or constructs is expected to offer substantial advantages in the treatment of chronic and recurrent human diseases that require repeated administration of compounds, such as inflammation, autoimmunity, and cancer.

[0037] One approach toward this goal has been to engineer mouse strains deficient in mouse antibody production with large fragments of the human Ig loci, with the expectation that such mice would produce a large repertoire of human antibodies in the absence of mouse antibodies. Large human Ig fragments would preserve the large variable gene diversity and proper regulation of antibody production and expression. By utilizing the mouse machinery for antibody diversification and selection and the lack of immune tolerance to human proteins, the recapitulated human antibody repertoire in these mouse strains should yield high-affinity antibodies against any antigen of interest, including human antigens. Using hybridoma technology, antigen-specific human mAbs with desired specificity could be readily produced and selected. This general strategy was demonstrated in connection with the development of the first XenoMouse™ mouse strain (see Green et al. Nature Genetics 7:13-21 (1994)). This XenoMouse™ strain was engineered with yeast artificial chromosomes (YACs) containing 245 kb and 190 kb germline-configured fragments of the human heavy chain and kappa light chain loci, respectively, that contained the core sequences of the variable and constant regions. The human Ig-containing YACs proved compatible with the mouse system for both antibody rearrangement and expression and were able to replace inactivated mouse Ig genes. This was demonstrated by their ability to induce B cell development to produce an adult-like human repertoire of fully human antibodies and to generate antigen-specific human mAbs. These results also suggested that the introduction of a large portion of the human Ig locus, containing multiple V genes, additional regulatory elements, and human Ig constant regions, could recapitulate a virtually complete repertoire characterized by the human humoral response to infection and immunization. Green et al.'s work has been extended to the introduction of over 80% of the human antibody repertoire by introducing megabase-sized germline-configured YAC fragments of the human heavy chain and kappa light chain loci, respectively. See Mendez et al. Nature Genetics 15:146-156 (1997) and U.S. Patent Application Publication No. 08 / 759,620.

[0038] The generation of the XenoMouse™ model is described in U.S. Patent Application Publication Nos. 07 / 466,008, 07 / 610,515, 07 / 919,297, 07 / 922,649, 08 / 031,801, 08 / 112,848, 08 / 234,145, 08 / 376,279, 08 / 430,938, 08 / 464,584, 08 / 464,582, 08 / 463,191, 08 / 462,837 ... This is further discussed and detailed in U.S. Pat. Nos. 08 / 486,853, 08 / 486,857, 08 / 486,859, 08 / 462,513, 08 / 724,752, 08 / 759,620 and U.S. Pat. Nos. 6,162,963; 6,150,584; 6,114,598; 6,075,181 and 5,939,598 and Japanese Patent Publications Nos. 3068180B2, 3068506B2 and 3068507B2. See also Mendez et al. Nature Genetics 15:146-156 (1997) and Green and Jakobovits J. Exp. Med. 188:483-495 (1998), EP 0463151 B1, WO 94 / 02602, WO 96 / 34096, WO 98 / 24893, WO 00 / 76310, and WO 03 / 47336.

[0039] In another approach, other companies, including, among others, GenPharm International, Inc., utilize a "minilocus" approach. In the minilocus method, an exogenous Ig locus is mimicked through the inclusion of pieces (individual genes) from the Ig locus. Thus, one or more VH genes, one or more DH genes, one or more JH genes, a μ constant region, and a second constant region (preferably a γ constant region) are formed as a construct for insertion into an animal. This approach is described in U.S. Pat. No. 5,545,807 to Surani et al. and U.S. Pat. Nos. 5,545,806, 5,625,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429, 5,789,650, 5,814,318, 5,877,397, 5,874,299, and 6,255,458 to Lonberg and Kay, respectively; U.S. Pat. Nos. 5,591,669 and 6,023,010 to Krimpenfort and Berns; U.S. Patent Nos. 5,612,205, 5,721,367, and 5,789,215 to Choi and Dunn et al., and U.S. Patent No. 5,643,763 to GenPharm and U.S. Patent Application Publication Nos. 07 / 574,748, 07 / 575,962, 07 / 810,279, 07 / 853,408, 07 / 904,068, 07 / 990,860, 08 / 053,131, 08 / 096,762, 08 / 155,301, 08 / 161,739, 08 / 165,699, and 08 / 209,741 to International.See also EP 0546073B1, WO 92 / 03918, WO 92 / 22645, WO 92 / 22647, WO 92 / 22670, WO 93 / 12227, WO 94 / 00569, WO 94 / 25585, WO 96 / 14436, WO 97 / 13852 and WO 98 / 24884, and U.S. Pat. No. 5,981,175. See also Taylor et al. (1992), Chen et al. (1993), Tuaillon et al. (1993), Choi et al. (1993), Lonberg et al. (1994), Taylor et al. (1994), and Tuaillon et al. (1995), Fishwild et al. (1996).

[0040] Kirin has also demonstrated the production of human antibodies from mice into which large chromosome fragments or entire chromosomes have been introduced by microcell fusion. See European Patent Applications 773288 and 843961. Xenerex Biosciences is developing a technology for the potential production of human antibodies. In this technology, SCID mice are reconstituted with human lymphocytes, e.g., B cells and / or T cells. The mice can then be immunized with an antigen to generate an immune response against the antigen. See U.S. Patent Nos. 5,476,996, 5,698,767, and 5,958,765.

[0041] In some embodiments, the constructs of the invention are "isolated" or "substantially pure" constructs. "Isolated" or "substantially pure," when used to describe constructs disclosed herein, refers to a construct that has been identified, separated, and / or recovered from components of its production environment. Preferably, the construct is free from or substantially free from association with all other components from its production environment. Contaminating components of the production environment, such as those resulting from recombinant transfected cells, are materials that may interfere with the diagnostic or therapeutic use of the construct and can include enzymes, hormones, and other proteinaceous or non-proteinaceous compounds. It is understood that an isolated or substantially pure construct may represent 5% to 99.9% by weight of the total protein / polypeptide content in a given sample, depending on the circumstances. The desired construct may be produced at significantly higher concentrations using inducible or high-expression promoters. This definition encompasses the production of constructs in a wide variety of organisms and / or host cells known in the art. In certain embodiments, the construct will be purified (1) sufficiently to obtain at least 15 residues of N-terminal or internal amino acid sequence using a spinning cup sequenator, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver staining. However, typically, an isolated construct will be prepared by at least one purification step.

[0042] According to one embodiment, the entire construct and / or binding domain is in the form of one or more polypeptides or proteins. In addition to the proteinaceous portion, such polypeptides or proteins may contain non-proteinaceous portions (e.g., chemical linkers or chemical cross-linking agents, such as glutaraldehyde).

[0043] Peptides are short chains of amino acid monomers linked by covalent peptide (amide) bonds. As such, peptides are classified within the broad chemical class of biological oligomers and polymers. The amino acids that are part of a peptide or polypeptide chain are called "residues" and can be numbered consecutively. All peptides, except cyclic peptides, have an N-terminal residue at one end and a C-terminal residue at the other end. Oligopeptides consist of only a small number of amino acids (usually 2-20). Polypeptides are longer, continuous, unbranched peptide chains. Peptides are distinguished from proteins based on size and can be understood to contain approximately 50 amino acids or less as an arbitrary standard. Proteins typically consist of one or more polypeptides arranged in a biologically functional manner. While the experimental techniques applied to peptides, polypeptides, and proteins differ (e.g., details of electrophoresis, chromatography, etc.), the size boundary that distinguishes peptides from polypeptides and proteins is not absolute. Therefore, in the context of the present invention, the terms "peptide," "polypeptide," and "protein" can be used interchangeably, with the term "polypeptide" often being preferred.

[0044] Polypeptides can further form multimers, such as dimers, trimers, and higher oligomers, consisting of two or more polypeptide molecules, as described above. The polypeptide molecules forming such dimers, trimers, etc. may be identical or non-identical. The corresponding higher-order structures of such multimers are consequently referred to as homodimers or heterodimers, homotrimers or heterotrimers, etc. An example of a heteromultimer is an antibody molecule or immunoglobulin molecule, which, in its naturally occurring form, consists of two identical polypeptide light chains and two identical polypeptide heavy chains. The terms "peptide," "polypeptide," and "protein" also refer to naturally modified peptides / polypeptides / proteins, where the modification is achieved, for example, by post-translational modifications such as glycosylation, acetylation, phosphorylation, etc. "Peptides," "polypeptides," or "proteins," as referred to herein, may also be chemically modified, such as by pegylation. Such modifications are well known in the art and are described herein below.

[0045] The terms "selectively" and "preferably, selectively," "bind (specifically or immunospecifically)," "recognize (specifically or immunospecifically)," or "react (specifically or immunospecifically)," mean, in accordance with the present invention, that a construct or binding domain selectively interacts or (immunologically) specifically interacts with a given epitope on a target molecule (antigen), here CD3, respectively. This selective interaction or association may occur more frequently, more rapidly, with a longer duration, with higher affinity, or some combination of these parameters, for an epitope on a particular target (herein CD3ε) compared to an alternative substance (non-target molecule, here CD3γ, etc.). However, due to sequence similarity between homologous proteins in different species, a construct or binding domain that selectively and / or immunospecifically binds to a target (e.g., a human target) may cross-react with a homologous target molecule from a different species (e.g., from a non-human primate). Thus, the terms "selectively bind," "specific / immunospecific binding," etc. may include binding of a construct or binding domain to more than one species of epitope or structurally related epitopes. In the context of the present invention, a polypeptide of the invention binds in a specific manner to its respective target structure. Preferably, a polypeptide according to the present invention comprises one paratope per binding domain that "specifically or immunospecifically binds to," "(specifically or immunospecifically) recognizes," or "(specifically or immunospecifically) reacts" with each target structure. This means that, according to the present invention, a polypeptide or its binding domain interacts or (immuno)specifically interacts with a given epitope on the target molecule (antigen), i.e., CD3ε, and, in a particular embodiment, with a given epitope on at least one further target molecule, such as a second and / or third target molecule. This interaction or association occurs more frequently, more rapidly, of longer duration, with greater affinity, or some combination of these parameters, for an epitope on a particular target compared to an alternative substance (non-target molecule).However, due to sequence similarities between homologous proteins in different species, an antibody construct or binding domain that immunospecifically binds to its target (such as a human target) may cross-react with homologous target molecules from different species (such as non-human primates). Thus, the term "specific / immunospecific binding" can include binding of an antibody construct or binding domain to epitopes in multiple species and / or structurally related epitopes. The term "(immuno)selectively binds" excludes binding to structurally related epitopes.

[0046] In the context of the present invention, the term "epitope" refers to a portion or region of an antigen that is selectively / immunospecifically recognized by a binding structure, i.e., a paratope. An "epitope" is antigenic, and therefore the term epitope may also be referred to herein as an "antigenic structure" or "antigenic determinant." The portion of a binding domain that binds to an epitope is called a paratope. Specific binding is believed to be achieved by a specific motif in the amino acid sequence of the binding domain and the antigen. Thus, binding is achieved due to its primary, secondary, and / or tertiary structure, as well as potential secondary modifications of said structure. When a paratope specifically interacts with its antigenic determinant, the site may simply bind to the antigen. In some cases, the specific interaction may instead or additionally result in the initiation of a signal, for example, due to the induction of a conformational change in the antigen, oligomerization of the antigen, etc.

[0047] Epitopes of protein antigens are classified into two categories, conformational epitopes and linear epitopes, based on their structure and interaction with the paratope. Conformational epitopes consist of discontinuous sections of the antigen's amino acid sequence. These epitopes interact with the paratope based on the antigen's three-dimensional surface features and shape or tertiary structure (folding). Methods for determining epitope conformation include, but are not limited to, X-ray crystallography, two-dimensional nuclear magnetic resonance (2D-NMR) spectroscopy, and site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy. In contrast, linear epitopes interact with the paratope based on their primary structure. Linear epitopes are formed by a contiguous sequence of amino acids from the antigen, typically containing at least three or at least four, more commonly at least five, at least six, or at least seven, e.g., about 8 to about 10, amino acids in a unique sequence.

[0048] A method for epitope mapping for a given human target protein is described below. A predetermined region (a consecutive stretch of amino acids) within the given human target protein is exchanged / replaced with the corresponding region of a target protein paralog (as long as the binding domain does not cross-react with the paralog used). These human target / paralog chimeras are expressed on the surface of host cells (such as CHO cells). Binding of the antibody or construct can be tested via FACS analysis. If binding of the antibody or construct to the chimeric molecule is completely abolished or if a significant reduction in binding is observed, it can be concluded that the region of the human target removed from the chimeric molecule is involved in immunospecific epitope-paratope recognition. The reduction in binding is preferably at least 10%, 20%, 30%, 40%, or 50%, more preferably at least 60%, 70%, or 80%, and most preferably 90%, 95%, or even 100%, compared to binding to the human (wild-type) target, thereby setting binding to the human target at 100%. Alternatively, the epitope mapping analysis described above can be modified by introducing one or several point mutations into the sequence of the human target, which may reflect, for example, differences between the human target and its paralog.

[0049] A further method for determining the contribution of specific residues of a target antigen to recognition by a construct or binding domain is alanine scanning, in which each residue being analyzed is replaced with alanine, for example, by site-directed mutagenesis (see, e.g., Morrison KL & Weiss GA. Curr Opin Chem Biol. 2001 Jun;5(3):302-7). Alanine is used because it has a non-bulky, chemically inert methyl functional group that still mimics the secondary structure criteria of many amino acids other than alanine. If it is desired to preserve the size of the mutated residue, bulky amino acids such as valine or leucine can be used.

[0050] The interaction between a binding domain and an epitope of a target antigen implies that the binding domain exhibits recognizable or significant affinity for the epitope / target antigen (herein CD3) and generally does not exhibit significant affinity for proteins or antigens other than the target antigen, even if there is cross-reactivity with, for example, homologous targets from other species, as discussed above. "Significant affinity" includes those having a recognizable or significant affinity for the epitope / target antigen (herein CD3), and those having a recognizable or significant affinity for the target antigen. -6 Preferably, the binding is with an affinity (dissociation constant, KD) of 10 or less. -7 M or less, 10 -8 M or less, 10 -9 M or less, 10 -10 M or less or even 10 -11 M or less or 10 -12 A binding domain is considered specific when its affinity is M or less. Whether a binding domain (immune) specifically reacts with or binds to a target can be easily tested, for example, by comparing the affinity of the binding domain for its desired target protein or antigen with the affinity of the binding domain for a non-target protein or antigen (here, a protein other than CD3, respectively). Preferably, the constructs of the invention do not significantly bind to proteins or antigens other than CD3 (i.e., a CD3-binding domain does not bind to a protein other than CD3), unless any additional binding domain for an additional target is intentionally introduced into the construct of the invention, in which case binding of the binding domain to its specific target is also provided by the invention.

[0051] The affinity of the first domain is envisioned to be 100 nM or less, 90 nM or less, 80 nM or less, 70 nM or less, 60 nM or less, 50 nM or less, 40 nM or less, 30 nM or less, or 20 nM or less for human CD3ε. These values ​​are preferably measured in a cell-based assay, such as a Scatchard assay. Other methods for determining affinity are also known. These values ​​are preferably measured in a surface plasmon resonance assay, such as a Biacore assay.

[0052] The terms "does not bind significantly" and "does not bind selectively" mean that the construct or binding domain of the invention, when expressed on the surface of a cell, does not bind to a protein or antigen other than CD3. Thus, the construct exhibits a reactivity of 30% or less, preferably 20% or less, more preferably 10% or less, and particularly preferably 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% or less with a protein or antigen other than CD3 (when the protein or antigen is expressed on the surface of a cell) (wherein binding to CD3 is taken as 100%). "Reactivity" can be expressed, for example, as an affinity value (see above).

[0053] It is envisioned that the constructs of the present invention (more particularly the domains comprising the paratope / binding domain that binds to human CD3ε) do not bind or do not significantly bind to CD3ε analogs, more particularly human CD3ε analogs and / or macaque / cynomolgus CD3ε analogs. It is also envisioned that the constructs do not bind or do not significantly bind to (human or macaque / cynomolgus) CD3ε paralogs on the surface of target cells.

[0054] The VH region as part of the binding domain binding to the extracellular epitope of the human CD3ε chain comprises the CDR-H1, CDR-H2, and CDR-H3 sequences described above. The CDR sequences contain placeholders, indicated as "X" for a given amino acid residue that can be substituted for the placeholder, as is common practice in the art. The placeholders are numbered consecutively in the format of X1, X2, X3, etc. for a given CDR sequence. The amino acid residues that can be substituted for the placeholders in the listed CDR sequences are preferably, for example, in other embodiments, unless otherwise specified, with the first listed amino acid being most preferred, the second listed amino acid being second most preferred, and so on. When a sequence contains two or more placeholders, the same applies with regard to amino acid preference; i.e., the first amino acid residue combination listed for each placeholder is most preferred, the second amino acid residue combination listed for each placeholder is second most preferred, and so on. This preference hierarchy does not exclude combinations of amino acid residues for each placeholder independent of the preference hierarchy, such that different preferred amino acid residues may be combined. The same hierarchy applies to all amino acid residues listed herein below that substitute for a placeholder in the amino acid sequences described herein below. Thus, the above also applies to the placeholder designated as "X" in the sequences of the VH and VL sequences, as well as any other sequences listed throughout this specification.

[0055] The CDR-H1 sequence comprises the amino acid sequence of X1YAX2N, where X1 is K, V, S, G, R, T, or I; and X2 is M or I. Following the hierarchy described above for the placeholder amino acids, the amino acids for placeholders X1 and X2 are listed according to their preference, with the first listed amino acid being most preferred, the second listed amino acid being second most preferred, and so on. For X1, the amino acid K is most preferred, and for X2, the amino acid M is most preferred. Preferred combinations are M for X2 combined with any of the amino acid residues for X1, such as combinations of X1 and X2 selected from K and M, V and M, and S and M, respectively, with the most preferred combination being K and M. Another preferred combination is K and I for X1 and X2, respectively. Preferred examples of CDR-H1 sequences are selected from KYAMN, VYAMN, SYAMN, GYAMN, RYAMN, TYAMN, IYAMN, and KYAIN, with KYAMN or KYAIN being most preferred.

[0056] The CDR-H2 sequence comprises the amino acid sequence RIRSKYNNYATYYADX1VKX2, where X1 is S or Q; and X2 is D, G, K, S, or E. For X1, the amino acid S is most preferred, and for X2, the amino acid D is most preferred. Preferred combinations are S for X1 combined with any of the amino acid residues for X2, such as combinations for X1 and X2 selected from S and D, S and G, S and K, and S and S, respectively, with the combination S and D being most preferred. Preferred examples of CDR-H2 sequences are selected from RIRSKYNNYATYYADSVKD, RIRSKYNNYATYYADSVKK, RIRSKYNNYATYYADSVKS, RIRSKYNNYATYYADSVKE, RIRSKYNNYATYYADSVKG, and RIRSKYNNYATYYADQVKD, with RIRSKYNNYATYYADSVKD being most preferred.

[0057] The CDR-H3 sequence comprises the amino acid sequence HX1NFGNSYX2SX3X4AY, where X1 is G, R, or A; X2 is I, L, V, or T; X3 is Y, W, or F; and X4 is W, F, or Y. For X1, the amino acid G is most preferred; for X2, the amino acid I is most preferred; for X3, the amino acid Y is most preferred; and for X4, the amino acid W is most preferred. Preferred examples of CDR-H3 sequences are selected from HGNFGNSYISYWAY, HGNFGNSYLSWWAY, HGNFGNSYTSYYAY, HRNFGNSYLSWFAY, HGNFGNSYVSFFAY, HGNFGNSYISWWAY, HGNFGNSYVSWWAY, HGNFGNSYLSYFAY, HGNFGNSYLSFWAY, HANFGNSYISYWAY, and HGNFGNFGNSYVSWFAY, with HGNFGNSYISYWAY being most preferred.

[0058] The CDR-L1 sequence comprises the amino acid sequence of X1SSTGAVTX2X3X4YX5N, where X1 is G, R or A; X2 is S or T; X3 is G or S; X4 is N or Y; and X5 is P or A. For X1, the amino acid G is most preferred; for X2, the amino acid S is most preferred; for X3, the amino acid G is most preferred; for X4, the amino acid N is most preferred; and for X5, the amino acid P is most preferred. Preferred examples of CDR-L1 sequences are selected from GSSTGAVTSGNYPN, RSSTGAVTSGYYPN, GSSTGAVTSGYYPN, ASSTGAVTSGNYPN and RSSTGAVTTSNYAN, with GSSTGAVTSGNYPN being most preferred.

[0059] The CDR-L2 sequence comprises the amino acid sequence X1TX2X3X4X5X6, where X1 is G or A; X2 is K, D, or N; X3 is F, M, or K; X4 is L or R; X5 is A, P, or V; and X6 is P or S. For X1, the amino acid G is most preferred; for X2, the amino acid K is most preferred; for X3, the amino acid F is most preferred; for X4, the amino acid L is most preferred; for X5, the amino acid A is most preferred, and for X6, the amino acid P is most preferred. Preferred examples of CDR-L2 sequences are selected from GTKFLAP, ATDMRPS, GTKFLVP, and GTNKRAP, with GTKFLAP being most preferred.

[0060] The CDR-L3 sequence comprises the amino acid sequence of X1LWYSNX2WV, where X1 is V, A, or T; and X2 is R or L. For X1, the amino acid V is most preferred, and for X2, the amino acid R is most preferred. Preferred combinations are R for X2 combined with any of the amino acid residues for X1, such as combinations of X1 and X2 selected from V and R, A and R, and T and R, respectively, with the most preferred combination being V and R. Preferred examples of CDR-L3 sequences are selected from VLWYSNRWV, ALWYSNRWV, TLWYSNRWV, and ALWYSNLWV, with VLWYSNRWV being most preferred.

[0061] In a preferred embodiment, the VH region of i) comprises a CDR-H1 sequence comprising or consisting of the amino acid sequence of X1YAX2N (X1 is K, V, S, G, R, or I; X2 is M or I); a CDR-H2 sequence of RIRSKYNNYATYYADX1VKX2 (X1 is S or Q; X2 is D, G, K, S, or E); and a CDR-H3 sequence of HX1NFGNSYX2SX3X4AY (X1 is G, R, or A; X2 is I, L, V, or T; X3 is Y, W, or F; and X4 is W, F, or Y); and the VL region of ii) comprises a CDR-L1 sequence of X1SSTGAVTSGX2YPN (X1 is G, R, or A; X2 is N or Y); and a CDR-L3 sequence of X1LWYSNRWV (X1 is V, A, or T); wherein the one or more CDR sequences of the VH region of i) and / or the VL region of ii) comprise one or more amino acid substitutions selected from X24V or X24F in CDR-H1; D15 (preferably E), X116A in CDR-H2; H1 (preferably A or N), X12E, F4 (preferably I) in CDR-H3, and / or N6 (preferably S or T) in CDR-H3; and W93 (preferably Y) in CDR-L3.

[0062] The CDR-H1 sequence comprises or consists of the amino acid sequence of X1YAX2N, where X1 is K, V, S, G, R, or I; and X2 is M or I. For X1, the amino acid K is most preferred, and for X2, the amino acid M is most preferred. Preferred combinations are M for X2 combined with any amino acid residue for X1, such as combinations of X1 and X2 selected from K and M, V and M, or S and M, respectively, with K and M being the most preferred combination. Another preferred combination is K and I for X1 and X2, respectively. Preferred examples of CDR-H1 sequences are selected from KYAMN, VYAMN, SYAMN, GYAMN, RYAMN, IYAMN, and KYAIN, with KYAMN or KYAIN being preferred, and KYAMN being most preferred.

[0063] The CDR-H2 sequence comprises or consists of the amino acid sequence RIRSKYNNYATYYADX1VKX2, where X1 is S or Q; and X2 is D, G, K, S, or E. For X1, the amino acid S is most preferred, and for X2, the amino acid D is most preferred. Preferred combinations are S for X1 combined with any of the amino acid residues for X2, such as combinations for X1 and X2 selected from S and D, S and G, S and K, and S and S, respectively, with the combination S and D being most preferred. Preferred examples of CDR-H2 sequences are selected from RIRSKYNNYATYYADSVKD, RIRSKYNNYATYYADSVKK, RIRSKYNNYATYYADSVKS, RIRSKYNNYATYYADSVKE, RIRSKYNNYATYYADSVKG, and RIRSKYNNYATYYADQVKD, with RIRSKYNNYATYYADSVKD being most preferred.

[0064] The CDR-H3 sequence comprises the amino acid sequence HX1NFGNSYX2SX3X4AY, where X1 is G, R, or A; X2 is I, L, V, or T; X3 is Y, W, or F; and X4 is W, F, or Y. For X1, the amino acid G is most preferred; for X2, the amino acid I is most preferred; for X3, the amino acid Y is most preferred; and for X4, the amino acid W is most preferred. Preferred examples of CDR-H3 sequences are selected from HGNFGNSYISYWAY, HGNFGNSYLSWWAY, HGNFGNSYTSYYAY, HRNFGNSYLSWFAY, HGNFGNSYVSFFAY, HGNFGNSYISWWAY, HGNFGNSYVSWWAY, HGNFGNSYLSYFAY, HGNFGNSYLSFWAY, HANFGNSYISYWAY, and HGNFGNFGNSYVSWFAY, with HGNFGNSYISYWAY being most preferred.

[0065] The CDR-L1 sequence comprises the amino acid sequence of X1SSTGAVTSGX2YPN, where X1 is G, R, or A; and X2 is N or Y. For X1, the amino acid G is most preferred; for X2, the amino acid N is preferred. Preferred examples of CDR-L1 sequences are selected from GSSTGAVTSGNYPN, RSSTGAVTSGYYPN, GSSTGAVTSGYYPN, and ASSTGAVTSGNYPN, with GSSTGAVTSGNYPN or ASSTGAVTSGNYPN being preferred, and GSSTGAVTSGNYPN being most preferred.

[0066] The CDR-L2 sequence comprises the amino acid sequence X1TX2X3X4X5X6, where X1 is G or A; X2 is K or D; X3 is F or M; X4 is L or R; X5 is A, P or V; and X6 is P or S. For X1, the amino acid G is most preferred; for X2, the amino acid K is most preferred; for X3, the amino acid F is most preferred; for X4, the amino acid L is most preferred; for X5, the amino acid A is most preferred, and for X6, the amino acid P is most preferred. Preferred examples of CDR-L2 sequences are selected from GTKFLAP, ATDMRPS, and GTKFLVP, with GTKFLAP or GTKFLVP being preferred, and GTKFLAP being most preferred.

[0067] The CDR-L3 sequence comprises the amino acid sequence of X1LWYSNRWV, where X1 is V, A or T, with the amino acid V being most preferred. Preferred examples of CDR-L3 sequences are selected from VLWYSNRWV, ALWYSNRWV and TLWYSNRWV, with VLWYSNRWV or TLWYSNRWV being more preferred, and VLWYSNRWV being most preferred.

[0068] In a further preferred embodiment, the VH region of i) comprises a CDR-H1 sequence comprising or consisting of the amino acid sequence of X1YAX2N (X1 is K, V, S, R, or I; X2 is M or I); a CDR-H2 sequence of RIRSKYNNYATYYADX1VKX2 (X1 is S or Q; X2 is D, G, K, or S); and a CDR-H3 sequence of HX1NFGNSYX2SX3X4AY (X1 is G or A; X2 is I, L, V, or T; X3 is Y, W, or F; and X4 is W, F, or Y); and the VL region of ii) comprises a CDR-H1 sequence comprising or consisting of the amino acid sequence of X1SSTGAVTSGX2YPN the CDR-L2 sequence of GTKFLX1P (X1 is A or V); and the CDR-L3 sequence of X1LWYSNRWV (X1 is V, A, or T); the one or more CDR sequences of i) the VH region and / or ii) the VL region contain one or more amino acid substitutions selected from X24V or X24F in CDR-H1; D15 (preferably E), X116A in CDR-H2; H1 (preferably A or N), X12E, F4 (preferably I), and / or N6 (preferably S or T) in CDR-H3; and W93 (preferably Y) in CDR-L3.

[0069] The CDR-H1 sequence comprises or consists of the amino acid sequence of X1YAX2N, where X1 is K, V, S, R, or I; and X2 is M or I. For X1, the amino acid K is most preferred, and for X2, the amino acid M is most preferred. Preferred combinations are M for X2 combined with any amino acid residue for X1, such as combinations of X1 and X2 selected from K and M, V and M, and S and M, respectively, with K and M being the most preferred combination. Another preferred combination is K and I for X1 and X2, respectively. Preferred examples of CDR-H1 sequences are selected from KYAMN, VYAMN, SYAMN, RYAMN, IYAMN, and KYAIN, with KYAMN or KYAIN being preferred, and KYAMN being most preferred.

[0070] The CDR-H2 sequence comprises or consists of the amino acid sequence RIRSKYNNYATYYADX1VKX2, where X1 is S or Q; and X2 is D, G, K, or S. For X1, the amino acid S is most preferred, and for X2, the amino acid D is most preferred. Preferred combinations are S for X2 combined with any of the amino acid residues for X1, such as combinations for X1 and X2 selected from S and D, S and G, S and K, S and S, respectively, most preferably the combination S and D. Preferred examples of CDR-H2 sequences are selected from RIRSKYNNYATYYADSVKD, RIRSKYNNYATYYADSVKK, RIRSKYNNYATYYADSVKS, RIRSKYNNYATYYADSVKG, and RIRSKYNNYATYYADQVKD, where RIRSKYNNYATYYADSVKD or RIRSKYNNYATYYADQVKD are preferred, and RIRSKYNNYATYYADSVKD is most preferred.

[0071] The CDR-H3 sequence comprises or consists of the amino acid sequence HX1NFGNSYX2SX3X4AY, where X1 is G or A; X2 is I, L, V, or T; X3 is Y, W, or F; and X4 is W, F, or Y. For X1, the amino acid G is most preferred; for X2, the amino acid I is most preferred; for X3, the amino acid Y is most preferred; and for X4, the amino acid W is most preferred. Preferred examples of CDR-H3 sequences are selected from HGNFGNSYISYWAY, HGNFGNSYLSWWAY, HGNFGNSYTSYYAY, HGNFGNSYVSFFAY, HGNFGNSYISWWAY, HGNFGNSYVSWWAY, HGNFGNSYLSYFAY, HGNFGNSYLSFWAY, and HANFGNSYISYWAY, with HGNFGNSYISYWAY or HANFGNSYISYWAY being preferred, and HGNFGNSYISYWAY being most preferred.

[0072] The CDR-L1 sequence comprises or consists of the amino acid sequence X1SSTGAVTSGX2YPN, where X1 is G or A; and X2 is N or Y. For X1, the amino acid G is most preferred; for X2, the amino acid N is preferred. Preferred examples of CDR-L1 sequences are selected from GSSTGAVTSGNYPN, GSSTGAVTSGYYPN, and ASSTGAVTSGNYPN, with GSSTGAVTSGNYPN or ASSTGAVTSGNYPN being preferred, and GSSTGAVTSGNYPN being most preferred.

[0073] The CDR-L2 sequence comprises or consists of the amino acid sequence of GTKFLX1P; X1 is A or V. For X1, the amino acid A is preferred. The CDR-L2 sequence is GTKFLAP or GTKFLVP, with GTKFLAP being preferred.

[0074] The CDR-L3 sequence comprises or consists of the amino acid sequence of X1LWYSNRWV, where X1 is V, A or T, with the amino acid V being most preferred. Preferred examples of CDR-L3 sequences are selected from VLWYSNRWV, ALWYSNRWV and TLWYSNRWV, with VLWYSNRWV or TLWYSNRWV being more preferred, and VLWYSNRWV being most preferred.

[0075] In an even more preferred embodiment, the VH region of i) comprises a CDR-H1 sequence comprising or consisting of the amino acid sequence of X1YAMN (X1 is K or S); a CDR-H2 sequence of RIRSKYNNYATYYADSVKX1 (X1 is D or G); and a CDR-H3 sequence of HGNFGNSYX1SX2WAY (X1 is I or V; X2 is Y or W); and the VL region of ii) comprises a CDR-L1 sequence of GSSTGAVTSGX1YPN (X1 is N or Y); and a CDR-L3 sequence of X1LWYSNRWV (X1 is V or A); wherein the one or more CDR sequences of the VH region of i) and / or the VL region of ii) comprise one or more amino acid substitutions selected from M4V or M4F in CDR-H1; D15 (preferably E), S16A in CDR-H2; H1 (preferably A or N), G2E, F4 (preferably I), and / or N6 (preferably S or T) in CDR-H3; and W93 (preferably Y) in CDR-L3.

[0076] The CDR-H1 sequence comprises or consists of the amino acid sequence of X1YAMN, where X1 is K or S, with the amino acid K being preferred. The CDR-H1 sequence is KYAMN or SYAMN, with KYAMN being most preferred.

[0077] The CDR-H2 sequence comprises or consists of the amino acid sequence RIRSKYNNYATYYADSVKX1, where X1 is D or G. For X1, the amino acid D is preferred. The CDR-H2 sequence is RIRSKYNNYATYYADSVKD or RIRSKYNNYATYYADSVKG, where RIRSKYNNYATYYADSVKD is preferred.

[0078] The CDR-H3 sequence comprises or consists of the amino acid sequence HGNFGNSYX1SX2WAY, where X1 is I or V; and X2 is Y or W. For X1, the amino acid I is preferred; and for X2, the amino acid Y is preferred. The CDR-H3 sequence is HGNFGNSYISYWAY or HGNFGNSYVSWWAY, with HGNFGNSYISYWAY being preferred.

[0079] The CDR-L1 sequence comprises or consists of the amino acid sequence GSSTGAVTSGX1YPN, where X1 is N or Y. For X1, the amino acid N is preferred. The CDR-L1 sequence is GSSTGAVTSGNYPN or GSSTGAVTSGYYPN, with GSSTGAVTSGNYPN being preferred.

[0080] The CDR-L2 sequence comprises or consists of the amino acid sequence of GTKFLAP.

[0081] The CDR-L3 sequence comprises or consists of the amino acid sequence of X1LWYSNRWV, where X1 is V or A, preferably V. The CDR-L3 sequence is VLWYSNRWV or ALWYSNRWV, preferably VLWYSNRWV.

[0082] Each of the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 sequences can be freely combined in the form of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, CDR-L3 (this is the preferred orientation of the VH and VL regions) or CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3, and exhibits the listed binding to the extracellular epitope of the human CD3 epsilon chain.

[0083] Preferred combinations of CDR-L1 to L3 sequences of the VL region and preferred combinations of CDR-H1 to H3 sequences of the VH region are listed in Table 1 below.

[0084] [Table 1]

[0085] Preferably, any of the above-listed combinations of CDR-L1 to L3 is combined with any of the above-listed combinations of CDR-H1 to H3 as a portion of the binding domain that binds to the extracellular space of the human CD3ε chain. In other words, the VL region has the following CDR-L1, CDR-L2, and CDR-L3 sequences in this order: GSSTGAVTSGYYPN, GTKFLAP, ALWYSNRWV; RSSTGAVTSGYYPN, ATDMRPS, ALWYSNRWV; GSSTGAVTSGNYPN, GTKFLAP, VLWYSNRWV; ASSTGAVTSGNYPN, GTKFLVP, TLWYSNRWV; or RSSTGAVTTSNYAN, GTNKRAP, ALWYSNLWV comprising or consisting of; and The VL region is composed of CDR-H1, CDR-H2, and CDR-H3 sequences, in this order: IYAMN, RIRSKYNNYATYYADSVKS, HGNFGNSYVSFFAY; KYAMN, RIRSKYNNYATYYADSVKD, HGNFGNSYISYWAY; SYAMN, RIRSKYNNYATYYADSVKG, HGNFGNSYLSFWAY; RYAMN, RIRSKYNNYATYYADSVKG, HGNFGNSYLSYFAY; VYAMN, RIRSKYNNYATYYADSVKK, HGNFGNSYLSWWAY; KYAMN, RIRSKYNNYATYYADSVKS, HGNFGNSYTSYYAY; GYAMN, RIRSKYNNYATYYADSVKE, HRNFGNSYLSWFAY; VYAMN, RIRSKYNNYATYYADSVKK, HGNFGNSYISWWAY; SYAMN, RIRSKYNNYATYYADSVKG, HGNFGNSYVSWWAY; KYAIN, RIRSKYNNYATYYADQVKD, HANFGNSYISYWAY; or TYAMN, RIRSKYNNYATYYADSVKD, HGNFGNSYVSWFAY It comprises or consists of:

[0086] According to the present invention, preferred combinations of CDR sequences of VH and VL regions, listed in the order of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, are as defined in SEQ ID NOs: 118 to 123, 166 to 171, 356 to 361, 546 to 551, 724 to 729, 922 to 927, 1100 to 1105, 1290 to 1295, 1468 to 1473, 1658 to 1663, and 1848 to 1853.

[0087] Most preferably, the VL region comprises the following CDR-L1, CDR-L2, CDR-L3 sequences, in that order: GSSTGAVTSGNYPN, GTKFLAP, VLWYSNRWV and The VL region is composed of CDR-H1, CDR-H2, and CDR-H3 sequences, in this order: As shown in SEQ ID NOs: 1848 to 1853, KYAMN, RIRSKYNNYATYYADSVKD, HGNFGNSYISYWAY Preferably, the orientation of the CDRs is from VH to VL, i.e., the orientation of the variable regions is from N-terminus to C-terminus, VH to VL.

[0088] According to the present invention, the CDR sequences of the VH and / or VL regions comprise one or more amino acid substitutions. As is common in the art, the location and nature of an amino acid substitution is indicated herein by indicating the original amino acid residue at the substituted position and the amino acid used for the substitution, e.g., in the format F4I, where "F" indicates the original amino acid residue occurring at position "4" of the given amino acid sequence and "I" indicates the amino acid residue that has substituted "F" at position 4. If the substitution is with any amino acid residue (other than the original amino acid residue), the amino acid residue is not indicated, but only the original amino acid residue and its position in the given amino acid sequence is indicated, thus the previous example is simply indicated as "F4". Preferably, only naturally occurring amino acids are used for the substitution.

[0089] The VH region (i) and / or the VL region (ii) contain one or more amino acid substitutions as specified herein in one or more of the CDR sequences listed herein above. Thus, at least one of the listed amino acid substitutions in the variable region is included in the binding domain that binds to an extracellular epitope of the human CD3ε chain. The at least one or more amino acid substitutions may be present in the CDR sequences of the VH region or the VL region if only one amino acid substitution is present, or may be present in the CDRs of both the VH region and the VL region if two or more amino acid substitutions are present, or may be limited to the CDR sequences of the VH region or the VL region only. In certain embodiments where two or more amino acid substitutions are present, the substitutions are present in both the VH region and the VL region, or in only one of the variable regions. Preferably, the substitutions are present in the CDRs of the VH region and the VL region.

[0090] If CDR-H1 contains an amino acid substitution at position 4, it is preferably a substitution of the original amino acid with amino acid V. Preferably, the CDR-H1 sequence is KYAIN or KYAMN, i.e., it does not contain any of the above listed amino acid substitutions.

[0091] In CDR-H2, the amino acid substitution at position 15 is preferably D15E, or the amino acid substitution at position 16 is amino acid A. Preferably, CDR-H1 contains an amino acid substitution at position 16 with amino acid A; in this case, the preferred amino acid for the last amino acid of CDR-H2 is D or G, with D being most preferred. Therefore, a preferred CDR-H2 sequence is RIRSKYNNYATYYADAVKD or RIRSKYNNYATYYADAVKG, with RIRSKYNNYATYYADAVKD being more preferred. When CDR-H2 contains two amino acid substitutions, a combination of amino acid E at position 15 and amino acid A at position 16 is preferred; in this case, the preferred amino acid for the last amino acid of CDR-H2 is G. Therefore, a preferred CDR-H2 sequence for the combination of amino acid E at position 15 and amino acid A at position 16 is RIRSKYNNYATYYAEAVKG.

[0092] In CDR-H3, the amino acid substitutions are at position 1, preferably with the amino acid A or N, at position 2 with the amino acid E, at position 4, preferably with the amino acid I, and at position 6, preferably with the amino acid S or T. Preferably, CDR-H3 comprises an amino acid A or N at position 1 and an amino acid S or T at position 6; in this case, and if a placeholder is present at the indicated position in the CDR-H3 sequence, the preferred amino acid for X1 is G or A, most preferably G; X2 is I; X3 is Y; and X4 is W or Y. When CDR-H3 comprises two amino acid substitutions, the preferred combinations are an amino acid A at position 1 and an amino acid S at position 6, and an amino acid N at position 1 and an amino acid T at position 6; in this case, and if a placeholder is present at the indicated position in the CDR-H3 sequence, the preferred amino acid for X1 is G or A, most preferably G; X2 is I, X3 is Y, and X4 is W or F. When CDR-H3 contains three amino acid substitutions, the preferred combinations are amino acid A at position 1, amino acid I at position 4, and amino acid S at position 6; and amino acid N at position 1, amino acid I at position 4, and amino acid T at position 6, in which case and when a placeholder is present at the indicated position in the CDR-H3 sequence, the preferred amino acid for X2 is I, X3 is Y, and X4 is W and F. When CDR-H3 contains four amino acid substitutions, the preferred combinations are amino acid N at position 1, amino acid E at position 2, amino acid I at position 4, and amino acid T at position 6, in which case and when a placeholder is present at the indicated position in the CDR-H3, the preferred amino acid for X2 is I, X3 is Y, and X4 is W. Thus, preferred CDR-H3 sequences are selected from AGNFGSSYISYWAY, NENIGTSYISYWAY, AGNFGTSYISYWAY, NANFGTSYISYFAY and AGNFGSSYISYFAY, with AGNFGSSYIWAY and AGNFGSSYISYFAY being most preferred.

[0093] In CDR-L3, the amino acid substitution at position 1 is by amino acid L in the sequence X1LWYSNX2WV, or the amino acid substitution is W93, preferably W93Y (also referred to as X193Y), where X1 is V, A, or T; and X2 is R or L. Preferably, when CDR-L3 contains only one amino acid substitution, the CDR-L3 sequence is VLYYSNRWV. However, it is preferred that CDR-L3 does not contain said X11L and W93 amino acid substitutions, in which case the preferred CDR-L3 sequence is VLYYSNRWV.

[0094] Without wishing to be bound by any particular scientific theory, each of the amino acid substitutions listed above results in an increase in the thermal stability of a binding domain that binds to an extracellular epitope of the human CD3ε chain, comprising or consisting of a VH region linked to a VL region described herein, compared to a binding domain that binds to an extracellular epitope of the human CD3ε chain, comprising or consisting of a VH region linked to a VL region sequence that is not modified, i.e., does not contain the amino acid residue substitution. Said increase in thermal stability is preferably measured by the well-known method of differential scanning fluorimetry (DSF), also described herein. The method is described, for example, in Wen et al., "Nano differential scanning fluorimetry for comparability studies of therapeutic proteins", Analytical Biochemistry, Volume 593, 2020, 113581, ISSN 0003-2697, or Dart, ML, et al. (2018) "Homogeneous Assay for Target Engagement Utilizing Bioluminescent Thermal Shift", ACS Medical Chemistry Letters, 9(6), 546-551. As is evident from the Examples section, the amino acid substitutions or combinations thereof defined herein result in increased thermal stability (see Example 1, Tables 2 and 3) compared to the unmodified CD3 binding domain defined herein ("I2C"), while maintaining cytotoxic activity, demonstrating that this is not the case with prior art methods for improving thermal stability, namely the CC44 / 100 cis-clamp, in which an additional disulfide bond was engineered to stabilize (Reiter, Y. et al., 1994) by substituting a cysteine ​​residue at position 44 in the heavy chain variable region and at position 100 in the light chain variable region (numbering scheme according to Kabat et al., 1991) of the unmodified CD3 binding domain defined herein (VH and VL SEQ ID NOs: 1854 and 1855, respectively; see Example 2, Table 5).Thus, the combination of VH and VL regions of the CD3ε binding domains described herein are temperature stabilized compared to VH and VL region sequences that do not contain the given substitutions introduced at the positions defined herein.

[0095] In a preferred embodiment of the polypeptide or polypeptide construct according to claim 1 of the present invention, in addition to the single amino acid substitution or combination thereof defined in iii), in the CDR-H1 sequence, X2 is I; in the CDR-H2 sequence, X2 is G; in the CDR-H3 sequence, X1 is A and X4 is F; and / or in the CDR-L3 sequence, X1 is A. The single amino acid substitution or combination thereof defined herein is further combined with a specific amino acid at a specific position in a CDR. Thus, at least one of the amino acid substitutions listed in iii) is present, and in the CDR-H2 sequence, at least one of X2 is G; in the CDR-H3, X1 is A and X4 is F; and / or in the CDR-L3 sequence, X1 is A, further present in the CDR. As previously stated herein above, when there is only one additional amino acid in a CDR, a preferred amino acid for X2 is I in the CDR-H1 sequence, or most preferably X2 is G in the CDR-H2 sequence and X2 is I in the CDR-H1 sequence. Two preferred combinations of the additional amino acids are X2 is I in the CDR-H1 sequence and X4 is F in the CDR-H3 sequence; or X2 is G in the CDR-H2 sequence and X4 is F in the CDR-H3 sequence. Three preferred combinations of the additional amino acids are X2 is I in the CDR-H1 sequence, X1 is A and X4 is F in the CDR-H3 sequence; or X2 is I in the CDR-H1 sequence, X4 is F in the CDR-H3 sequence, and X1 is A in the CDR-L3 sequence. Since the latter amino acids will further be present, it is understood that the additional amino acids listed that overlap in position with the amino acid substitutions in iii) cannot replace the overlapping amino acid substitutions.For example, if the only amino acid substitution according to iii) is either X24V or X24F in CDR-H1, then said amino acid cannot be replaced with amino acid I in said CDR-H1 sequence, since there are no remaining amino acid substitutions defined in iii) in the binding domain. In other words, there must always be at least one amino acid substitution defined in iii) included in a polypeptide or polypeptide construct of the invention.

[0096] It is also understood that, depending on the actual sequence of the CDR, one or more of said additional amino acids may already be part of a CDR as defined herein above, or if not, those CDRs are modified to present said additional amino acids in combination with one or a combination of amino acid substitutions as listed under iii).

[0097] In other preferred embodiments, the polypeptide or polypeptide construct of the invention comprises or consists of said amino acid substitutions as defined in iii), 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more amino acid substitutions or a combination of up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 amino acid substitutions. Preferably, only substitutions as defined herein are introduced into the VH and VL region sequences. This applies to all embodiments recited herein, such that the VH and VL region sequences consist of the unmodified amino acid sequences with substitutions as defined herein.

[0098] More specifically, i) the one amino acid substitution is selected from a. X24V or X24F in CDR-H1; H1A in CDR-H3; and b. X11L and W3Y in CDR-H3; ii) the combination of two or more amino acid substitutions is selected from X116A in CDR-H2 and N6S in CDR-H3; and X116A in CDR-H2 and N6T in CDR-H3; iii) the combination of three or more amino acid substitutions is selected from a. X24V or X24F in CDR-H1; H1A in CDR-H3; and b. X11L and W3Y in CDR-H3; X116A in CDR-H2 and H1A and N6S in CDR-H3; X116A in CDR-H2, H1A and N6T in CDR-H3; X116A in CDR-H2, H1N and N6T in CDR-H3; iv) the combination of four or more amino acid substitutions is selected from: X116A in CDR-H2, H1A, N6S in CDR-H3 and W3Y in CDR-H3; D15E, X116A in CDR-H2, H1N, N6T in CDR-H3 X116A in CDR-H2, H1A, F4I and N6S in CDR-H3; X116A in CDR-H2, H1A, F4I and N6S in CDR-H3; v) the combination of five or six amino acid substitutions is selected from X116A in CDR-H2, H1A, X12E, F4I and N6T in CDR-H3; X116A in CDR-H2, H1N, X12E, F4I and N6S in CDR-H3. 6S; D15E, X116A in CDR-H2, H1A, N6S in CDR-H3; and W3Y in CDR-L3; D15E, X116A in CDR-H2, H1N, X12E, F4I and N6T in CDR-H3; D15E, X116A in CDR-H2, H1A, X12E, F4I and N6T in CDR-H3; D15E, X116A in CDR-H2, X12E, F4I and N6T in CDR-H3.

[0099] In accordance with the foregoing, the amino acid substitution H1A in CDR-H3 is a preferred amino acid substitution when there is only one amino acid substitution in the CD3ε-binding domain within the CDR sequences or VH region sequences defined herein. Furthermore, in the case of a combination of amino acid substitutions as defined in iii), H1A in CDR-H3 is preferably one of the amino acid substitutions in said combination.

[0100] In one embodiment, the invention relates to a polypeptide or polypeptide construct comprising a binding domain that binds to an extracellular epitope of the human CD3 epsilon chain, the binding domain comprising or consisting of a VH region and a VL region, i) the VH region is [ka] X1 is Q or K; X2 is V or L; X3 is V or E; X4 is G or K; X5 is K, V, S, G, R, T or I; X6 is M or I; X7 is S or Q; X8 is D, G, K, S or E; X9 is K or Q; 10 is N or S; X 11 is T or I; X 12 is A or L; X 13 is V or M; X 14 is G, R or A; X 15 is I, L, V or T; X 16 is Y, W or F; X 17 is W, F or Y; X 18 is S or A; and ii) the VL region is [ka] X1 is T or A; X2 is P or S; X3 is S or A; X4 is V or T; X5 is G or E; X6 is G, R or A; X7 is S or T; X8 is G or S; X9 is N or Y; 10 is P or A; X 11 is Q or E; X 12 is G or D; X 13is Q or H; X 14 is A or L; X 15 is P or F; X 16 is R or T; X 17 is G or A; X 18 is K or D; X 19 is F or M; X 20 is L or R; X 21 is A, P or V; X 22 is P or S; X 23 is T or V; X 24 is L or I; X 25 is G or D; X 26 is L or I; X 27 is S or T; X 28 is V or A; X 29 is P or T; X 30 is E or I; X 31 is Y or F; X 32 is V, A or T; X 33 is R or L; and iii) the VH region sequence and / or the VL region sequence are ai), N30 (preferably S), X634V, X634F, Q39 (E, K, R, D, preferably E), L45 (M or V, preferably M), D64 (preferably E), X765A, X 12 81V, X 12 81T, X 12 81I, V99 (A or L, preferably A), H101 (preferably A or N), X 14 102E, F104 (preferably I) and N106 (preferably S or T); and b.ii) in the VL region sequence L20 (preferably I or M), V38 (preferably I), X 11 40R, X 11 40K, X 24 69 (S or E, preferably S), X 32 91L, X 33 93 (preferably Y) and G102 (preferably S) The present invention relates to a polypeptide or polypeptide construct comprising one or more amino acid substitutions selected from:

[0101] In this embodiment, preferred VH and VL region sequences that are included in the binding domain and mediate binding to the human CD3ε chain are described. As is clear from the above, the VH and VL region sequences of i) and ii) contain various placeholders, and the same hierarchy and nomenclature applies to the placeholders listed herein above in relation to the CDRs contained in the VH and VL regions of i) and ii). As is clear from the VH and VL region sequences, the CDRs described herein above are part of the VH and VL region sequences. Therefore, the preferred embodiments of the CDRs of i) and ii) herein above also apply to this preferred embodiment.

[0102] i) The VH region is [ka] X1 is Q or K; X2 is V or L; X3 is V or E; X4 is G or K; X5 is K, V, S, G, R, T or I; X6 is M or I; X7 is S or Q; X8 is D, G, K, S or E; X9 is K or Q; 10 is N or S; X 11 is T or I; X 12 is A or L; X 13 is V or M; X 14 is G, R or A; X 15 is I, L, V or T; X 16 is Y, W or F; X 17 is W, F or Y; X 18 is S or A.

[0103] The CDR sequences within the VH region are as follows: a CDR-H1 sequence comprising or consisting of X5YAX6N; a CDR-H2 sequence comprising or consisting of RIRSKYNNYATYYADX7VKX8; and 14 NFGNSYX 15 SX 16 X17 A CDR-H3 sequence comprising or consisting of AY. Preferred examples of the CDR-H1 sequence X5YAX6N are selected from KYAMN, VYAMN, SYAMN, GYAMN, RYAMN, TYAMN, IYAMN and KYAIN, with KYAMN or KYAIN being most preferred. Preferred examples of the CDR-H2 sequence RIRSKYNNYATYYADX7VKX8 are selected from RIRSKYNNYATYYADSVKD, RIRSKYNNYATYYADSVKK, RIRSKYNNYATYYADSVKS, RIRSKYNNYATYYADSVKE, RIRSKYNNYATYYADSVKG and RIRSKYNNYATYYADQVKD, with RIRSKYNNYATYYADSVKD being most preferred. The CDR-H3 sequence HX 14 NFGNSYX 15 SX 16 X 17 Preferred examples of AY are selected from HGNFGNSYISYWAY, HGNFGNSYLSWWAY, HGNFGNSYTSYYAY, HRNFGNSYLSWFAY, HGNFGNSYVSFFAY, HGNFGNSYISWWAY, HGNFGNSYVSWWAY, HGNFGNSYLSYFAY, HGNFGNSYLSFWAY, HANFGNSYISYWAY and HGNFGNSYVSWFAY, with HGNFGNSYISYWAY being most preferred.

[0104] For X1, the amino acid Q is preferred. For X2, the amino acid V is preferred. For X3, the amino acid V is preferred. For X4, the amino acid G is preferred. For X5, the amino acid K is most preferred, and for X6, the amino acid M is most preferred. A preferred combination for X5 and X6 is M for X6 combined with any amino acid residue for X5, such as combinations of X5 and X6 selected from K and M, V and M, and S and M, respectively, with the most preferred combination being K and M. Another preferred combination is K and I for X5 and X6, respectively. Preferred examples of the CDR-H1 sequence X5YAX6N are selected from KYAMN, VYAMN, SYAMN, GYAMN, RYAMN, TYAMN, IYAMN, and KYAIN, with KYAMN or KYAIN being most preferred. For X7, the amino acid S is most preferred, and for X8, the amino acid D is most preferred. Preferred combinations are S at X7 combined with any of the amino acid residues at X8, such as combinations of X7 and X8 selected from S and D, S and G, S and K, S and S, respectively, with the combination S and D being most preferred. Preferred examples of the CDR-H2 sequence RIRSKYNNYATYYADX7VKX8 are selected from RIRSKYNNYATYYADSVKD, RIRSKYNNYATYYADSVKK, RIRSKYNNYATYYADSVKS, RIRSKYNNYATYYADSVKK, RIRSKYNNYATYYADSVKG and RIRSKYNNYATYYADQVKD, with RIRSKYNNYATYYADSVKD being most preferred. For X9, the amino acid K is preferred. 10 For X, the amino acid N is preferred. 11 For X, the amino acid T is preferred. 12 For X, the amino acid A is preferred. 13 For X, amino acid V is preferred. 14 For X, the amino acid G is most preferred; 15 For 16 For 17 For the CDR-H3 sequence HX, the amino acid W is most preferred. 14 NFGNSYX 15 SX16 X 17 Preferred examples of AY are selected from HGNFGNSYISYWAY, HGNFGNSYLSWWAY, HGNFGNSYTSYYAY, HRNFGNSYLSWFAY, HGNFGNSYVSFFAY, HGNFGNSYISWWAY, HGNFGNSYVSWWAY, HGNFGNSYLSYFAY, HGNFGNSYLSFWAY, HANFGNSYISYWAY and HGNFGNSYVSWFAY, with HGNFGNSYISYWAY being most preferred. 18 For the amino acid sequence S is preferred.

[0105] ii) The VL region is [ka] X1 is T or A; X2 is P or S; X3 is S or A; X4 is V or T; X5 is G or E; X6 is G, R or A; X7 is S or T; X8 is G or S; X9 is N or Y; 10 is P or A; X 11 is Q or E; X 12 is G or D; X 13 is Q or H; X 14 is A or L; X 15 is P or F; X 16 is R or T; X 17 is G or A; X 18 is K or D; X 19 is F or M; X 20 is L or R; X 21 is A, P or V; X 22 is P or S; X 23 is T or V; X 24 is L or I; X 25 is G or D; X 26 is L or I; X 27 is S or T; X 28 is V or A; X 29 is P or T; X 30 is E or I; X 31is Y or F; X 32 is V, A, or T; and X 33 is R or L.

[0106] The CDR sequences within the VL region are as follows: X6SSTGAVTX7X8X9YX 10 a CDR-L1 sequence comprising or consisting of N; X 17 TX 18 X 19 X 20 X 21 X 22 a CDR-L2 sequence comprising or consisting of: 32 LWYSNX 33 A CDR-L3 sequence comprising or consisting of WV. Preferred examples of CDR-L1 sequences are selected from GSSTGAVTSGNYPN, RSSTGAVTSGYYPN, GSSTGAVTSGYYPN, ASSTGAVTSGNYPN, and RSSTGAVTTSNYAN, with GSSTGAVTSGNYPN being most preferred. Preferred examples of CDR-L2 sequences are selected from GTKFLAP, ATDMRPS, GTKFLVP, and GTNKRAP, with GTKFLAP being most preferred. Preferred examples of CDR-L3 sequences are selected from VLWYSNRWV, ALWYSNRWV, TLWYSNRWV, and ALWYSNLWV, with VLWYSNRWV being most preferred.

[0107] For X1, the amino acid T is preferred. For X2, the amino acid P is preferred. For X3, the amino acid S is preferred. For X4, the amino acid V is preferred. For X5, the amino acid G is preferred. For X6, the amino acid G is most preferred. For X7, the amino acid S is most preferred. For X8, the amino acid G is most preferred. For X9, the amino acid N is most preferred. 10 The amino acid P is most preferred. 10is a part of the CDR-L1 sequence in the VL region. Preferred examples of the CDR-L1 sequence are selected from GSSTGAVTSGNYPN, RSSTGAVTSGYYPN, GSSTGAVTSGYYPN, ASSTGAVTSGNYPN and RSSTGAVTTSNYAN, with GSSTGAVTSGNYPN being the most preferred. 11 For X, the amino acid Q is preferred. 12 For X, the amino acid G is preferred. 13 For X, the amino acid Q is preferred. 14 For X, the amino acid A is preferred. 15 For X, the amino acid P is preferred. 16 For X, the amino acid R is preferred. 17 For X, the amino acid G is preferred. 18 For X, the amino acid K is preferred. 19 For X, the amino acid F is preferred. 20 For X, the amino acid L is preferred. 21 For X, the amino acid A is preferred. 22 For X, the amino acid P is preferred. 17 ~X 22 is a part of the CDR-L2 sequence of the VL region. Preferred examples of the CDR-L2 sequence are selected from GTKFLAP, ATDMRPS, GTKFLVP and GTNKRAP, with GTKFLAP being the most preferred. 23 For X, the amino acid T is preferred. 24 For X, the amino acid L is preferred. 25 For X, the amino acid G is preferred. 26 For X, the amino acid L is preferred. 27 For X, the amino acid S is preferred. 28 For X, amino acid V is preferred. 29 For X, the amino acid P is preferred. 30 For X, the amino acid E is preferred. 31 For X, the amino acid Y is preferred. 32 For X, amino acid V is preferred. 33 For X, the amino acid R is preferred. 32 and X 33is a part of the CDR-L3 sequence of the VL region. Preferred examples of the CDR-L3 sequence are selected from VLWYSNRWV, ALWYSNRWV, TLWYSNRWV and ALWYSNLWV, with VLWYSNRWV being the most preferred.

[0108] In a more preferred embodiment, the VH region of i) is [ka] X is V or E; X is K, V, S, G, R, or I; X is M or I; X is S or Q; X is D, G, K, S, or E; X is G, R, or A; X is I, L, V, or T; X is Y, W, or F; and X is W or F; and ii) the VL region comprises or consists of the sequence QTVVTQEPSLTVSPGGTVTLTCX1SSTGAVTSGX2YPNWVQQKP X4 is K or D; X5 is F or M; X6 is L or R; X7 is A, P or V; X8 is P or S; and X9 is V, A or T.

[0109] i) The VH region is [ka] X is V or E; X is K, V, S, G, R or I; X is M or I; X is S or Q; X is D, G, K, S or E; X is G, R or A; X is I, L, V or T; X is Y, W or F; and X is W or F.

[0110] The CDR sequences within the VH region are as follows: a CDR-H1 sequence comprising or consisting of X2YAX3N; a CDR-H2 sequence comprising or consisting of RIRSKYNNYATYYADX4VKX5; and a CDR-H3 sequence comprising or consisting of HX6NFGNSYX7SX8X9AY. Preferred examples of the CDR-H1 sequence X2YAX3N are selected from KYAMN, VYAMN, SYAMN, GYAMN, RYAMN, TYAMN, IYAMN and KYAIN, with KYAMN or KYAIN being more preferred, and KYAMN being most preferred. Preferred examples of said CDR-H2 sequence RIRSKYNNYATYYADX4VKX5 are selected from RIRSKYNNYATYYADSVKD, RIRSKYNNYATYYADSVKK, RIRSKYNNYATYYADSVKS, RIRSKYNNYATYYADSVKE, RIRSKYNNYATYYADSVKG and RIRSKYNNYATYYADQVKD, with RIRSKYNNYATYYADSVKD or RIRSKYNNYATYYADQVKD being more preferred, and RIRSKYNNYATYYADSVKD being most preferred. Preferred examples of said CDR-H3 sequence HX6NFGNSYX7SX8X9AY are selected from HGNFGNSYISYWAY, HGNFGNSYLSWWAY, HGNFGNSYTSYYAY, HRNFGNSYLSWFAY, HGNFGNSYVSFFAY, HGNFGNSYISWWAY, HGNFGNSYVSWWAY, HGNFGNSYLSYFAY, HGNFGNSYLSFWAY, HANFGNSYISYWAY and HGNFGNSYVSWFAY, with HGNFGNSYISYWAY being the most preferred.

[0111] For X1, the amino acid V is preferred. For X2, the amino acid K is most preferred, and for X3, the amino acid M is most preferred. A preferred combination for X2 and X3 is M for X3 combined with any of the amino acid residues for X2, such as combinations of X2 and X3 selected from K and M, V and M, and S and M, respectively, with the most preferred combination being K and M. Another preferred combination is K and I for X2 and X3, respectively. Preferred examples of the CDR-H1 sequence X2YAX3N are selected from KYAMN, VYAMN, SYAMN, GYAMN, RYAMN, TYAMN, IYAMN, and KYAIN, with KYAMN or KYAIN being more preferred, and KYAMN being most preferred. For X4, the amino acid S is preferred. For X5, the amino acid D is most preferred. A preferred combination is S for X4 combined with any of the amino acid residues for X5, such as combinations for X4 and X5 selected from S and D, S and G, S and K, and S and S, respectively, with the most preferred combination being S and D. Preferred examples of the CDR-H2 sequence RIRSKYNNYATYYADX4VKX5 are selected from RIRSKYNNYATYYADSVKD, RIRSKYNNYATYYADSVKK, RIRSKYNNYATYYADSVKS, RIRSKYNNYATYYADSVKG and RIRSKYNNYATYYADQVKD, with RIRSKYNNYATYYADSVKD or RIRSKYNNYATYYADQVKD being preferred, and RIRSKYNNYATYYADSVKD being most preferred. For X6, the amino acid G is most preferred; for X7, the amino acid I is most preferred; for 8, the amino acid Y is most preferred; and for X9, the amino acid W is most preferred. Preferred examples of said CDR-H3 sequence HX6NFGNSYX7SX8X9AY are selected from HGNFGNSYISYWAY, HGNFGNSYLSWWAY, HGNFGNSYTSYYAY, HRNFGNSYLSWFAY, HGNFGNSYVSFFAY, HGNFGNSYISWWAY, HGNFGNSYVSWWAY, HGNFGNSYLSYFAY, HGNFGNSYLSFWAY, HANFGNSYISYWAY and HGNFGNSYVSWFAY, with HGNFGNSYISYWAY being the most preferred.

[0112] ii) The VL region comprises or consists of the sequence QTVVTQEPSLTVSPGGTVTLTCX1SSTGAVTSGX2YPNWVQQKPGQAPRGLIGX3TX4X5X6X7X8GTPARFSGSLLGGKAALTLSGVQPEDEAEYYCX9LWYSNRWVFGGGTKLTVL, wherein X1 is G, R, or A; X2 is N or Y; X3 is G or A; X4 is K or D; X5 is F or M; X6 is L or R; X7 is A, P, or V; X8 is P or S; and X9 is V, A, or T.

[0113] The CDR sequences within the VL region are as follows: a CDR-L1 sequence comprising or consisting of X1SSTGAVTSGX2YPN; a CDR-L2 sequence comprising or consisting of X3TX4X5X6X7X8; and a CDR-L3 sequence comprising or consisting of X9LWYSNRWV. Preferred examples of CDR-L1 sequences are selected from GSSTGAVTSGNYPN, RSSTGAVTSGYYPN, GSSTGAVTSGYYPN, ASSTGAVTSGNYPN, and RSSTGAVTTSNYAN, with GSSTGAVTSGNYPN being most preferred. Preferred examples of CDR-L2 sequences are selected from GTKFLAP, ATDMRPS, GTKFLVP, and GTNKRAP, with GTKFLAP being most preferred. Preferred examples of CDR-L3 sequences are selected from VLWYSNRWV, ALWYSNRWV, TLWYSNRWV, and ALWYSNLWV, with VLWYSNRWV being most preferred.

[0114] For X1, the amino acid G is most preferred. For X2, the amino acid N is preferred. X1 and X2 are part of the CDR-L1 sequence X1SSTGAVTSGX2YPN in the VL region. Preferred examples of the CDR-L1 sequence are selected from GSSTGAVTSGNYPN, RSSTGAVTSGYYPN, GSSTGAVTSGYYPN, ASSTGAVTSGNYPN, and RSSTGAVTTSNYAN, with GSSTGAVTSGNYPN being most preferred. For X3, the amino acid G is preferred. For X4, the amino acid K is preferred. For X5, the amino acid F is preferred. For X6, the amino acid L is preferred. For X7, the amino acid A is most preferred. For X8, the amino acid P is preferred. X3 to X8 are part of the CDR-L2 sequence X3TX4X5X6X7X8 in the VL region. Preferred examples of the CDR-L2 sequence are selected from GTKFLAP, ATDMRPS, GTKFLVP, and GTNKRAP, with GTKFLAP being most preferred. For X9, the amino acid V is most preferred. X9 is part of the CDR-L3 sequence X9LWYSNRWV of the VL region. Preferred examples of the CDR-L3 sequence are selected from VLWYSNRWV, ALWYSNRWV, TLWYSNRWV and ALWYSNLWV, with VLWYSNRWV being the most preferred.

[0115] In another preferred embodiment, the VH region of ii) is [ka] X1 is V or E; X2 is K, V, S, R, or I; X3 is M or I; X4 is S or Q; X5 is D, G, K, or S; X6 is G or A; X7 is I, L, V, or T; X8 is Y, W, or F; and X9 is W or F; and ii) the VL region comprises or consists of a sequence of QTVVTQEPSLTVSPGGTVTLTCX1SSTGAVTSGX2YPNWVQQKPGQAPRGLIGGTKFLX3PGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCX4LWYSNRWVFGGGTKLTVL or

[0116] ii) The VH region is [ka] X is V or E; X is K, V, S, R or I; X is M or I; X is S or Q; X is D, G, K or S; X is G or A; X is I, L, V or T; X is Y, W or F; and X is W or F.

[0117] The CDR sequences within the VH region are as follows: a CDR-H1 sequence comprising or consisting of X2YAX3N; a CDR-H2 sequence comprising or consisting of RIRSKYNNYATYYADX4VKX5; and a CDR-H3 sequence comprising or consisting of HX6NFGNSYX7SX8X9AY. Preferred examples of the CDR-H1 sequence X2YAX3N are selected from KYAMN, VYAMN, SYAMN, RYAMN, IYAMN, and KYAIN, with KYAMN or KYAIN being more preferred, and KYAMN being most preferred. Preferred examples of said CDR-H2 sequence RIRSKYNNYATYYADX4VKX5 are selected from RIRSKYNNYATYYADSVKD, RIRSKYNNYATYYADSVKK, RIRSKYNNYATYYADSVKS, RIRSKYNNYATYYADSVKG and RIRSKYNNYATYYADQVKD, with RIRSKYNNYATYYADSVKD or RIRSKYNNYATYYADQVKD being more preferred, and RIRSKYNNYATYYADSVKD being most preferred. Preferred examples of the CDR-H3 sequence HX6NFGNSYX7SX8X9AY are selected from HGNFGNSYISYWAY, HGNFGNSYLSWWAY, HGNFGNSYTSYYAY, HRNFGNSYLSWFAY, HGNFGNSYVSFFAY, HGNFGNSYISWWAY, HGNFGNSYVSWWAY, HGNFGNSYLSYFAY, HGNFGNSYLSFWAY, HANFGNSYISYWAY and HGNFGNSYVSWFAY, with HGNFGNSYISYWAY or HANFGNSYISYWAY being more preferred, and HGNFGNSYISYWAY being most preferred.

[0118] For X1, the amino acid V is preferred. For X2, the amino acid K is most preferred, and for X3, the amino acid M is most preferred. A preferred combination for X2 and X3 is M for X3 combined with any of the amino acid residues for X2, such as combinations of X2 and X3 selected from K and M, V and M, and S and M, respectively, with the most preferred combination being K and M. Another preferred combination is K and I for X2 and X3, respectively. Preferred examples of the CDR-H1 sequence X2YAX3N are selected from KYAMN, VYAMN, SYAMN, RYAMN, IYAMN, and KYAIN, with KYAMN or KYAIN being more preferred, and KYAMN being most preferred. For X4, the amino acid S is preferred. For X5, the amino acid D is most preferred. A preferred combination is S for X4 combined with any of the amino acid residues for X5, such as combinations for X4 and X5 selected from S and D, S and G, S and K, and S and S, respectively, with the most preferred combination being S and D. Preferred examples of the CDR-H2 sequence RIRSKYNNYATYYADX4VKX5 are selected from RIRSKYNNYATYYADSVKD, RIRSKYNNYATYYADSVKK, RIRSKYNNYATYYADSVKG and RIRSKYNNYATYYADQVKD, with RIRSKYNNYATYYADSVKD or RIRSKYNNYATYYADQVKD being more preferred, and RIRSKYNNYATYYADSVKD being most preferred, and selected from RIRSKYNNYATYYADSVKD, RIRSKYNNYATYYADSVKK, RIRSKYNNYATYYADSVKS, RIRSKYNNYATYYADSVKE, RIRSKYNNYATYYADSVKG and RIRSKYNNYATYYADQVKD being most preferred. For X6, the amino acid G is most preferred; for X7, the amino acid I is most preferred; for 8, the amino acid Y is most preferred; and for X9, the amino acid W is most preferred.Preferred examples of said CDR-H3 sequence HX6NFGNSYX7SX8X9AY are selected from HGNFGNSYISYWAY, HGNFGNSYLSWWAY, HGNFGNSYTSYYAY, HRNFGNSYLSWFAY, HGNFGNSYVSFFAY, HGNFGNSYISWWAY, HGNFGNSYVSWWAY, HGNFGNSYLSYFAY, HGNFGNSYLSFWAY, HANFGNSYISYWAY and HGNFGNSYVSWFAY, with HGNFGNSYISYWAY being the most preferred.

[0119] ii) The VL region comprises or consists of the sequence QTVVTQEPSLTVSPGGTVTLTCX1SSTGAVTSGX2YPNWVQQKPGQAPRGLIGGTKFLX3PGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCX4LWYSNRWVFGGGTKLTVL, wherein X1 is G or A; X2 is N or Y; X3 is A or V; and X4 is V, A, or T.

[0120] The CDR sequences within the VL region are as follows: a CDR-L1 sequence comprising or consisting of X1SSTGAVTSGX2YPN; a CDR-L2 sequence comprising or consisting of GTKFLX3P; and a CDR-L3 sequence comprising or consisting of X4LWYSNRWV. Preferred examples of CDR-L1 sequences are selected from GSSTGAVTSGNYPN, RSSTGAVTSGYYPN, GSSTGAVTSGYYPN, ASSTGAVTSGNYPN, and RSSTGAVTTSNYAN, with GSSTGAVTSGNYPN being most preferred. Preferred examples of CDR-L2 sequences are selected from GTKFLAP, ATDMRPS, GTKFLVP, and GTNKRAP, with GTKFLAP being most preferred. Preferred examples of CDR-L3 sequences are selected from VLWYSNRWV, ALWYSNRWV, TLWYSNRWV, and ALWYSNLWV, with VLWYSNRWV being most preferred.

[0121] For X1, the amino acid G is preferred. For X2, the amino acid N is preferred. X1 and X2 are part of the CDR-L1 sequence X1SSTGAVTSGX2YPN in the VL region. Preferred examples of the CDR-L1 sequence are selected from GSSTGAVTSGNYPN, RSSTGAVTSGYYPN, GSSTGAVTSGYYPN, ASSTGAVTSGNYPN, and RSSTGAVTTSNYAN, with GSSTGAVTSGNYPN being most preferred. X3 is part of the CDR-L2 sequence GTKFLX3P in the VL region. The CDR-L2 sequence can be either GTKFLAP or GTKFLVP, with GTKFLAP being preferred. For X3, the amino acid A is preferred. X4 is part of the CDR-L3 sequence X4LWYSNRWV in the VL region. Preferred examples of the CDR-L3 sequence are selected from VLWYSNRWV, ALWYSNRWV, and TLWYSNRWV, with VLWYSNRWV being most preferred. For X4, the amino acid V is most preferred.

[0122] In a further preferred embodiment, the VH region of i) is [ka] X1 is K or S; X2 is D or G; X3 is I or V; and X4 is Y or W; and ii) the VL region comprises or consists of the sequence QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGX1YPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCX2LWYSNRWVFGGGTKLTVL, where X1 is N or Y; and X2 is V or A.

[0123] i) The VH region is [ka] wherein X1 is K or S, with K being preferred; X2 is D or G, with D being preferred; X3 is I or V, with I being preferred; and X4 is Y or W, with Y being preferred.

[0124] The CDR sequences within the VH region are as follows: a CDR-H1 sequence comprising or consisting of X1YAMN; a CDR-H2 sequence comprising or consisting of RIRSKYNNYATYYADSVKX2; and a CDR-H3 sequence comprising or consisting of HGNFGNSYX3SX4WAY. The CDR-H1 sequence X1YAMN is selected from KYAMN or SYAMN, with KYAMN being preferred. A preferred example of the CDR-H2 sequence RIRSKYNNYATYYADSVKX2 is selected from RIRSKYNNYATYYADSVKD and RIRSKYNNYATYYADSVKG, with RIRSKYNNYATYYADSVKD being preferred. The CDR-H3 sequence HGNFGNSYX3SX4WAY is HGNFGNSYISYWAY or HGNFGNSYVSWWAY, with HGNFGNSYISYWAY being preferred.

[0125] ii) The VL region comprises or consists of the sequence QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGX1YPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCX2LWYSNRWVFGGGTKLTVL, wherein X1 is N or Y, with N being preferred; and X2 is V or A, with V being preferred.

[0126] The CDR sequences within the VL region are as follows: a CDR-L1 sequence comprising or consisting of GSSTGAVTSGX1YPN; a CDR-L2 sequence comprising or consisting of GTKFLAP; and a CDR-L3 sequence comprising or consisting of X2LWYSNRWV. The CDR-L1 sequence is GSSTGAVTSGNYPN or GSSTGAVTSGYYPN, with GSSTGAVTSGNYPN being preferred. The CDR-L3 sequence is VLWYSNRWV or ALWYSNRWV, with VLWYSNRWV being preferred.

[0127] In a preferred embodiment, the VH region of i) comprises or consists of the sequence defined in SEQ ID NO: 124, 172, 362, 552, 730, 928, 1106, 1296, 1474, 1664 or 1854, and the VL region of ii) comprises or consists of the sequence defined in SEQ ID NO: 125, 173, 363, 553, 731, 929, SEQ ID NO: 1107, SEQ ID NO: 1297, SEQ ID NO: 1475, SEQ ID NO: 1665 or SEQ ID NO: 1855. Preferred combinations of VH and VL regions of i) and ii) are defined in SEQ ID NOs: 124 and 125 (the binder combination designated "2B2"), 172 and 173 ("A2J"), 362 and 363 ("E1L"), 552 and 553 ("E2M"), 730 and 731 ("F12Q"), 928 and 929 ("F6A"), 1106 and 1107 ("F7O"), 1296 and 1297 ("G4H"), 1474 and 1475 ("H1E"), 1664 and 1665 ("H2C") or 1854 and 1855 ("I2C"). Preferably, the CD3ε binding domain comprises or consists of a combination of VH and VL regions i) and ii) selected from the group consisting of SEQ ID NOs: 124 and 125, 172 and 173, 362 and 363, 552 and 553, 730 and 731, 928 and 929, 1106 and 1107, 1296 and 1297, 1474 and 1475, 1664 and 1665 and 1854 and 1855. More preferably, the CD3ε binding domain comprises or consists of a combination of VH and VL regions i) and ii) selected from the group consisting of SEQ ID NOs: 124 and 125, 362 and 363, 730 and 731, 928 and 929, 1106 and 1107, 1296 and 1297, 1474 and 1475, 1664 and 1665, and 1854 and 1855. Even more preferably, the CD3ε binding domain comprises or consists of a combination of VH and VL regions i) and ii) selected from the group consisting of SEQ ID NOs: 124 and 125, 730 and 731, 1664 and 1665, and 1854 and 1855.In an even more preferred embodiment, the CD3ε binding domain comprises or consists of a combination of VH and VL regions of i) and ii) selected from the group consisting of SEQ ID NOs: 730 and 731, 1664 and 1665 and 1854 and 1855.

[0128] In a most preferred embodiment, the VH region of i) is [ka] and ii) the VL region comprises or consists of the sequence QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL (SEQ ID NO: 1855).

[0129] As outlined herein above in relation to amino acid substitutions in the CDR sequences and also applicable to this embodiment, the VH region (i) and / or the VL region (ii) contain one or more amino acid substitutions as specified herein above. Accordingly, at least one of the recited amino acid substitutions in the variable regions is included in the binding domain that binds to an extracellular epitope of the human CD3ε chain. The at least one or more amino acid substitutions may be present in the CDR sequences of the VH region or the VL region, if only one amino acid substitution is present, or in the CDRs of both the VH and VL regions, if two or more amino acid substitutions are present, or may be limited to the CDR sequences of the VH region or the VL region only. In certain embodiments where two or more amino acid substitutions are present, the substitutions are present in both the VH and VL regions or in only one variable region. Preferably, the substitutions are present in the VH and VL regions.

[0130] Specifically, the VH and / or VL region sequences of i) and / or ii) are ai), N30 (preferably S), X634V, X634F, Q39 (E, K, R, D, preferably E), L45 (M or V, preferably M), D64 (preferably E), X765A, X 12 81V, X 12 81T, X 12 81I, V99 (A or L, preferably A), H101 (preferably A or N), X 14 102E, F104 (preferably I), N106 (preferably S or T); and b.ii) in the VL region sequence L20 (preferably I or M), V38 (I, L, M, F or Y, preferably I), X 11 40R, X 11 40K, X 24 69 (S or E, preferably S), X 32 91L, X 33 93 (preferably Y), G102 (preferably S) It contains one or more amino acid substitutions selected from:

[0131] In the VH region sequence, the amino acid substitution at position 30 is preferably S (and therefore can be written as N30S; similarly for subsequent substitutions given according to position number). The amino acid substitution at position 34 is V or F. Preferably, position 34 of the VH region sequence contains I or M, most preferably I. The amino acid substitution at position 39 is E, K, R, or D, preferably E. The amino acid substitution at position 45 is preferably M or V, more preferably M. The amino acid substitution at position 64 is preferably E. The amino acid substitution at position 65 is A. The amino acid substitution at position 81 is V, T, or I, preferably V. The amino acid substitution at position 99 is preferably A or L, more preferably A. The amino acid substitution at position 101 is preferably A or N, more preferably A (H1A). The amino acid substitution at position 102 is E. The amino acid substitution at position 104 is preferably I. The amino acid substitution at position 106 is preferably S or T, more preferably S.

[0132] Notwithstanding the above, in the CDR-H2 sequence within the VH region sequence, the amino acid substitution at position 64 is preferably D15E, or the amino acid substitution at position 65 is with amino acid A. Preferably, the CDR-H2 contains an amino acid substitution at position 65 with amino acid A; in this case, the preferred amino acid for the last amino acid of CDR-H2 is D or G, preferably D. Therefore, preferred CDR-H2 sequences are RIRSKYNNYATYYADAVKD or RIRSKYNNYATYYADAVKG, with RIRSKYNNYATYYADAVKD being more preferred. When CDR-H2 contains two amino acid substitutions, the preferred combination is amino acid E at position 64 and amino acid A at position 64; in this case, the preferred amino acid for the last amino acid of CDR-H2 is G. Therefore, a preferred CDR-H2 sequence for the combination of amino acid E at position 64 and amino acid A at position 65 is RIRSKYNNYATYYAEAVKG.

[0133] In the CDR-H3 sequence of the VH sequence, the amino acid substitutions are at position 101, preferably with amino acid A or N, at position 102 with amino acid E, at position 104, preferably with amino acid I, and at position 106, preferably with amino acid S or T. Preferably, the CDR-H3 comprises amino acid A or N at position 101 and amino acid S or T at position 106; in this case and when a placeholder is present at the indicated position in the CDR-H3 sequence, X 14 Preferred amino acids of X are G or A, most preferred is G; 15 is I;X 16 is Y;X 17 is W or Y. When the CDR-H3 contains two amino acid substitutions, the preferred combinations are amino acid A at position 101 and amino acid S at position 106; and amino acid N at position 101 and amino acid T at position 106; in this case and when a placeholder is present at the indicated position in the CDR-H3 sequence, X 14 Preferred amino acids of X are G or A, most preferred is G; 15 is I and X 16 is Y and X 17are W and F. When the CDR-H3 contains three amino acid substitutions, the preferred combinations are amino acid A at position 101, amino acid I at position 104, and amino acid S at position 106; and amino acid N at position 101, amino acid I at position 104, and amino acid T at position 106, in which case and when a placeholder is present at the indicated position in the CDR-H3 sequence, X 15 The preferred amino acid of X is I, 16 is Y and X 17 are W and F. If CDR-H3 contains four amino acid substitutions, the preferred combination is amino acid N at position 101, amino acid E at position 102, amino acid I at position 104 and amino acid T at position 106; in this case and when a placeholder is present in the indicated position of CDR-H3, X 15 The preferred amino acid of X is I, 16 is Y and X 17 is W. Thus, preferred CDR-H3 sequences are selected from AGNFGSSYISYWAY, NENIGTSYISYWAY, AGNFGTSYISYWAY, NANFGTSYISYFAY and AGNFGSSYISYFAY, with AGNFGSSYISYWAY and AGNFGSSYISYFAY being most preferred.

[0134] In the VL region sequence, the amino acid substitution at position 20 is preferably I or M, more preferably I. The amino acid substitution at position 38 is preferably with I, L, M, F or Y, more preferably I. The amino acid substitution at position 40 is preferably K or R, more preferably K. The amino acid substitution at position 69 is preferably S or E, more preferably S. The amino acid substitution at position 91 is L. The amino acid substitution at position 93 is preferably Y. The amino acid substitution at position 102 is preferably S.

[0135] Notwithstanding the above, in the CDR-L3 sequence within the VL region sequence, the amino acid substitution at position 1 is with amino acid L, or the amino acid substitution is W93, preferably W93Y. Preferably, when CDR-L3 contains an amino acid substitution, the CDR-L3 sequence is VLYYSNRWV. However, it is preferred that CDR-L3 does not contain the X11L and W93 amino acid substitutions, in which case the preferred CDR-L3 sequence is VLYYSNRWV.

[0136] In accordance with the foregoing, when there is only one amino acid substitution in the CD3ε-binding domain within the VH and / or VL region sequences defined herein, it is preferred that the amino acid substitution H101A in the VH region (part of CDR-H3) is that amino acid substitution. Furthermore, in the case of a combination of amino acid substitutions as defined in iii), it is preferred that H101A in the VH region sequence is one of the amino acid substitutions in said combination.

[0137] In a preferred embodiment of the polypeptide or polypeptide construct of the present invention, in addition to the single amino acid substitution or combination thereof defined in iii), X6 is I; X8 is G; and in the VH region sequence, X 12 is L and X 14 is A and X 17 is F; and / or X 32 In a preferred embodiment of the polypeptide or polypeptide construct of claim 1 of the present invention, in addition to the single amino acid substitution or combination thereof as defined in iii), X6 is I; X8 is G; and in the VH region sequence, X 14 is A and X 17 is F; and / or in said VL region sequence, X 32 is A. One or a combination of amino acid substitutions defined herein may be further combined with a particular amino acid at a particular position in a CDR. Thus, if at least one of the amino acid substitutions listed under iii) is present and at least one of X8 is G; 14 is A and X17 is F; and / or in said VL region sequence, X 32 is A and is further present in the CDR. As previously mentioned herein above, when only one additional amino acid is present in the CDR, the preferred amino acid for X6 in said VH region sequence is I, or X8 is G, and most preferably X5 in said VH region sequence is I. Two preferred combinations of said additional amino acids are when X6 is I and X8 is G in said VH region sequence. 17 or in said VH region sequence, X8 is G and X 17 Three preferred combinations of the additional amino acids are: X6 is I and X 14 is A and X 17 or in the VH region sequence, X6 is I and X 17 is F, and in the VL region sequence X 32 is A. Since the latter amino acid will also be present, it is understood that the additional amino acids listed that overlap in position with the amino acid substitution in iii) cannot replace the overlapping amino acid substitution. For example, if the only amino acid substitution in iii) is either X634V or X634F in CDR-H1, then said amino acid cannot be replaced with amino acid I in said CDR-H1 sequence, since there is no remaining amino acid substitution defined in iii) in the binding domain. In other words, at least one of the amino acid substitutions defined in iii) must always be present in the polypeptide or polypeptide construct of the present invention.

[0138] It is also understood that depending on the actual sequence of the VH and / or VL region sequences, one or more of said additional amino acids may already be part of the given variable sequence as defined herein above, or if not, said given variable sequence will be altered to present said additional amino acids in combination with one or a combination of amino acid substitutions as listed under iii).

[0139] In a preferred embodiment of the polypeptide or polypeptide construct of the invention, the polypeptide or polypeptide construct comprises a combination of said amino acid substitutions as defined in iii) of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more amino acid substitutions.

[0140] In a preferred embodiment of the polypeptide or polypeptide construct of the invention, i) said one amino acid substitution is a. X634V, Q39E, Q39K, Q39R, Q39D, L45M, L45V, X634V in the VH region 12 81V, X 12 81T, X 12 81I, V99A, V99L, H101A, H101N; and b.V38I, V38L, V38M, V38F, V38Y, X 11 40R, X 11 40K, X 24 69S, X 24 69E, X 32 91L, X 33 ii) a combination of two or more amino acid substitutions is selected from X634F and A81V (X634F and A81V) in the VH region; 12 Q39E in the VH region and X in the VL region; 11 40K; Q39E in the VH region and X in the VL region 11 40R; Q39D in the VH region and X in the VL region 11 40K; Q39D in the VH region and X in the VL region 11 40R; L45V in the VH region and V38F in the VL region; L45V in the VH region and V38Y in the VL region; L45M in the VH region and X in the VL region 11 40K; L20I and G102S in the VL region; A81V in the VH region and X in the VL region 11 iii) the combination of three or more amino acid substitutions is selected from Q39E in the VH region, X39E in the VL region, 11 40K and G102S; Q39E in the VH region, X in the VL region 11 40R and G102S; Q39D in the VH region, X in the VL region11 40K and G102S; Q39D in the VH region, X in the VL region 11 40R and G102S; L45M in the VH region, X in the VL region 11 40K and G102S; X765A, H101A and N106S in the VH region; L20I, X in the VL region 11 iv) the combination of four or more amino acid substitutions is selected from Q39E in the VH region, L20I in the VL region, X 11 40K and G102S; Q39E in the VH region, L20I in the VL region, X 11 40R and G102S; Q39D in the VH region, L20I in the VL region, X 11 40K and G102S; Q39D in the VH region, L20I in the VL region, X 11 40R and G102S; L45M in the VH region, L20I in the VL region, X 11 40K and G102S; L20I, X in the VL region 11 40K, X 24 69S and G102S; A81V in the VH region, L20I in the VL region, X 11 and / or v) said combination of 5, 6, 7, 8, 9, 10 or more amino acid substitutions is selected from X765A, A81V, V99A, N106S in the VH region, L20I, X765A, A81V, V99A, N106S in the VL region, 11 40K, X 24 69S and G102S; D64E, X765A, A81V, X in the VH region 14 102E, F104I, N106T, L20I in the VL region, X 24 69S and G102S; L45M, X765A, N106T in the VH region, L20I, X in the VL region 11 40K, X 24 69S and G102S; X765A, A81V, V99A, H101A, F104I, N106S in the VH region, L20I, X in the VL region 11 40K, X 2469S, G102S; X765A, A81V, V99A, H101A, N106S in the VH region; A81V, V99A in the VH region, L20I, X in the VL region 11 40K, X 24 69S, G102S; Q39E, A81V in VH region, L20I, X in VL region 11 40K, G102S; L45V in VH region, L20I, V38F, X in VL region 11 Selected from 40K and G102S.

[0141] In a more preferred embodiment of the polypeptide or polypeptide construct of the invention, said combination of amino acid substitutions is i) in the VH region, a.X765A, A81V, V99A, H101A and N106S; b.D64E, X765A, A81V, H101N, X 14 102E, F104I and N106S; c.L45M, X765A, H101A and N106T; d.L45M, X765A, H101A and N106S; e.Q39E, X765A, H101N and N106T; f.D64E, X765A, V99A, H101A and N106T; g.X765A, A81V, V99A, H101A, X 14 102E, F104I and N106T; h.X765A, A81V, H101N, X 14 102E, F104I and N106S; i.D64E, X765A, A81V, H101A and N106S; j.D64E, X765A, H101A and N106T; k.X765A, V99A, H101A and N106T; l.D64E, X765A, H101A and N106S; m.D64E, X765A, A81V, V99A, H101A and N106S; n.X765A, H101A and N106S; o.N30S, Q39E, D64E, X765A, A81V, H101A, X 14 102E, F104I and N106T; p.L45M, D64E, X765A, H101A and N106T; q.N30S, L45M, X765A, A81V, H101A and N106T; r.N30S, L45M, D64E, X765A, A81V, H101A and N106S; ii) in the VL region, a.L20I, X 11 40K, X 24 69S and G102S; b.L20I, X 24 69S and G102S; c.L20I, V38I, X 11 40K, X 24 69E, G102S and W93Y; dX 11 40K and G102S; e.L20I,X 11 40K, X 24 69S, G102S and W93Y; f.L20M, X 11 40K and X 24 69E; g.L20I, V38I, X 11 40K, X 24 69E and G102S; hX 11 40K, X 24 69S and W93Y; iX 11 40K and X 24 69S; and iii) a combination of one amino acid substitution in i) and ii). is selected from.

[0142] In yet another preferred embodiment of the polypeptide or polypeptide construct of the invention, said combination of amino acid substitutions iii) is a. X765A, A81V, V99A, H101A, N106S in the VH region, L20I, X in the VL region11 40K, X 24 69S and G102S; b. D64E, X765A, A81V, H101N, and X in the VH region 14 102E, F104I, N106S, L20I in the VL region, X 24 69S and G102S; c. L45M, X765A, H101A, N106T in the VH region, L20I, X in the VL region 11 40K, X 24 69S and G102S; d. L45M, X765A, H101A, N106S in the VH region, L20I, V38I, X in the VL region 11 40K, X 24 69E, G102S and W93Y; e. Q39E, X765A, H101N, N106T in the VH region, X in the VL region 11 40K and G102S; f. D64E, X765A, V99A, H101A, N106T in the VH region, X in the VL region 11 40K and G102S; g. X765A, A81V, V99A, H101A, and X in the VH region 14 102E, F104I, N106T, L20I in the VL region, X 11 40K, X 24 69S and G102S; h. X765A, A81V, H101N, and X in the VH region 14 102E, F104I, N106S, L20I in the VL region, X 24 69S and G102S; i. D64E, X765A, A81V, H101A, N106S in the VH region, L20I, X in the VL region 24 69S and G102S; j. D64E, X765A, H101A, N106T in the VH region, X in the VL region 11 40K and G102S; k. X765A, V99A, H101A, N106T in the VH region, X in the VL region11 40K and G102S; l. D64E, X765A, H101A, N106S in the VH region, X in the VL region 11 40K and G102S; m. D64E, X765A, A81V, V99A, H101A, N106S in the VH region, L20I, X in the VL region 11 40K, X 24 69S, G102S and W93Y; n. X765A, H101A, N106S in the VH region, X in the VL region 11 40K and G102S; o.N30S, Q39E, D64E, X765A, A81V, H101A, X in the VH region 14 102E, F104I, N106T, L20M in the VL region, X 11 40K and X 24 69E; p.L45M, D64E, X765A, H101A, N106T in the VH region, L20I, V38I, X in the VL region 11 40K, X 24 69E and G102S; q. N30S, L45M, X765A, A81V, H101A, N106T in the VH region, X in the VL region 11 40K and X 24 69S; and r. N30S, L45M, D64E, X765A, A81V, H101A, N106S in the VH region, X in the VL region 11 40K and X 24 69S is selected from.

[0143] In another preferred embodiment of the polypeptide or polypeptide construct of the invention, in said combination of defined amino acid substitution combinations: In a. and m., X6 is I in the VH region sequence; In b. and r., X8 is G in the VH region sequence; In c., X 12 is L in the VH region; d. In the VH region, X 12 is L and X 17 is F; e., in the VH region sequence, X6 is I and X 14 is A, and X 17 is F; f. In the VH domain sequence, X6 is I; and in the VL domain sequence, X 32 is A; g., in the VH region sequence, X6 is I; h. and i. in said VH region sequence, X8 is G; In j., l. and n., in the VH region sequence, X6 is I, and X 17 is F; and in said VL region sequence, X 32 is A; In k., in the VH domain sequence, X6 is I; and in the VL domain sequence, X 32 is A; p., in the VH region, X 12 is L; or In q., in the VH region sequence, X8 is G and X 17 is F.

[0144] Exemplary VH and VL region sequences as defined in this embodiment are defined by SEQ ID NOs: 2012 and 2013; 2020 and 2021; 2028 and 2029; 2036 and 2037; 2044 and 2045; 26 and 27; 34 and 35; 42 and 43; 50 and 51; 58 and 59; 66 and 67; 74 and 75; 82 and 83; 90 and 91; 98 and 99; 100 and 101; 108 and 109; 116 and 117. Table 2 demonstrates the increased temperature stability of the aforementioned VH and VL region sequence combinations of the CD3ε binding domain over the CD3ε binding domain comprising the VH and VL combination defined as "I2C" (SEQ ID NOs: 1854 and 1855, respectively).

[0145] According to the present invention, the binding domain of the polypeptide or polypeptide construct of the present invention, which binds to an extracellular epitope of the human CD3 epsilon chain and comprises or consists of a VH region and a VL region as defined herein, comprises at least one of the following CDR sequences or combinations thereof as defined in SEQ ID NOs: 1878, 1908, 1989 and 2003, said sequences comprising one amino acid substitution or combinations thereof as defined in iii) and, in some instances, also comprising said further amino acids as defined herein above. Preferred combinations of said CDR sequences are listed below in the order CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3: 1878-1883; 1908-1913; 1984-1989; 1998-2003.

[0146] Combinations of CDR sequences in the order of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 defined in SEQ ID NOs: 126-131; 134-139; 142-147; 150-155; and 158-163 are also within the scope of the present invention.

[0147] According to the present invention, combinations of CDR sequences defined by SEQ ID NOs (listed in the order of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, CDR-L3): 2006-2011; 2014-2019; 2022-2027; 2030-2035; 2038-2043; 20-25; 36-41; 44-49; 52-57; 60-65; 68-73; 76-81; 84-89; 92-97; 102-107; 110-115 are also more preferred. Further examples include SEQ ID NOs: 282-287; 290-295; 298-303; 306-311; 314-319; 472-477; 480-485; 488-493; 496-501; 504-509; 650-655; 658-663; 666-671; 674-679; 682-687; 840-845; 848-853; 856-861; 864-869; 872-877; 1026-1031; 1034-1039; 1042-1047; 1050-1055; 1060-1061; 1062-1063; 1064-1065; 1066-1067; 1068-1069; 1070-1071; 1072-1073; 1074-1075; 1076-1077; 1078-1079; 1080-1081; 1082-1083; 1084-1085; 1086-1087; 1088-1089; 1090-1091; 1092-1093; 1094-1095; 1096-1097; 1098-1099; 2000-2001; 2002-2003; 2003-2004; 2004-2005; 2005-2006; 20 058~1063;1216~1221;1224~1229;1232~1237;1240~1245;1248~1253;1394~1399;1402~1407;1410~1415;1418~1423;1426~1431;1584~1589;1592~1597;1600~1605;1608~1613;1616~1621;1774~1779;1782~1787;1790~1795;1798~1803;1806~1811.

[0148] According to the present invention, a binding domain of a polypeptide or polypeptide construct of the invention that binds to an extracellular epitope of the human CD3 epsilon chain comprises or consists of a VH region sequence as defined in the following SEQ ID NOs: 1856, 1860, 1862, 1864, 1872, 1884, 1894, 1896, 1898, 1900, 1902, 1904, 1906, 1914, 1916, 1918, 1920, 1922, 1924, 1926, 1928, 1930, 1932, 1934, 1936, 1938, 1940, 1942, 1944, 1946, 1948, 1950, said sequence comprising one amino acid substitution or a combination thereof as defined herein above, and in some instances also comprising said further amino acid as defined herein above. This also applies to the following embodiments, which feature SEQ ID NO: CD3ε binding domains resulting from the amino acid residue exchanges specified herein.

[0149] According to the present invention, a binding domain of a polypeptide or polypeptide construct of the invention which binds to an extracellular epitope of the human CD3 epsilon chain comprises or consists of a VL region sequence as defined in the following SEQ ID NOs: 1867, 1869, 1871, 1875, 1877, 1899, 1901, 1903, 1905, 1907, 1919, 1921, 1923, 1925, 1927, 1929, 1931, 1933, 1935, 1937, 1939, 1941, 1943, 1961, 1963, 1965, 1967, 1969, 1971, 1975, 1991, 1993, 1995, 1997 or 2005.

[0150] Preferred combinations of VH and VL region sequences of said binding domains of a polypeptide or polypeptide construct of the invention that binds to an extracellular epitope of the human CD3 epsilon chain are represented by the following SEQ ID NOs: 1856 and 1857; 1860 and 1861; 1862 and 1863; 1864 and 1865; 1866 and 1867; 1868 and 1869; 1870 and 1871 ;1872 and 1873;1874 and 1875;1876 and 1877;1884 and 1885;1894 and 1895;1896 and 1897;1898 and 1899;1900 and 1901;1902 and 1903;1904 and 1905;1906 and 1907;1914 and 1915;1916 and 1917;1918 and 1919;19 20 and 1921; 1922 and 1923; 1924 and 1925; 1926 and 1927; 1928 and 1929; 1930 and 1931; 1932 and 1933; 1934 and 1935; 1936 and 1937; 1938 and 1939; 1940 and 1941; 1942 and 1943; 1944 and 1945; 1946 and 1947; 1948 and and 1949; 1950 and 1951; 1960 and 1961; 1962 and 1963; 1964 and 1965; 1966 and 1967; 1968 and 1969; 1970 and 1971; 1974 and 1975; 1990 and 1991; 1992 and 1993; 1994 and 1995; 1996 and 1997; or 2004 and 2005.

[0151] The VH region sequences defined in SEQ ID NOs: 132, 140, 148, 156 and 164 are also in accordance with the present invention.

[0152] The VL region sequences defined in SEQ ID NOs: 133, 141, 149, 157 and 165 are also in accordance with the present invention.

[0153] Any combination of the above particularly preferred VH and VL region sequences is also in accordance with the present invention. The following VH and VL combinations defined by the following SEQ ID NOs are preferred: SEQ ID NOs: 132 and 133; 140 and 141; 148 and 149; 156 and 157; and 164 and 165.

[0154] The VH region sequences defined by SEQ ID NOs: 2012, 2020, 2028, 2036, 2044, 26, 34, 42, 50, 58, 66, 74, 82, 90, 98, 100, 108 and 116 are also in accordance with the invention and are more preferred. Further examples are SEQ ID NOs: 288, 296, 304, 312, 320, 478, 486, 494, 502, 510, 656, 664, 672, 680, 688, 846, 854, 862, 870, 878, 1032, 1040, 1048, 1056, 1064, 1222, 1230, 1238, 1246, 1254, 1400, 1408, 1416, 1424, 1432, 1590, 1598, 1606, 1614, 1622, 1780, 1788, 1796, 1804 and 1812.

[0155] The VL region sequences defined by SEQ ID NOs: 2013, 2021, 2029, 2037, 2045, 27, 35, 43, 51, 59, 67, 75, 83, 91, 99, 101, 109 and 117 are also in accordance with the invention and are more preferred. Further examples are SEQ ID NOs: 289, 297, 305, 313, 321, 479, 487, 495, 503, 511, 657, 665, 673, 681, 689, 847, 855, 863, 871, 879, 1033, 1041, 1049, 1057, 1065, 1223, 1231, 1239, 1247, 1255, 1401, 1409, 1417, 1425, 1433, 1591, 1599, 1607, 1615, 1623, 1781, 1789, 1797, 1805 and 1813.

[0156] Any combination of the above particularly preferred VH and VL domain sequences is also in accordance with the invention and is preferred. The following VH and VL combinations defined by the following SEQ ID NOs are preferred: 2012 and 2013; 2020 and 2021; 2028 and 2029; 2036 and 2037; 2044 and 2045; 26 and 27; 34 and 35; 42 and 43; 50 and 51; 58 and 59; 66 and 67; 74 and 75; 82 and 83; 90 and 91; 98 and 99; 100 and 101; 108 and 109; and 116 and 117. Further examples include SEQ ID NOs: 288 and 289; 296 and 297; 304 and 305; 312 and 313; 320 and 321; 478 and 479; 486 and 487; 494 and 495; 502 and 503; 510 and 511; 656 and 657; 664 and 665; 672 and 673; 680 and 681; 688 and 689; 846 and 847; 854; 855; 862 and 863; 870 and 871; 878 and 879; 1032 and 1033; 1040 and 1041; 1048 and 1049; 1056 and 1057; 1058 and 1059; 1060 and 1061; 1062 and 1063; 1064 and 1065; 1066 and 1067; 1068 and 1069; 1070 and 1071; 1072 and 1073; 1074 and 1075; 1076 and 1077; 1078 and 1079; 1080 and 1081; 1082 and 1083; 1084 and 1085; 1086 and 1087; 1088 and 1089; 1090 and 1091; 1092 and 1093; 1094 and 1095; 1096 and 1097; 1098 and 1099; 2 064 and 1065; 1222 and 1223; 1230 and 1231; 1238 and 1239; 1246 and 1247; 1254 and 1255; 1400 and 1401; 1408 and 1409; 1416 and 1417; 1424 and 1425; 1432 and 1433; 1590 and 1591; 1598 and 1599; 1606 and 1607; 1614 and 1615; 1622 and 1623; 1780 and 1781; 1788 and 1789; 1796 and 1797; 1804 and 1805; and 1812 and 1813.

[0157] According to the invention, the binding domain of a polypeptide or polypeptide construct of the invention that binds to an extracellular epitope of the human CD3 epsilon chain is selected from the group consisting of SEQ ID NOs: 2012, 2020, 2028, 2036, 2044, 26, 34, 42, 50, 58, 66, 74, 82, 90, 98, 100, 108, 116, 288, 296, 304, 312, 320, 478, 486, 494, 502, 510, 656, 664, 672, 680, 688, 846, 854, 862, 870, 878, 1032, 1040, 1048, 1056, 1064, 1222, 1230, 1238, 1246, 1254, 140 0, 1408, 1416, 1424, 1432, 1590, 1598, 1606, 1614, 1622, 1780, 1788, 1796, 1804, 1812, 1856, 1860, 1862, 1864, 1872, 1884, 1894, 1896, 1898, 1900, 1902, 1904, 1 a VH region sequence selected from 906, 1914, 1916, 1918, 1920, 1922, 1924, 1926, 1928, 1930, 1932, 1934, 1936, 1938, 1940, 1942, 1944, 1946, 1948, 1950, 132, 140, 148, 156, and 164;and / or SEQ ID NOs: 2013, 2021, 2029, 2037, 2045, 27, 35, 43, 51, 59, 67, 75, 83, 91, 99, 101, 109 and 117, 289, 297, 305, 313, 321, 479, 487, 495, 503, 511, 657, 665, 673, 681, 689, 847, 855, 863, 871, 879, 1033, 1041, 1049, 1057, 1065, 1223, 1231, 1239, 1247, 1255, 1401, 1409, 1417, 1425, 1433, 1591, 1599, 1607, 1615 , 1623, 1781, 1789, 1797, 1805, 1813, 1867, 1869, 1871, 1875, 1877, 1899, 1901, 1903, 1905, 1907, 1919, 1921, 1923, 1925, 1927, 1929, 1931, 1933, 1935, 1937, 1939, 1941, 1943, 1961, 1963, 1965, 1967, 1969, 1971, 1975, 1991, 1993, 1995, 1997, 2005, 133, 141, 149, 157, and 165;

[0158] According to the present invention, preferably, the polypeptide or polypeptide construct comprises a binding domain that binds to an extracellular epitope of said human CD3 epsilon chain, comprising or consisting of i) a VH region and ii) a VL region, wherein said VH and VL regions i) and ii) are selected from the VH and VL region combinations defined in SEQ ID NOs: 124 and 125, 172 and 173, 362 and 363, 552 and 553, 730 and 731, 928 and 929, 1106 and 1107, 1296 and 1297, 1474 and 1475, 1664 and 1665 and 1854 and 1855, and wherein said single amino acid substitution or combination thereof in said VH and / or VL region sequence results in a VH and / or VL region sequence having the following amino acid residues: i. I at position 34, A at position 65, V at position 81, A at position 99, A at position 101, S at position 106 in the VH domain sequence, I at position 20, K at position 40, S at position 69, and S at position 102 in the VL domain sequence; M at position 45, A at position 65, L at position 81, A at position 101, T at position 106 in the VH region sequence, I at position 20, K at position 40, S at position 69, and S at position 102 in the VL region sequence; M at position 45, A at position 65, L at position 81, A at position 101, S at position 106, F at position 112 in the VH region sequence, I at position 20, I at position 38, K at position 40, E at position 69, S at position 102, and Y at position 93 in the VL region sequence; I at position 34, E at position 39, A at position 65, N at position 101, A at position 102, T at position 106, F at position 112 in the VH region sequence, K at position 40 and S at position 102 in the VL region sequence; I at position 34, E at position 64, A at position 65, A at position 99, A at position 101, T at position 106 in the VH region sequence, K at position 40, A at position 91, and S at position 102 in the VL region sequence; I at position 34, A at position 65, V at position 81, A at position 99, A at position 101, E at position 102, I at position 104, T at position 106 in the VH region sequence, I at position 20, K at position 40, S at position 69, and S at position 102 in the VL region sequence; E at position 64, A at position 65, G at position 68, V at position 81, A at position 101, S at position 106 in the VH region sequence, I at position 20, S at position 69, and S at position 102 in the VL region sequence; I at position 34, E at position 64, A at position 65, A at position 101, T at position 106, F at position 112 in the VH region sequence, K at position 40, A at position 91, and S at position 102 in the VL region sequence; I at position 34, E at position 64, A at position 65, A at position 101, S at position 106, F at position 112 in the VH region sequence, K at position 40, A at position 91, and S at position 102 in the VL region sequence; I at position 34, E at position 64, A at position 65, V at position 81, A at position 99, A at position 101, S at position 106 in the VH region sequence, I at position 20, K at position 40, S at position 69, S at position 102, and Y at position 93 in the VL region sequence; I at position 34, A at position 65, A at position 101, S at position 106, F at position 112 in the VH region sequence, K at position 40, A at position 91, and S at position 102 in the VL region sequence; M at position 45, E at position 64, A at position 65, L at position 81, A at position 101, T at position 106 in the VH region sequence, I at position 20, I at position 38, K at position 40, E at position 69, and S at position 102 in the VL region sequence; S at position 30, M at position 45, A at position 65, G at position 68, V at position 81, A at position 101, T at position 106, F at position 112 in the VH region sequence, K at position 40, S at position 69, and Y at position 93 in the VL region sequence; and S at position 30, M at position 45, E at position 64, A at position 65, G at position 68, V at position 81, A at position 101, S at position 106 in the VH region sequence, K at position 40 and S at position 69 in the VL region sequence; ii. E at position 64, A at position 65, G at position 68, V at position 81, N at position 101, E at position 102, I at position 104, T at position 106 in the VH domain sequence, I at position 20, S at position 69, and S at position 102 in the VL domain sequence; A at position 65, G at position 68, V at position 81, N at position 101, E at position 102, I at position 104, S at position 106 in the VH region sequence, I at position 20, S at position 69, and S at position 102 in the VL region sequence; an I at position 34, an A at position 65, an A at position 99, an A at position 101, a T at position 106 in the VH region sequence, a K at position 40, an A at position 91, and an S at position 102 in the VL region sequence; and S at position 30, E at position 39, E at position 64, A at position 65, V at position 81, A at position 101, E at position 102, I at position 104, T at position 106 in the VH region sequence, M at position 20, K at position 40, and E at position 69 in the VL region sequence; iii. A at position 101 in the VH region sequence; iv. V at position 81, A at position 99 in the VH domain sequence, I at position 20, K at position 40, S at position 69, and S at position 102 in the VL domain sequence; I at position 34, V at position 81 in the VH region sequence, I at position 20, K at position 40, and S at position 102 in the VL region sequence; I at position 34 in the VH region sequence, I at position 20, K at position 40, and S at position 102 in the VL region sequence; I at position 34, E at position 39, V at position 81 in the VH region sequence, I at position 20, K at position 40, and S at position 102 in the VL region sequence; v. I at position 34 in the VH region sequence, V at position 81, and K at position 40 in the VL region sequence; M at position 45 in the VH region sequence, I at position 20, K at position 40, and S at position 102 in the VH region sequence; I at position 34, A at position 65, A at position 101, and S at position 106 in the VH region sequence; I at position 20, K at position 40, S at position 69, and S at position 102 in the VL region sequence; F at position 34 and V at position 81 in the VH region sequence; M at position 45 in the VH region sequence and K at position 40 and S at position 102 in the VL region sequence; D at position 39 in the VH region sequence, I at position 20, K at position 40, and S at position 102 in the VL region sequence; I at position 34 and V at position 81 in the VH region sequence; E at position 39 in the VH region sequence, I at position 20, K at position 40, and S at position 102 in the VL region sequence; M at position 45 in the VH region sequence and K at position 40 in the VL region sequence; an I at position 34, an A at position 65, a V at position 81, an A at position 99, an A at position 101, and an S at position 106 in the VH region sequence; or vi. D at position 39 in the VH domain sequence, I at position 20, R at position 40, and S at position 102 in the VL domain sequence; I at position 34 in the VH region sequence; K at position 40 in the VL region sequence; D at position 39 in the VH region sequence and K at position 40 and S at position 102 in the VL region sequence; I at position 20 and S at position 102 in the VH region sequence; K at position 39 in the VH region sequence; E at position 39 in the VH region sequence and K at position 40 and S at position 102 in the VL region sequence; E at position 39 in the VH region sequence, I at position 20, R at position 40, and S at position 102 in the VL region sequence; D at position 39 in the VH region sequence and R at position 40 and S at position 102 in the VL region sequence; V at position 81 in the VH region sequence; D at position 39 in the VH region sequence and K at position 40 in the VL region sequence; F at position 112 in the VH region sequence; F at position 112 in the VH region sequence and I at position 38 in the VL region sequence; E at position 39 in the VH region sequence and K at position 40 in the VL region sequence; V at position 34 in the VH region sequence.

[0159] The above amino acid residues are present at a given position in the CD3ε-binding domain according to the present invention. If one of the VH and / or VL region sequences to be altered, i.e., the base VH and / or VL region sequence, already contains one of the above amino acid residues at a given position, then it cannot be altered, since the respective amino acid residue is already present in the VH and / or VL region of i) and / or ii).

[0160] More preferably, the CD3ε binding domain comprises or consists of a combination of VH and VL regions i) and ii) selected from the group consisting of SEQ ID NOs: 124 and 125, 362 and 363, 730 and 731, 928 and 929, 1106 and 1107, 1296 and 1297, 1474 and 1475, 1664 and 1665, and 1854 and 1855. Even more preferably, the CD3ε binding domain comprises or consists of a combination of VH and VL regions i) and ii) selected from the group consisting of SEQ ID NOs: 124 and 125, 730 and 731, 1664 and 1665, and 1854 and 1855. In an even more preferred embodiment, the CD3ε binding domain comprises or consists of a combination of VH and VL regions i) and ii) selected from the group consisting of SEQ ID NOs: 730 and 731, 1664 and 1665, and 1854 and 1855. Most preferred, said CD3ε binding domain comprises or consists of a combination of VH and VL regions i) and ii) of SEQ ID NOs: 1854 and 1855, respectively.

[0161] As is evident from the Examples section, the amino acid residues listed above at given positions have a favorable effect on increasing thermal stability as measured by the well-known and herein described method of DSF (differential scanning fluorimetry); for example, when introduced into the VH and VL sequences listed above in i) and ii), see Wen et al., "Nano differential scanning fluorimetry for comparability studies of therapeutic proteins," Analytical Biochemistry, Volume 593, 2020, 113581, ISSN 0003-2697; Dart, M. Let al. (2018) "Homogeneous Assay for Target Engagement Utilizing Bioluminescent Thermal Shift," ACS Medical Chemistry Letters, 9(6), 546-551. More specifically, the amino acid residues at given positions listed in sections i) and iv) show an increase in thermal stability of 6°C or more compared to the value measured for the unmodified sequences, i.e., the respective base VH and VL sequences. Sections ii. and v. exhibit an increase in temperature stability of 3°C or more compared to the value measured for the unmodified sequences, i.e., the respective basic VH and VL sequences, while sections iii. and vi. exhibit an increase in temperature stability of 1°C or more compared to the value measured for the unmodified sequences, i.e., the respective basic VH and VL sequences.

[0162] Preferably, the amino acid residue at a given position in sections i., ii., iv. and / or v. is present in the CD3ε-binding domain according to this embodiment. More preferably, the amino acid residue at a given position in sections i. and / or iv. is present in the CD3ε-binding domain according to the present invention. This preference also applies to the preferred embodiments listed below. As outlined herein, it is also preferred that the VH and VL regions are linked by a linker, preferably a peptide linker, most preferably a G4S or G4Q linker; preferably, said G4S or G4Q linker is repeated three times, i.e., a (G4S)3 or (G4Q)3 linker.

[0163] According to the present invention, there is provided a polypeptide or polypeptide construct comprising a binding domain that binds to an extracellular epitope of the human CD3 epsilon chain, comprising or consisting of a VH region and a VL region, a) the VH region comprises or consists of the sequence set forth in SEQ ID NO: 1854 and the VL region comprises or consists of the sequence set forth in SEQ ID NO: 1855; the VH and / or VL region sequences are i. M34I, S65A, A81V, V99A, H101A, N106S in the VH domain sequence (the resulting VH sequence defined in SEQ ID NO: 2012), L20I, Q40K, L69S, and G102S in the VL domain sequence (the resulting VL sequence defined in SEQ ID NO: 2013); L45M, S65A, A81L, H101A, N106T in the VH region sequence (SEQ ID NO: 2036), L20I, Q40K, L69S, and G102S in the VL region sequence (SEQ ID NO: 2037); L45M, S65A, A81L, H101A, N106S, W112F in the VH region sequence (SEQ ID NO: 2028), L20I, V38I, Q40K, L69E, G102S, and W93Y in the VL region sequence (SEQ ID NO: 2029); M34I, Q39E, S65A, H101N, G102A, N106T, W112F in the VH region sequence (SEQ ID NO: 2044), Q40K and G102S in the VL region sequence (SEQ ID NO: 2045); M34I, D64E, S65A, V99A, H101A, N106T in the VH region sequence (SEQ ID NO: 34), Q40K, V91A, and G102S in the VL region sequence (SEQ ID NO: 35); M34I, S65A, A81V, V99A, H101A, G102E, F104I, N106T in the VH region sequence (SEQ ID NO: 90), L20I, Q40K, L69S, and G102S in the VL region sequence (SEQ ID NO: 91); D64E, S65A, D68G, A81V, H101A, N106S in the VH region sequence (SEQ ID NO: 100), L20I, L69S, G102S in the VL region sequence (SEQ ID NO: 101); M34I, D64E, S65A, H101A, N106T, W112F in the VH region sequence (SEQ ID NO: 58), Q40K, V91A, and G102S in the VL region sequence (SEQ ID NO: 59); M34I, D64E, S65A, H101A, N106S, W112F in the VH region sequence (SEQ ID NO: 50), Q40K, V91A, and G102S in the VL region sequence (SEQ ID NO: 51); M34I, D64E, S65A, A81V, V99A, H101A, N106S in the VH region sequence (SEQ ID NO: 98), L20I, Q40K, L69S, G102S, and W93Y in the VL region sequence (SEQ ID NO: 99); M34I, S65A, H101A, N106S, W112F in the VH region sequence (SEQ ID NO: 42), Q40K, V91A, and G102S in the VL region sequence (SEQ ID NO: 43); L45M, D64E, S65A, A81L, H101A, N106T in the VH region sequence (SEQ ID NO: 116), L20I, V38I, Q40K, L69E, and G102S in the VL region sequence (SEQ ID NO: 117); N30S, L45M, S65A, D68G, A81V, H101A, N106T, W112F in the VH region sequence (SEQ ID NO: 82), Q40K, L69S, and W93Y in the VL region sequence (SEQ ID NO: 83); and N30S, L45M, D64E, S65A, D68G, A81V, H101A, N106S in the VH region sequence (SEQ ID NO: 74), Q40K and L69S in the VL region sequence (SEQ ID NO: 75); ii. D64E, S65A, D68G, A81V, H101N, G102E, F104I, N106T in the VH domain sequence (SEQ ID NO: 2020), L20I, L69S, and G102S in the VL domain sequence (SEQ ID NO: 2021); S65A, D68G, A81V, H101N, G102E, F104I, N106S in the VH region sequence (SEQ ID NO: 108), L20I, L69S, and G102S in the VL region sequence (SEQ ID NO: 109); M34I, S65A, V99A, H101A, N106T in the VH region sequence (SEQ ID NO: 26), Q40K, V91A, and G102S in the VL region sequence (SEQ ID NO: 27); and N30S, Q39E, D64E, S65A, A81V, H101A, G102E, F104I, N106T in the VH region sequence (SEQ ID NO: 66), L20M, Q40K, and L69E in the VL region sequence (SEQ ID NO: 67); iii. H101A in the VH region sequence (SEQ ID NO: 2560); iv. A81V, V99A in the VH region sequence, L20I, Q40K, L69S and G102S in the VL region sequence; M34I, A81V in the VH region sequence (SEQ ID NO: 1898), L20I, Q40K, and G102S in the VL region sequence (SEQ ID NO: 1899); M34I in the VH region sequence (SEQ ID NO: 1904), L20I, Q40K, and G102S in the VL region sequence (1905); M34I, Q39E, A81V in the VH region sequence (SEQ ID NO: 1906), L20I, Q40K, and G102S in the VL region sequence (SEQ ID NO: 1907); v. M34I, A81V in the VH region sequence (SEQ ID NO: 1900) and Q40K in the VL region sequence (SEQ ID NO: 1901); L45M in the VH region sequence (SEQ ID NO: 1866), L20I, Q40K, and G102S in the VH region sequence (SEQ ID NO: 1867); M34I, S65A, H101A, and N106S in the VH region sequence; L20I, Q40K, L69S, and G102S in the VL region sequence; M34F and A81V in the VH region sequence (SEQ ID NO: 1884); L45M in the VH region sequence (SEQ ID NO: 1870), Q40K and G102S in the VL region sequence (SEQ ID NO: 1871); Q39D in the VH region sequence (SEQ ID NO: 1918), L20I, Q40K, and G102S in the VL region sequence (SEQ ID NO: 1919); M34I and A81V in the VH region sequence (SEQ ID NO: 1869); Q39E in the VH region sequence (SEQ ID NO: 1932), L20I, Q40K, and G102S in the VL region sequence (SEQ ID NO: 1933); L45M in the VH region sequence (SEQ ID NO: 1868) and Q40K in the Vl region sequence (SEQ ID NO: 1869); M34I, S65A, A81V, V99A, H101A, and N106S in the VH region sequence; or vi. Q39D in the VH domain sequence (SEQ ID NO: 1928), L20I, Q40R, and G102S in the VL domain sequence (SEQ ID NO: 1929); M34I in the VH region sequence (SEQ ID NO: 1892); Q40K in the VL region sequence (SEQ ID NO: 1974); Q39D in the VH region sequence (SEQ ID NO: 1922), Q40K and G102S in the VL region sequence (SEQ ID NO: 1923); L20I and G102S in the VH region sequence; Q39K in the VH region sequence (SEQ ID NO: 1944); Q39E in the VH region sequence (SEQ ID NO: 1938), Q40K and G102S in the VL region sequence (SEQ ID NO: 1939); Q39E in the VH region sequence (SEQ ID NO: 1934), L20I, Q40R, and G102S in the VL region sequence (SEQ ID NO: 1935); Q39D in the VH region sequence (SEQ ID NO: 1926), Q40R and G102S in the VL region sequence (SEQ ID NO: 1926); A81V in the VH region sequence (SEQ ID NO: 1862); Q39D in the VH region sequence (SEQ ID NO: 1920) and Q40K in the VL region sequence (SEQ ID NO: 1921); W112F in the VH region sequence (SEQ ID NO: 1958); W112F in the VH region sequence (SEQ ID NO: 1960) and V38I in the VL region sequence (SEQ ID NO: 1961); Q39E in the VH region sequence (SEQ ID NO: 1936) and Q40K in the VL region sequence (SEQ ID NO: 1937); M34V in the VH region sequence (SEQ ID NO: 1914) comprising an amino acid substitution selected from: b) the VH region comprises or consists of the sequence defined in SEQ ID NO: 730 and the VL region comprises or consists of the sequence defined in SEQ ID NO: 731; the VH and / or VL region sequences are i. M34I, S65A, A81V, V99A, H101A, N106S in the VH domain sequence (the resulting VH sequence defined in SEQ ID NO: 846), L20I, Q40K, L69S, and G102S in the VL domain sequence (the resulting VL sequence defined in SEQ ID NO: 847); L45M, S65A, A81L, H101A, N106T in the VH region sequence (SEQ ID NO: 870), L20I, Q40K, L69S, and G102S in the VL region sequence (SEQ ID NO: 871); L45M, S65A, A81L, H101A, N106S, W112F in the VH region sequence (SEQ ID NO: 862), L20I, V38I, Q40K, L69E, G102S, and W93Y in the VL region sequence (SEQ ID NO: 863); M34I, Q39E, S65A, H101N, G102A, N106T, W112F in the VH region sequence (SEQ ID NO: 878), Q40K and G102S in the VL region sequence (SEQ ID NO: 879); M34I, D64E, S65A, V99A, H101A, N106T in the VH region sequence, Q40K, V91A, and G102S in the VL region sequence; M34I, S65A, A81V, V99A, H101A, G102E, F104I, N106T in the VH region sequence, L20I, Q40K, L69S, and G102S in the VL region sequence; D64E, S65A, A81V, H101A, N106S in the VH region sequence, L20I, L69S, G102S in the VL region sequence; M34I, D64E, S65A, H101A, N106T, W112F in the VH region sequence, Q40K, V91A, and G102S in the VL region sequence; M34I, D64E, S65A, H101A, N106S, W112F in the VH region sequence, Q40K, V91A, and G102S in the VL region sequence; M34I, D64E, S65A, A81V, V99A, H101A, N106S in the VH region sequence, L20I, Q40K, L69S, G102S, and W93Y in the VL region sequence; M34I, S65A, H101A, N106S, W112F in the VH region sequence, Q40K, V91A, and G102S in the VL region sequence; L45M, D64E, S65A, A81L, H101A, N106T in the VH region sequence, L20I, V38I, Q40K, L69E, and G102S in the VL region sequence; N30S, L45M, S65A, A81V, H101A, N106T, W112F in the VH region sequence, Q40K, L69S, and W93Y in the VL region sequence; and N30S, L45M, D64E, S65A, A81V, H101A, N106S in the VH region sequence, Q40K and L69S in the VL region sequence; ii. D64E, S65A, A81V, H101N, G102E, F104I, N106T in the VH domain sequence (SEQ ID NO: 854), L20I, L69S, and G102S in the VL domain sequence (SEQ ID NO: 855); S65A, A81V, H101N, G102E, F104I, N106S in the VH region sequence, L20I, L69S, G102S in the VL region sequence; M34I, S65A, V99A, H101A, N106T in the VH region sequence, Q40K, V91A, and G102S in the VL region sequence; and N30S, Q39E, D64E, S65A, A81V, H101A, G102E, F104I, N106T in the VH region sequence, L20M, Q40K, and L69E in the VL region sequence; iii. H101A in the VH region sequence; iv. A81V, V99A in the VH region sequence, L20I, Q40K, L69S and G102S in the VL region sequence; M34I, A81V in the VH region sequence (SEQ ID NO: 774), L20I, Q40K, and G102S in the VL region sequence (SEQ ID NO: 775); M34I in the VH region sequence (SEQ ID NO: 780), L20I, Q40K, and G102S in the VL region sequence (781); M34I, Q39E, A81V in the VH region sequence (SEQ ID NO: 782), L20I, Q40K, and G102S in the VL region sequence (SEQ ID NO: 783); v. M34I, A81V in the VH region sequence (SEQ ID NO: 776) and Q40K in the VL region sequence (SEQ ID NO: 777); L45M in the VH region sequence (SEQ ID NO: 742), L20I, Q40K, and G102S in the VH region sequence (SEQ ID NO: 743); M34I, S65A, H101A, and N106S in the VH region sequence; L20I, Q40K, L69S, and G102S in the VL region sequence; M34F and A81V in the VH region sequence (SEQ ID NO: 760); L45M in the VH region sequence (SEQ ID NO: 746), Q40K and G102S in the VL region sequence (SEQ ID NO: 747); Q39D in the VH region sequence (SEQ ID NO: 794), L20I, Q40K, and G102S in the VL region sequence (SEQ ID NO: 795); M34I and A81V in the VH region sequence (SEQ ID NO: 772); Q39E in the VH region sequence (SEQ ID NO: 808), L20I, Q40K, and G102S in the VL region sequence (SEQ ID NO: 809); L45M in the VH region sequence (SEQ ID NO: 744) and Q40K in the Vl region sequence (SEQ ID NO: 745); M34I, S65A, A81V, V99A, H101A, and N106S in the VH region sequence; or vi. Q39D in the VH domain sequence (SEQ ID NO: 796), L20I, Q40R, and G102S in the VL domain sequence (SEQ ID NO: 797); M34I in the VH region sequence (SEQ ID NO: 768); Q40K in the VL region sequence (SEQ ID NO: 891); Q39D in the VH region sequence (SEQ ID NO: 800), Q40K and G102S in the VL region sequence (SEQ ID NO: 801); L20I and G102S in the VH region sequence; Q39K in the VH region sequence (SEQ ID NO: 820); Q39E in the VH region sequence (SEQ ID NO: 814), Q40K and G102S in the VL region sequence (SEQ ID NO: 815); Q39E in the VH region sequence (SEQ ID NO: 810), L20I, Q40R, and G102S in the VL region sequence (SEQ ID NO: 811); Q39D in the VH region sequence (SEQ ID NO: 804), Q40R and G102S in the VL region sequence (SEQ ID NO: 805); A81V in the VH region sequence (SEQ ID NO: 738); Q39D in the VH region sequence (SEQ ID NO: 798) and Q40K in the VL region sequence (SEQ ID NO: 799); W112F in the VH region sequence (SEQ ID NO: 834); W112F in the VH region sequence (SEQ ID NO: 836) and V38I in the VL region sequence (SEQ ID NO: 837); Q39E in the VH region sequence (SEQ ID NO: 812) and Q40K in the VL region sequence (SEQ ID NO: 813); M34V in the VH region sequence (SEQ ID NO: 790) comprising an amino acid substitution selected from: the VH region comprises or consists of the sequence set forth in SEQ ID NO: 1664, and the VL region comprises or consists of the sequence set forth in SEQ ID NO: 1665; the VH and / or VL region sequences are i. M34I, S65A, A81V, V99A, H101A, N106S in the VH domain sequence (the resulting VH sequence defined in SEQ ID NO: 1780), L20I, Q40K, L69S, and G102S in the VL domain sequence (the resulting VL sequence defined in SEQ ID NO: 1781); L45M, S65A, A81L, H101A, N106T in the VH region sequence (SEQ ID NO: 1804), L20I, Q40K, L69S, and G102S in the VL region sequence (SEQ ID NO: 1805); L45M, S65A, A81L, H101A, N106S, W112F in the VH region sequence (SEQ ID NO: 1796), L20I, V38I, Q40K, L69E, G102S, and W93Y in the VL region sequence (SEQ ID NO: 1797); M34I, Q39E, S65A, H101N, G102A, N106T, W112F in the VH region sequence (SEQ ID NO: 1812), Q40K and G102S in the VL region sequence (SEQ ID NO: 1813); M34I, D64E, S65A, V99A, H101A, N106T in the VH region sequence, Q40K and G102S in the VL region sequence; M34I, S65A, A81V, V99A, H101A, G102E, F104I, N106T in the VH region sequence, L20I, Q40K, L69S, and G102S in the VL region sequence; D64E, S65A, D68G, A81V, H101A, N106S in the VH region sequence, L20I, L69S, G102S in the VL region sequence; M34I, D64E, S65A, H101A, N106T, W112F in the VH region sequence, Q40K and G102S in the VL region sequence; M34I, D64E, S65A, H101A, N106S, W112F in the VH region sequence, Q40K and G102S in the VL region sequence; M34I, D64E, S65A, A81V, V99A, H101A, N106S in the VH region sequence, L20I, Q40K, L69S, G102S, and W93Y in the VL region sequence; M34I, S65A, H101A, N106S, W112F in the VH region sequence, Q40K and G102S in the VL region sequence; L45M, D64E, S65A, A81L, H101A, N106T in the VH region sequence, L20I, V38I, Q40K, L69E, and G102S in the VL region sequence; N30S, L45M, S65A, D68G, A81V, H101A, N106T, W112F in the VH region sequence, Q40K, L69S, and W93Y in the VL region sequence; and N30S, L45M, D64E, S65A, D68G, A81V, H101A, N106S in the VH region sequence, Q40K and L69S in the VL region sequence; ii. D64E, S65A, D68G, A81V, H101N, G102E, F104I, N106T in the VH domain sequence (SEQ ID NO: 1788), L20I, L69S, and G102S in the VL domain sequence (SEQ ID NO: 1789); S65A, D68G, A81V, H101N, G102E, F104I, N106S in the VH region sequence, L20I, L69S, G102S in the VL region sequence; M34I, S65A, V99A, H101A, N106T in the VH region sequence, Q40K and G102S in the VL region sequence; and N30S, Q39E, D64E, S65A, A81V, H101A, G102E, F104I, N106T in the VH region sequence, L20M, Q40K, and L69E in the VL region sequence; iii. H101A in the VH region sequence; iv. A81V, V99A in the VH region sequence, L20I, Q40K, L69S and G102S in the VL region sequence; M34I, A81V in the VH region sequence (SEQ ID NO: 1708), L20I, Q40K, and G102S in the VL region sequence (SEQ ID NO: 1709); M34I in the VH region sequence (SEQ ID NO: 1714), L20I, Q40K, and G102S in the VL region sequence (1715); M34I, Q39E, A81V in the VH region sequence (SEQ ID NO: 1716), L20I, Q40K, and G102S in the VL region sequence (SEQ ID NO: 1717); v. M34I, A81V in the VH region sequence (SEQ ID NO: 1710) and Q40K in the VL region sequence (SEQ ID NO: 1711); L45M in the VH region sequence (SEQ ID NO: 1676), L20I, Q40K, and G102S in the VH region sequence (SEQ ID NO: 1677); M34I, S65A, H101A, and N106S in the VH region sequence; L20I, Q40K, L69S, and G102S in the VL region sequence; M34F and A81V in the VH region sequence (SEQ ID NO: 1694); L45M in the VH region sequence (SEQ ID NO: 1680), Q40K and G102S in the VL region sequence (SEQ ID NO: 1681); Q39D in the VH region sequence (SEQ ID NO: 1728), L20I, Q40K, and G102S in the VL region sequence (SEQ ID NO: 1729); M34I and A81V in the VH region sequence (SEQ ID NO: 1706); Q39E in the VH region sequence (SEQ ID NO: 1742), L20I, Q40K, and G102S in the VL region sequence (SEQ ID NO: 1743); L45M in the VH region sequence (SEQ ID NO: 1678) and Q40K in the Vl region sequence (SEQ ID NO: 1679); M34I, S65A, A81V, V99A, H101A, and N106S in the VH region sequence; or vi. Q39D in the VH domain sequence (SEQ ID NO: 1730), L20I, Q40R, and G102S in the VL domain sequence (SEQ ID NO: 1731); M34I in the VH region sequence (SEQ ID NO: 1702); Q40K in the VL region sequence (SEQ ID NO: 1755); Q39D in the VH region sequence (SEQ ID NO: 1734), Q40K and G102S in the VL region sequence (SEQ ID NO: 1735); L20I and G102S in the VH region sequence; Q39K in the VH region sequence (SEQ ID NO: 1754); Q39E in the VH region sequence (SEQ ID NO: 1748), Q40K and G102S in the VL region sequence (SEQ ID NO: 1749); Q39E in the VH region sequence (SEQ ID NO: 1744), L20I, Q40R, and G102S in the VL region sequence (SEQ ID NO: 1745); Q39D in the VH region sequence (SEQ ID NO: 1738), Q40R and G102S in the VL region sequence (SEQ ID NO: 1739); A81V in the VH region sequence (SEQ ID NO: 1672); Q39D in the VH region sequence (SEQ ID NO: 1732) and Q40K in the VL region sequence (SEQ ID NO: 1733); W112F in the VH region sequence (SEQ ID NO: 1768); W112F in the VH region sequence (SEQ ID NO: 1770) and V38I in the VL region sequence (SEQ ID NO: 1771); Q39E in the VH region sequence (SEQ ID NO: 1746) and Q40K in the VL region sequence (SEQ ID NO: 1747); M34V in the VH region sequence (SEQ ID NO: 1724) or d) the VH region comprises or consists of the sequence defined in SEQ ID NO: 124 and the VL region comprises or consists of the sequence defined in SEQ ID NO: 125; the VH and / or VL region sequences are i. Q65A, A81V, V99A, H101A, N106S in the VH domain sequence (the resulting VH sequence defined in SEQ ID NO: 132), L20I, Q40K, L69S, and G102S in the VL domain sequence (the resulting VL sequence defined in SEQ ID NO: 133); L45M, Q65A, A81L, H101A, N106T in the VH region sequence (SEQ ID NO: 156), L20I, Q40K, L69S, and G102S in the VL region sequence (SEQ ID NO: 157); L45M, Q65A, A81L, H101A, N106S, W112F in the VH region sequence (SEQ ID NO: 148), L20I, V38I, Q40K, L69E, G102S, and W93Y in the VL region sequence (SEQ ID NO: 149); Q39E, Q65A, H101N, N106T, W112F in the VH region sequence (SEQ ID NO: 164), Q40K and G102S in the VL region sequence (SEQ ID NO: 165); D64E, Q65A, V99A, H101A, N106T in the VH region sequence, Q40K, T91A, and G102S in the VL region sequence; Q65A, A81V, V99A, H101A, A102E, F104I, N106T in the VH region sequence, L20I, Q40K, L69S, and G102S in the VL region sequence; D64E, Q65A, D68G, A81V, H101A, N106S in the VH region sequence, L20I, L69S, G102S in the VL region sequence; D64E, Q65A, H101A, N106T, W112F in the VH region sequence, Q40K and G102S in the VL region sequence; D64E, Q65A, H101A, N106S, W112F in the VH region sequence, Q40K and G102S in the VL region sequence; D64E, Q65A, A81V, V99A, H101A, N106S in the VH region sequence, L20I, Q40K, L69S, G102S, and W93Y in the VL region sequence; Q65A, H101A, N106S, W112F in the VH region sequence, Q40K and G102S in the VL region sequence; L45M, D64E, Q65A, A81L, H101A, N106T in the VH region sequence, L20I, V38I, Q40K, L69E, and G102S in the VL region sequence; N30S, L45M, Q65A, D68G, A81V, H101A, N106T, W112F in the VH region sequence, Q40K, L69S, and W93Y in the VL region sequence; and N30S, L45M, D64E, Q65A, D68G, A81V, H101A, N106S in the VH region sequence, Q40K and L69S in the VL region sequence; ii. D64E, Q65A, D68G, A81V, H101N, A102E, F104I, N106T in the VH domain sequence (SEQ ID NO: 140), L20I, L69S, and G102S in the VL domain sequence (SEQ ID NO: 141); Q65A, D68G, A81V, H101N, A102E, F104I, N106S in the VH region sequence, L20I, L69S, G102S in the VL region sequence; Q65A, V99A, H101A, N106T in the VH region sequence, Q40K and G102S in the VL region sequence; and N30S, Q39E, D64E, Q65A, A81V, H101A, A102E, F104I, N106T in the VH region sequence, L20M, Q40K, and L69E in the VL region sequence; iii. H101A in the VH region sequence; iv. A81V, V99A in the VH region sequence, L20I, Q40K, L69S and G102S in the VL region sequence; A81V in the VH region sequence, L20I, Q40K, and G102S in the VL region sequence; L20I, Q40K, and G102S in the VL region sequence; Q39E, A81V in the VH region sequence, L20I, Q40K, and G102S in the VL region sequence; v. A81V in the VH region sequence and Q40K in the VL region sequence; L45M in the VH region sequence, L20I, Q40K, and G102S in the VH region sequence; Q65A, H101A, and N106S in the VH region sequence; L20I, Q40K, L69S, and G102S in the VL region sequence; I34F and A81V in the VH region sequence; L45M in the VH region sequence, Q40K and G102S in the VL region sequence; Q39D in the VH region sequence, L20I, Q40K, and G102S in the VL region sequence; Q39E in the VH region sequence, L20I, Q40K, and G102S in the VL region sequence; L45M in the VH region sequence and Q40K in the VL region sequence; Q65A, A81V, V99A, H101A, and N106S in the VH region sequence; or vi. Q39D in the VH region sequence, L20I, Q40R and G102S in the VL region sequence; Q40K in the VL region sequence; Q39D in the VH region sequence, Q40K and G102S in the VL region sequence; L20I and G102S in the VH region sequence; Q39K in the VH region sequence; Q39E in the VH region sequence, Q40K and G102S in the VL region sequence; Q39E in the VH region sequence, L20I, Q40R, and G102S in the VL region sequence; Q39D in the VH region sequence, Q40R and G102S in the VL region sequence; A81V in the VH region sequence; Q39D in the VH region sequence and Q40K in the VL region sequence; W112F in the VH region sequence; W112F in the VH region sequence and V38I in the VL region sequence; Q39E in the VH region sequence and Q40K in the VL region sequence; M34V in the VH region sequence The present invention relates to a polypeptide or polypeptide construct comprising an amino acid substitution selected from:

[0164] The CD3ε-binding domain of the polypeptide or polypeptide construct of the present invention is preferably selected from the options a) to c), with option a) being more preferred. The preferred linkers listed above, i.e., (G4S)3 or G4Q)3, are also preferred for this embodiment.

[0165] According to the present invention, the polypeptides or polypeptide constructs of the present invention may comprise linkers, half-life extending peptides and other structural moieties as disclosed in SEQ ID NOs: 1-19 and 2551. Details regarding the properties and functions of these structures can be found in the sequence listing disclosed herein.

[0166] The present invention provides an embodiment in which the polypeptide construct is in a format selected from the group consisting of scFv, (scFv)2, diabody, and any of the above-mentioned formats, provided that an additional binding domain is present. The term "in the format" does not exclude that the construct can be further modified, for example, by conjugation or fusion to other moieties described herein. According to one embodiment of the polypeptide construct of the present invention, the paratope-containing domain described herein is in the format of an scFv. In an scFv, the VH and VL regions are arranged in the order VH-VL or VL-VH (from N-terminus to C-terminus). It is envisioned that the VH and VL regions of the domain are connected via a linker, preferably a peptide linker. In a preferred embodiment, the peptide linker is a G4S or G4Q linker or a repeat thereof, for example, preferably a (G4S)3 (i.e., three repeats of G4S) or (G4Q)3 (i.e., three repeats of G4Q). According to one embodiment of a binding domain comprising a VH and a VL region described herein, the VH region is located at the N-terminus of the linker, and the VL region is located at the C-terminus of the linker. In other words, in one embodiment of a domain comprising a paratope described herein, the scFv comprises, from N-terminus to C-terminus, VH-linker-VL. When a polypeptide or polypeptide construct comprises at least one further binding domain in addition to the CD3ε-binding domain, it is further envisaged that the binding domains of the construct (including the paratope described herein) are linked via a linker, preferably a peptide linker. The construct may, for example, comprise the domains in the following order (from N-terminus to C-terminus): CD3-binding domain-linker-further binding domain. The reverse order (further binding domain-linker-CD3-binding domain) is also possible and is the preferred orientation.

[0167] The linker is preferably a peptide linker, more preferably a short-chain peptide linker. According to the present invention, a "peptide linker" comprises an amino acid sequence that connects the amino acid sequence of one domain of a construct to another (variable and / or binding) domain (e.g., a variable domain or a binding domain). An essential technical feature of such a peptide linker is that it does not contain polymerization activity. Suitable peptide linkers include those described in U.S. Pat. Nos. 4,751,180 and 4,935,233 or WO 88 / 09344. Peptide linkers can also be used to connect other domains, modules, or regions (such as half-life extending domains) to the constructs of the present invention. Examples of useful peptide linkers are shown in SEQ ID NOS: 1-11 and 2551. In this context, a "short" linker has 2 to 50 amino acids, preferably 3 to 35 amino acids, 4 to 30 amino acids, 5 to 25 amino acids, 6 to 20 amino acids, or 6 to 17 amino acids. The linker between the two variable regions of one binding domain may have a different length (e.g., it may be longer) than the linker between the two binding domains. For example, the linker between the two variable regions of one binding domain may have a length of 7 to 15 amino acids, preferably 9 to 13, and the linker between the two binding domains may have a length of 3 to 10 amino acids, preferably 4 to 8. Furthermore, it is contemplated that the peptide linker is a glycine / serine linker, such as those set forth in SEQ ID NOS: 1 to 11 and 2551. Most of the amino acids in the glycine / serine linker are selected from glycine and serine.

[0168] When a linker is used, it is preferable that the linker be of a length and sequence that ensures that each of the first and second domains retains its distinct binding specificity independently of the other. For peptide linkers connecting at least two binding domains (or two variable regions forming one binding domain) in a construct, peptide linkers containing only a small number of amino acid residues, for example, 12 or fewer, are contemplated. Thus, peptide linkers of 12, 11, 10, 9, 8, 7, 6, or 5 amino acid residues are preferred. Contemplated peptide linkers with fewer than 5 amino acids contain 4, 3, 2, or 1 amino acid, with Gly-rich linkers being preferred. In the context of the "peptide linker" above, a "single amino acid" linker is Gly. Another embodiment of the peptide linker is characterized by the amino acid sequence Gly-Gly-Gly-Gly-Ser, i.e., Gly4Ser (SEQ ID NO: 15), or a polymer thereof, i.e., (Gly4Ser)x, where x is an integer equal to or greater than 1 (e.g., 2 or 3), such as SEQ ID NOs: 1 and 2. Preferred linkers are shown in SEQ ID NOs: 2, 4, 5, 6, 8, 10, 11, and 2551. Another preferred linker comprises or consists of (Gly4Ser)6. The characteristics of such peptide linkers are known in the art and are described, for example, in Dall'Acqua et al. (Biochem. (1998) 37, 9266-9273), Cheadle et al. (Mol Immunol (1992) 29, 21-30), and Raag and Whitlow (FASEB (1995) 9(1), 73-80). Peptide linkers that do not promote secondary structure are preferred. The linkage of said domains to one another can be provided, for example, by genetic engineering.Methods for preparing fused and operably linked bispecific single-chain constructs and expressing them in mammalian cells or bacteria are known in the art (e.g., WO 99 / 54440 or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001). More preferably, the peptide linker is a G4S or G4Q linker or repeats thereof, such as (G4S)3 or (G4Q)3.

[0169] According to one embodiment of the present invention, the polypeptide construct of the present invention is a "single-chain construct" or "single-chain polypeptide." In the case of an additional binding domain, it is also envisioned that either the CD3 binding domain or the additional binding domain (also referred to as "second"), or both binding domains, may be in the format of a "single-chain Fv" (scFv). Although the two domains of an Fv fragment, VL and VH, are encoded by separate genes, they can be linked by an artificial linker (as described hereinabove) that allows them to be produced using recombinant methods as a single protein chain in which the VL and VH regions pair to form a monovalent molecule; see, for example, Huston et al. (1988) Proc. Natl. Acad. Sci USA 85:5879-5883. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are evaluated for function in the same manner as full-length antibodies or IgGs. Thus, single-chain variable fragments (scFv) are fusion proteins of the variable regions of the heavy (VH) and light (VL) chains of immunoglobulins, usually linked by a short linker peptide. The linker is usually rich in glycine for flexibility and serine or even threonine for solubility, and can connect the N-terminus of VH to the C-terminus of VL, or vice versa. The protein retains the specificity of the original immunoglobulin despite the removal of the constant regions and the introduction of the linker.

[0170] Bispecific single chain molecules are known in the art and are described in WO 99 / 54440, Mack, J. Immunol. (1997), 158, 3965-3970, Mack, PNAS, (1995), 92, 7021-7025, Kufer, Cancer Immunol. Immunother., (1997), 45, 193-197, Loeffler, Blood, (2000), 95, 6, 2098-2103, Bruehl, Immunol., (2001), 166, 2420-2426, Kipriyanov, J. Mol. Biol., (1999), 293, 41-56. Techniques described for producing single-chain constructs (see, inter alia, U.S. Pat. No. 4,946,778; Kontermann and Duebel (2010), supra; and Little (2009), supra) can be adapted to produce single-chain constructs that selectively, and preferably specifically, recognize a target of choice.

[0171] Bivalent (also called divalent) or bispecific single-chain variable fragments (bi-scFv or di-scFv) having the scFv2 format can be engineered by linking two scFv molecules (e.g., by a linker as described above). Linking can be achieved by creating a single polypeptide chain with two VH and two VL regions, resulting in a tandem scFv (see, e.g., Kufer, P. et al., (2004) Trends in Biotechnology 22(5):238-244). Another possibility is to create scFv molecules with a linker peptide (e.g., about 5 amino acids) that is too short for the two variable regions to fold together, forcing the scFv to dimerize. In this case, the VH and VL of the binding domains (binding either to CD3ε or to an additional target antigen) are not directly connected via a peptide linker. Thus, for example, the VH of a CD3 antigen-binding domain can be fused to the VL of an additional target antigen-binding domain via a peptide linker, and the VH of the additional target antigen-binding domain is fused to the VL of the CD3 binding domain via such a peptide linker. This type is known as a diabody (see, e.g., Hollinger, Philipp et al., (July 1993) Proceedings of the National Academy of Sciences of the United States of America 90(14):6444-8).

[0172] According to the present invention, the polypeptide or polypeptide construct of the present invention comprises at least one further binding domain. In other words, the polypeptide or polypeptide construct comprises a CD3ε-binding domain as defined herein above and at least one further binding domain. Said further binding domain may be a further CD3-binding domain, preferably one as defined herein. Thus, the polypeptide may comprise two of the same CD3-binding domains as defined herein. Alternatively or additionally (in constructs comprising at least three binding domains), at least one further binding domain binds to a different target, e.g., a cell surface antigen.

[0173] Preferably, the at least one additional binding domain binds to a cell surface antigen. As used herein, the term "cell surface antigen" refers to a molecule displayed on the surface of a cell. In most cases, the molecule is located inside or on the plasma membrane of the cell so that at least a portion of the molecule remains accessible from the outside of the cell in its tertiary form. A non-limiting example of a cell surface molecule located inside the plasma membrane is a transmembrane protein that contains hydrophilic and hydrophobic regions in its tertiary conformation. Here, at least one hydrophobic region allows the cell surface molecule to be embedded or inserted into the hydrophobic plasma membrane of the cell, while the hydrophilic regions extend into the cytoplasm and extracellular space, respectively, on either side of the plasma membrane. An extracellular epitope will be understood to refer to an epitope constituted by a portion of a protein located in the extracellular space, for example, a portion of a cell surface molecule that extends into the extracellular space when the cell surface molecule is in its natural configuration located, embedded, or inserted in the plasma membrane. Non-limiting examples of cell surface molecules located on the plasma membrane are proteins modified at a cysteine ​​residue to have a palmitoyl group, proteins modified at the C-terminal cysteine ​​residue to have a farnesyl group, or proteins modified at the C-terminus to have a glycosylphosphatidylinositol ("GPI") anchor.

[0174] The cell surface antigen is preferably a tumor antigen. As used herein, the term "tumor antigen" can be understood as those antigens presented on tumor cells. These antigens can be presented on the cell surface with an extracellular portion, and often have both transmembrane and cytoplasmic portions of the molecule. These antigens can sometimes be presented only by tumor cells and never by normal cells. Tumor antigens can be expressed exclusively on tumor cells or can exhibit tumor-specific mutations compared to normal cells. In this case, they are called tumor-specific antigens. More common antigens are antigens presented by tumor cells and normal cells, and they are called tumor-associated antigens. These tumor-associated antigens can be overexpressed compared to normal cells, or are accessible for antibody binding in tumor cells due to the less compact structure of tumor tissue compared to normal tissue.

[0175] In a preferred embodiment, the tumor antigen is BCMA (B cell maturation antigen), CD123 (interleukin-3 receptor alpha chain (IL-3R)), CD19 (B lymphocyte antigen CD19), CD20 (B lymphocyte antigen CD20), CD22 (cluster of differentiation-22), CD33 (Siglec-3), CD70 (cluster of differentiation 70), CDH19 (cadherin 19), CDH3 (cadherin 3), CLL1 (C-type lectin domain family 12 member A), CS1 (CCND3 subset 1), or a combination thereof. ), CLDN6 (claudin-6), CLDN18.2 (claudin 18.2), DLL3 (Delta-like ligand 3), EGFRvIII (epidermal growth factor receptor vIII), FLT3 (fms-like kinase 3), MAGEB2 (melanoma-associated antigen B2), MART1 (melanoma antigen 1 recognized by T cells), MSLN (mesothelin), MUC17 (mucin-17), PSMA (prostate-specific membrane antigen), and STEAP1 (metalloreductase STEAP1). These tumor antigens are well known in the art due to their expression on tumor cells.

[0176] Preferred CDR sequences and VH / VL region sequences for the BCMA binding domain as further binding domain of the polypeptide or polypeptide construct of the invention and the bispecific single chain molecule sequences of the polypeptide or polypeptide construct according to the invention having the BCMA binding domain as further binding domain (with and without half-life prolonging domain) are defined in SEQ ID NOs: 2072 to 2095.

[0177] Preferred CDR sequences and VH / VL region sequences for the CD123-binding domain as the further binding domain of the polypeptide or polypeptide construct of the invention and the bispecific single-chain molecule sequences of the polypeptide or polypeptide construct according to the invention having the CD123-binding domain as the further binding domain (with and without a half-life prolonging domain) are defined in SEQ ID NOs: 2096 to 2110.

[0178] Preferred CDR sequences and VH / VL region sequences for the CD19-binding domain as the further binding domain of the polypeptide or polypeptide construct of the invention and the bispecific single chain molecule sequences of the polypeptide or polypeptide construct according to the invention having the CD19-binding domain as the further binding domain (with and without a half-life prolonging domain) are defined in SEQ ID NOs: 2111 to 2125.

[0179] Preferred CDR sequences and VH / VL region sequences for a CD20-binding domain as an additional binding domain of a polypeptide or polypeptide construct of the invention and bispecific single-chain molecule sequences of a polypeptide or polypeptide construct according to the invention having a CD20-binding domain as an additional binding domain (with and without a half-life prolonging domain) are defined in SEQ ID NOs: 2126 to 2140.

[0180] Preferred CDR sequences and VH / VL region sequences for the CD22-binding domain as the further binding domain of the polypeptide or polypeptide construct of the invention and the bispecific single chain molecule sequences of the polypeptide or polypeptide construct according to the invention having the CD22-binding domain as the further binding domain (with and without a half-life prolonging domain) are defined in SEQ ID NOs: 2141 to 2154.

[0181] Preferred CDR sequences and VH / VL region sequences for the CD33-binding domain as the further binding domain of the polypeptide or polypeptide construct of the invention and the bispecific single-chain molecule sequences of the polypeptide or polypeptide construct according to the invention having the CD33-binding domain as the further binding domain (with and without a half-life prolonging domain) are defined in SEQ ID NOs: 2155 to 2178.

[0182] Preferred CDR sequences and VH / VL region sequences for a CD70-binding domain as an additional binding domain of a polypeptide or polypeptide construct of the invention and the bispecific single-chain molecule sequences of a polypeptide or polypeptide construct according to the invention having a CD70-binding domain as an additional binding domain (with and without a half-life prolonging domain) are defined in SEQ ID NOs: 2179 to 2192.

[0183] Preferred CDR sequences and VH / VL region sequences for the CDH19-binding domain as the further binding domain of the polypeptide or polypeptide construct of the invention and the bispecific single-chain molecule sequences of the polypeptide or polypeptide construct according to the invention having the CDH19-binding domain as the further binding domain (with and without a half-life prolonging domain) are defined in SEQ ID NOs: 2193 to 2212.

[0184] Preferred CDR sequences and VH / VL region sequences for the CDH3-binding domain as the further binding domain of the polypeptide or polypeptide construct of the invention and the bispecific single-chain molecule sequences of the polypeptide or polypeptide construct according to the invention having the CDH3-binding domain as the further binding domain (with and without a half-life prolonging domain) are defined in SEQ ID NOs: 2213 to 2256.

[0185] Preferred CDR sequences and VH / VL region sequences for the CLL1-binding domain as an additional binding domain of the polypeptide or polypeptide construct of the invention and the bispecific single chain molecule sequences of the polypeptide or polypeptide construct according to the invention having the CLL1-binding domain as an additional binding domain (with and without a half-life prolonging domain) are defined in SEQ ID NOs: 2257 to 2271.

[0186] Preferred CDR sequences and VH / VL region sequences for the CS1-binding domain as an additional binding domain of the polypeptide or polypeptide construct of the invention and the bispecific single-chain molecule sequences of the polypeptide or polypeptide construct according to the invention having the CS1-binding domain as an additional binding domain (with and without a half-life prolonging domain) are defined in SEQ ID NOs: 2272 to 2285.

[0187] Preferred CDR sequences and VH / VL region sequences for the CLDN6-binding domain as the further binding domain of the polypeptide or polypeptide construct of the present invention, and the bispecific single-chain molecule sequences of the polypeptide or polypeptide construct according to the present invention having the CLDN6-binding domain as the further binding domain (with and without a half-life prolonging domain) are defined in SEQ ID NOs: 2286 to 2299.

[0188] Preferred CDR sequences and VH / VL region sequences for the CLDN18.2-binding domain as the further binding domain of the polypeptide or polypeptide construct of the present invention and the bispecific single-chain molecule sequences of the polypeptide or polypeptide construct according to the present invention having the CLDN18.2-binding domain as the further binding domain (with and without a half-life prolonging domain) are defined in SEQ ID NOs: 2300 to 2314.

[0189] Preferred CDR sequences and VH / VL region sequences for the DLL3-binding domain as an additional binding domain of the polypeptide or polypeptide construct of the invention and bispecific single chain molecule sequences of the polypeptide or polypeptide construct according to the invention having a DLL3-binding domain as an additional binding domain (with and without a half-life prolonging domain) are defined in SEQ ID NOs: 2315 to 2328.

[0190] Preferred CDR sequences and VH / VL region sequences for an EGFRvIII binding domain as an additional binding domain of a polypeptide or polypeptide construct of the invention and bispecific single chain molecule sequences of a polypeptide or polypeptide construct according to the invention having an EGFRvIII binding domain as an additional binding domain (with and without a half-life prolonging domain) are defined in SEQ ID NOs: 2329 to 2342.

[0191] Preferred CDR sequences and VH / VL region sequences for the FLT3-binding domain as an additional binding domain of the polypeptide or polypeptide construct of the present invention and bispecific single-chain molecule sequences of the polypeptide or polypeptide construct according to the present invention having the FLT3-binding domain as an additional binding domain (with and without a half-life extending domain) are defined in SEQ ID NOs: 2343 to 2357.

[0192] Preferred CDR sequences and VH / VL region sequences for a MAGEB2-binding domain as an additional binding domain of a polypeptide or polypeptide construct of the invention and bispecific single chain molecule sequences of a polypeptide or polypeptide construct according to the invention having a MAGEB2-binding domain as an additional binding domain (with and without a half-life prolonging domain) are defined in SEQ ID NOs: 2358 to 2378.

[0193] Preferred CDR sequences and VH / VL region sequences for the MART1 binding domain as a further binding domain of the polypeptide or polypeptide construct of the present invention are defined in SEQ ID NOs: 2379-2387.

[0194] Preferred CDR sequences and VH / VL region sequences for the MSLN-binding domain as an additional binding domain of the polypeptide or polypeptide construct of the invention and the bispecific single chain molecule sequences of the polypeptide or polypeptide construct according to the invention having the MSLN-binding domain as an additional binding domain (with and without a half-life prolonging domain) are defined in SEQ ID NOs: 2388 to 2431.

[0195] Preferred CDR sequences and VH / VL region sequences for the MUC17-binding domain as an additional binding domain of the polypeptide or polypeptide construct of the invention and the bispecific single chain molecule sequences of the polypeptide or polypeptide construct according to the invention having the MUC17-binding domain as an additional binding domain (with and without a half-life prolonging domain) are defined in SEQ ID NOs: 2432 to 2445.

[0196] Preferred CDR sequences and VH / VL region sequences for the PSMA-binding domain as an additional binding domain of the polypeptide or polypeptide construct of the invention and the bispecific single chain molecule sequences of the polypeptide or polypeptide construct according to the invention having the PSMA-binding domain as an additional binding domain (with and without a half-life prolonging domain) are defined in SEQ ID NOs: 2446 to 2475.

[0197] Preferred bispecific single chain molecule sequences of polypeptides or polypeptide constructs according to the invention having CD20 and CD22 binding domains (with and without an additional CD3 binding domain as defined herein) as further binding domains and a half-life prolonging domain are defined in SEQ ID NOs: 2505 to 2516.

[0198] Preferred bispecific single chain molecule sequences of a polypeptide or polypeptide construct according to the invention having a CS1- and a BCMA-binding domain as further binding domains and a half-life prolonging domain are defined in SEQ ID NOs: 2517 to 2522.

[0199] Preferred bispecific single chain molecule sequences of a polypeptide or polypeptide construct according to the invention having an MSLN- and CDH3-binding domain as further binding domain and a half-life prolonging domain are defined in SEQ ID NOs: 2529-2536.

[0200] As explained above, the polypeptide construct of the present invention comprises a binding domain that binds to CD3 on the surface of T cells. "CD3" (cluster of differentiation 3) is a T cell coreceptor composed of four chains. In mammals, the CD3 protein complex contains the CD3γ (gamma) chain, the CD3δ (delta) chain, and two CD3ε (epsilon) chains. These four chains associate with the T cell receptor (TCR) and the so-called ζ (zeta) chain to form the "T cell receptor complex," which generates activation signals in T lymphocytes. The CD3γ (gamma), CD3δ (delta), and CD3ε (epsilon) chains are highly related cell surface proteins of the immunoglobulin superfamily, each containing a single extracellular immunoglobulin domain. The intracellular tail of the CD3 molecule contains a single conserved motif known as an immunoreceptor tyrosine-based activation motif (ITAM), which is essential for the signal transduction ability of the TCR. The CD3ε molecule is a polypeptide encoded by the CD3ε gene, located on chromosome 11 in humans. In the context of the present invention, CD3 is understood as a protein complex and T cell coreceptor involved in the activation of both cytotoxic T cells (CD8+ naive T cells) and T helper cells (CD4+ naive T cells). CD3 is typically composed of four different chains. In particular, in mammals, this complex contains the CD3γ chain, the CD3δ chain, and two CD3ε chains. These chains associate with the T cell receptor (TCR) and the ζ chain (zeta chain) to generate an activation signal in T lymphocytes. The TCR, ζ chain, and CD3 molecule together constitute the TCR complex.

[0201] Lysis of target cells redirected via recruitment of T cells with constructs that bind to CD3 on T cells and target proteins on target cells generally involves cytolytic synapse formation and delivery of perforin and granzymes. The engaged T cells possess a range of target cell lysis capabilities and are not susceptible to immune escape mechanisms that prevent peptide antigen processing and presentation or clonal T cell differentiation; see, e.g., WO 2007 / 042261.

[0202] The cytotoxicity mediated by a given tumor antigen × CD3 construct can be measured in various ways. The "half-maximal effective concentration" (EC50) is commonly used as a measure of the potency of biologically active molecules, such as the constructs of the present invention. It can be expressed in molar units. In this case of measuring cytotoxicity, the EC50 value refers to the concentration of the construct that induces a cytotoxic response (lysis of target cells) halfway between the baseline and maximum. Effector cells in cytotoxicity assays can be, for example, stimulated enriched (human) CD8+ T cells or unstimulated (human) peripheral blood mononuclear cells (PBMCs). EC50 values ​​can typically be expected to be lower when stimulated / enriched CD8+ T cells are used as effector cells compared to unstimulated PBMCs. When target cells are of macaque origin or transfected with a given macaque tumor antigen, the effector cells should also be of macaque origin, such as a macaque T cell line, e.g., 4119LnPx. The target cells express the tumor antigen on their cell surface. The target cells can be a cell line (such as CHO) stably or transiently transfected with the tumor antigen. Alternatively, the target cells can be a tumor antigen-positive naturally expressing cell line, such as a human cancer line. Typically, the EC50 value is expected to be lower when using target cells that express a higher level of the tumor antigen on their cell surface compared to target cells with a lower target expression rate.

[0203] The effector to target cell (E:T) ratio in cytotoxicity assays is usually about 10:1, but can vary. The cytotoxic activity of tumor antigen x CD3 constructs can be measured in a 51-chromium release assay (e.g., using an incubation time of about 18 hours) or a FACS-based cytotoxicity assay (e.g., using an incubation time of about 48 hours). Modifications of the incubation time (cytotoxic response) are also contemplated. Other methods for measuring cytotoxicity are well known and include MTT or MTS assays, ATP-based assays including bioluminescence assays, sulforhodamine B (SRB) assays, WST assays, clonogenic assays, and ECIS techniques.

[0204] According to one embodiment, the cytotoxic activity mediated by the tumor antigen x CD3 constructs of the present invention is measured in a cell-based cytotoxicity assay. This can also be measured in a 51-chromium release assay. The EC50 value of the constructs of the present invention is expected to be 300 pM or less, 280 pM or less, 260 pM or less, 250 pM or less, 240 pM or less, 220 pM or less, 200 pM or less, 180 pM or less, 160 pM or less, 150 pM or less, 140 pM or less, 120 pM or less, 100 pM or less, 90 pM or less, 80 pM or less, 70 pM or less, 60 pM or less, 50 pM or less, 40 pM or less, 30 pM or less, 20 pM or less, 15 pM or less, 10 pM or less, or 5 pM or less.

[0205] The above-mentioned given EC50 value can be measured under different conditions in different assays. For example, when human PBMCs are used as effector cells and tumor antigen transfected cells such as CHO cells are used as target cells, the EC50 value of the tumor antigen x CD3 construct is expected to be 500pM or less, 400pM or less, 300pM or less, 280pM or less, 260pM or less, 250pM or less, 240pM or less, 220pM or less, 200pM or less, 180pM or less, 160pM or less, 150pM or less, 140pM or less, 120pM or less, 100pM or less, 90pM or less, 80pM or less, 70pM or less, 60pM or less, 50pM or less, 40pM or less, 30pM or less, 20pM or less, 15pM or less, 10pM or less, or 5pM or less. When human PBMCs are used as effector cells, and therefore when the target cells are a CLDN6-positive cell line, the EC50 value of the CLDN6xCD3 construct is expected to be 300 pM or less, 280 pM or less, 260 pM or less, 250 pM or less, 240 pM or less, 220 pM or less, 200 pM or less, 180 pM or less, 160 pM or less, 150 pM or less, 140 pM or less, 120 pM or less, 100 pM or less, 90 pM or less, 80 pM or less, 70 pM or less, 60 pM or less, 50 pM or less, 40 pM or less, 30 pM or less, 20 pM or less, 15 pM or less, 10 pM or less, or 5 pM or less.

[0206] According to one embodiment, the tumor antigen x CD3 polypeptide / polypeptide construct of the present invention does not induce / mediate lysis or essentially does not induce / mediate lysis of cells that do not express the given tumor antigen on their surface (tumor antigen-negative cells), such as CHO cells. The terms "does not induce lysis," "essentially does not induce lysis," "does not mediate lysis," or "essentially does not mediate lysis" mean that the construct of the present invention does not induce or mediate lysis of more than 30%, preferably not more than 20%, more preferably not more than 10%, and particularly preferably not more than 9%, 8%, 7%, 6%, or 5% of tumor antigen-negative cells, thereby achieving 100% lysis of tumor antigen-expressing target cells (cells transformed or transfected with the tumor antigen or naturally expressing cell lines, such as human cancer lines). This typically applies to construct concentrations of up to 500 nM. Measuring cytolysis is a routine technique. Moreover, specific instructions for measuring cytolysis are provided herein.

[0207] The difference in cytotoxic activity between the monomeric and dimeric isoforms of a particular tumor antigen × CD3 polypeptide / polypeptide construct is referred to as the "potency gap." This potency gap can be calculated, for example, as the ratio between the EC50 value of the monomeric form of the molecule and the EC50 value of the dimeric form. One method for determining this gap is to perform an 18-hour 51-chromium release assay or a 48-hour FACS-based cytotoxicity assay using the purified monomer and dimer of the construct, as described below. Effector cells are stimulated enriched human CD8+ T cells or unstimulated human PBMCs. Target cells are CHO cells transfected with a human tumor antigen. The effector-to-target cell (E:T) ratio is 10:1. The potency gap of the tumor antigen × CD3 constructs of the present invention is preferably 5 or less, more preferably 4 or less, even more preferably 3 or less, even more preferably 2 or less, and most preferably 1 or less.

[0208] The binding domain of the polypeptide construct of the invention is preferably cross-species specific for members of the mammalian order of primates, such as macaques. According to one embodiment, the additional binding domain, in addition to binding to a human tumor antigen, will also bind to said tumor antigen in primates, including (but not limited to) New World primates (such as common marmosets (Callithrix jacchus), cotton-top tamarins (Saguinus Oedipus), or squirrel monkeys (Saimiri sciureus)), Old World primates (such as baboons and macaques), gibbons, orangutans, and non-human hominidae. It is envisioned that the domain that binds to human CD3 on the surface of T cells of the invention also binds to at least macaque CD3. A preferred macaque is the cynomolgus monkey (Macaca fascicularis). Rhesus monkeys (Macaca mulatta) (Rhesus monkey) are also envisioned. The polypeptide or polypeptide construct of the invention comprises a domain that binds to human CD3 epsilon and at least macaque CD3 on the surface of T cells.

[0209] In one embodiment, the affinity gap of a construct according to the invention for binding human CD3 compared to macaque CD3 (as determined, for example, by BiaCore or by Scatchard analysis) [KD ma CD3:KD hu CD3] is between 0.01 and 100, preferably between 0.1 and 10, more preferably between 0.2 and 5, more preferably between 0.3 and 4, even more preferably between 0.5 and 3 or between 0.5 and 2.5 and most preferably between 0.5 and 1.

[0210] As detailed herein above, said binding domain of the polypeptide or polypeptide construct of the invention binds to human CD3 epsilon (or human CD3 epsilon on the surface of T cells), preferably to CD3 epsilon of marmoset (Callithrix jacchus) or squirrel monkey (Saimiri sciureus). More particularly, said domain binds to an extracellular epitope of human CD3ε. It is also envisaged that said domain binds to an extracellular epitope of human and macaque CD3ε chain. Said extracellular epitope of CD3 epsilon is comprised within amino acid residues 1 to 27 of the extracellular domain of human CD3 epsilon (see amino acid residues 1 to 27 of SEQ ID NO: 2552; SEQ ID NO: 2553). Even more particularly, the epitope comprises at least the amino acid sequence Gln-Asp-Gly-Asn-Glu. The marmoset (Callithrix jacchus) is a New World primate belonging to the family Marmosets (Callitrichidae), while the squirrel monkey (Saimiri sciureus) is a New World primate belonging to the family Capuchin monkeys (Cebidae).

[0211] In a preferred embodiment, the polypeptide or polypeptide construct of the invention is a single-chain polypeptide that is at least bispecific. In this embodiment, the CD3-binding domain according to the invention is preferably present in the polypeptide or polypeptide construct as an scFv.

[0212] It is also envisioned that the polypeptide constructs of the present invention, in addition to their function of binding to CD3, will in certain embodiments have the further function of binding at least an additional binding domain. In this format, the constructs may be trifunctional or multifunctional constructs by providing additional functions such as targeting of target cells, preferably through tumor antigen binding, mediating cytotoxic T cell activity through CD3 binding, and providing a means or domain for enhancing or extending serum half-life, a fully functional or modified Fc constant domain for mediating cytotoxicity through the recruitment of effector cells, a therapeutic agent such as a label (such as fluorescent), a toxin or a radionuclide.

[0213] Examples of means or domains for extending the serum half-life of the polypeptides / polypeptide constructs of the present invention include peptides, proteins or protein domains that are fused or otherwise attached to the polypeptides / polypeptide constructs. The group of peptides, proteins or protein domains includes peptides that bind to other proteins with favorable pharmacokinetic profiles in the human body, such as serum albumin (see WO 2009 / 127691). An alternative concept for such half-life extending peptides includes peptides that bind to neonatal Fc receptors (FcRn, see WO 2007 / 098420), which can also be used in the constructs of the present invention. The concept of attaching larger domains of proteins or complete proteins includes fusions with human serum albumin, human serum albumin (see WO 2011 / 051489, WO 2012 / 059486, WO 2012 / 150319, WO 2013 / 135896, WO 2014 / 072481, WO 2013 / 075066) or variants or mutants of domains thereof, as well as fusions with immunoglobulin constant regions (Fc domains) and variants thereof. Such variants of the Fc domain are called Fc-based domains and can be optimized / modified, for example, to allow the desired pairing of dimers or multimers, to abrogate Fc receptor binding (e.g., to avoid ADCC or CDC), or for other reasons. A further concept known in the art for increasing the half-life of substances or molecules in the human body is the pegylation of those molecules (such as the constructs of the present invention).

[0214] In one embodiment, a polypeptide / polypeptide construct according to the invention is linked (e.g., via a peptide bond) to a fusion partner (e.g., a protein, polypeptide, or peptide, etc.), e.g., to extend the serum half-life of the construct. These fusion partners can be selected from human serum albumin ("HSA" or "HALB") and sequence variants thereof, peptides that bind to HSA, peptides that bind to FcRn ("FcRn BP"), or constructs comprising an Fc region (derived from an antibody). Exemplary sequences of these fusion partners are shown in SEQ ID NOs: 16, 18, and 19. Generally, the fusion partner can be linked to the N- or C-terminus of a construct according to the invention either directly (e.g., via a peptide bond) or through a peptide linker such as (GGGGS)n or (GGGGQ)n (where "n" is an integer equal to or greater than 1, e.g., 2, 3, or 4). Suitable peptide linkers are discussed above and are shown in SEQ ID NOs: 2 and 2551.

[0215] A polypeptide construct according to the invention comprises at least a bispecific single-chain polypeptide, said polypeptide comprising, in the following order from N-terminus to C-terminus: a) VL (comprising a portion of the cell surface antigen-binding domain / paratope)-(G4S)3 or (G4Q)3-VH (comprising a portion of the cell surface antigen-binding domain / paratope)-peptide linker (SG4S) or (SG4Q)-VH (comprising a portion of the CD3 epsilon-binding domain / paratope)-(G4S)3 or (G4Q)3-VL (comprising a portion of the CD3 epsilon-binding domain / paratope); b) VH (comprising a portion of the cell surface antigen-binding domain / paratope)-(G4S)3 or (G4Q)3-VL (comprising a portion of the cell surface antigen-binding domain / paratope)-peptide linker (SG4S) or (SG4Q)-VH (comprising a portion of the CD3 epsilon-binding domain / paratope)-(G4S)3 or (G4Q)3-VL (comprising a portion of the CD3 epsilon-binding domain / paratope); c) VL (comprising a portion of the cell surface antigen binding domain / paratope)-(G4S)3 or (G4Q)3-VH (comprising a portion of the cell surface antigen binding domain / paratope)-peptide linker (SG4S) or (SG4Q)-VH (comprising a portion of the CD3 epsilon binding domain / paratope)-(G4S)3 or (G4Q)3-VL (comprising a portion of the CD3 epsilon binding domain / paratope)-peptide linker (G4)-Fc monomer (part of the HLE domain)-(G4S)6 or (G4Q)6-Fc monomer (part of the HLE domain); d) VH (comprising a portion of the cell surface antigen binding domain / paratope)-(G4S)3 or (G4Q)3-VL (comprising a portion of the cell surface antigen binding domain / paratope)-peptide linker (SG4S) or (SG4Q)-VH (comprising a portion of the CD3 epsilon binding domain / paratope)-(G4S)3 or (G4Q)3-VL (comprising a portion of the CD3 epsilon binding domain / paratope)-peptide linker (G4)-Fc monomer (part of the HLE domain)-(G4S)6 or (G4Q)6-Fc monomer (part of the HLE domain); e) VH (comprising a portion of a first cell surface antigen binding domain / paratope)-(G4S)3 or (G4Q)3-VL (comprising a portion of a first cell surface antigen binding domain / paratope)-peptide linker (SG4S) or (SG4Q)-VL (comprising a portion of a second cell surface antigen binding domain / paratope)-(G4S)3 or (G4Q)3-VH (comprising a portion of a second cell surface antigen binding domain / paratope)-peptide linker (SG4S) or (SG4Q)-VH (comprising a portion of a CD3 epsilon binding domain / paratope)-(G4S)3 or (G4Q)3-VL (comprising a portion of a CD3 epsilon binding domain / paratope)-peptide linker (G4)-Fc monomer (part of an HLE domain)-(G4S)6 or (G4Q)6 or -Fc monomer (part of an HLE domain); f) VH (comprising a portion of a first cell surface antigen binding domain / paratope)-(G4S)3 or (G4Q)3-VL (comprising a portion of a first cell surface antigen binding domain / paratope)-peptide linker (SG4S) or (SG4Q)-VH (comprising a portion of a second cell surface antigen binding domain / paratope)-(G4S)3 or (G4Q)3-VL (comprising a portion of a second cell surface antigen binding domain / paratope)-peptide linker (SG4S) or (SG4Q)-VH (comprising a portion of a CD3 epsilon binding domain / paratope)-(G4S)3 or (G4Q)3-VL (comprising a portion of a CD3 epsilon binding domain / paratope)-peptide linker (G4)-Fc monomer (part of an HLE domain)-(G4S)6 or (G4Q)6 or -Fc monomer (part of an HLE domain); g) VL (comprising a portion of a first cell surface antigen binding domain / paratope)-(G4S)3 or (G4Q)3-VH (comprising a portion of a first cell surface antigen binding domain / paratope)-peptide linker (SG4S) or (SG4Q)-VL (comprising a portion of a second cell surface antigen binding domain / paratope)-(G4S)3 or (G4Q)3-VH (comprising a portion of a second cell surface antigen binding domain / paratope)-peptide linker (SG4S) or (SG4Q)-VH (comprising a portion of a CD3 epsilon binding domain / paratope)-(G4S)3 or (G4Q)3-VL (comprising a portion of a CD3 epsilon binding domain / paratope)-peptide linker (G4)-Fc monomer (part of an HLE domain)-(G4S)6 or (G4Q)6 or -Fc monomer (part of an HLE domain) h) VL (comprising a portion of a first cell surface antigen binding domain / paratope)-(G4S)3 or (G4Q)3-VH (comprising a portion of a first cell surface antigen binding domain / paratope)-peptide linker (SG4S) or (SG4Q)-VH (comprising a portion of a second cell surface antigen binding domain / paratope)-(G4S)3 or (G4Q)3-VL (comprising a portion of a second cell surface antigen binding domain / paratope)-peptide linker (SG4S) or (SG4Q)-VH (comprising a portion of a CD3 epsilon binding domain / paratope)-(G4S)3 or (G4Q)3-VL (comprising a portion of a CD3 epsilon binding domain / paratope)-peptide linker (G4)-Fc monomer (part of an HLE domain)-(G4S)6 or (G4Q)6 or -Fc monomer (part of an HLE domain); or i) Binding domain 1 ((VL (comprising a portion of the first cell surface antigen binding domain / paratope)-(G4S)3 or (G4Q)3-VH (comprising a portion of the first cell surface antigen binding domain / paratope)) or (VH (comprising a portion of the first cell surface antigen binding domain / paratope)-(G4S)3 or (G4Q)3-VL (comprising a portion of the first cell surface antigen binding domain / paratope)))-peptide linker (G4S) or (G4Q)-CD3 binding domain 1 (VH (comprising a portion of the first CD3 epsilon binding domain / paratope)-(G4S)3 or (G4Q)3-VL (comprising a portion of the first CD3 epsilon binding domain / paratope))-peptide linker (G4)-Fc monomer (part of the HLE domain)-(G4S)6 or (G4Q)6-Fc monomer (part of the HLE domain)-peptide linker (G4)-binding domain 2 ((VL (including part of the second cell surface antigen binding domain / paratope)-(G4S)3 or (G4Q)3-VH (including part of the first cell surface antigen binding domain / paratope)) or (VH (including part of the second cell surface antigen binding domain / paratope)-(G4S)3 or (G4Q)3-VL (including part of the second cell surface antigen binding domain / paratope)))-peptide linker (G4S) or (G4Q)-CD3 binding domain 2 (VH (including part of the second CD3 epsilon binding domain / paratope)-(G4S)3 or (G4Q)3-VL (including part of the second CD3 epsilon binding domain / paratope)). It comprises or consists of:

[0216] As is clear from the above, the orientation of the sequences of the VH and VL regions of the cell surface antigen-binding domain can be VH-VL or VL-VH. Preferably, the cell surface antigen is a tumor antigen as detailed hereinabove. In particular, the HLE domain sequence composed of the Fc monomer and the connecting linker is preferably selected from the sequences defined in SEQ ID NOs: 18 and 19. The two CD3-binding domains of the polypeptide construct of item i) are preferably the same CD3-binding domain, for example, CD3-binding domains preferably having the VH and VL region sequences of SEQ ID NOs: 2028 and 2029. Peptide linkers (SG4S) or (SG4Q) are preferred at the indicated positions, but can also be replaced by (G4S) or (G4Q) linkers.

[0217] Covalent modifications of polypeptides / polypeptide constructs are also within the scope of the present invention and are generally, but not necessarily, performed post-translationally. For example, several types of covalent modifications of constructs are introduced into molecules by reacting specific amino acid residues of the construct with organic derivatizing agents that can react with selected side chains or N- or C-terminal residues. Derivatization with bifunctional agents is useful for crosslinking the constructs of the present invention to water-insoluble support matrices or surfaces for use in various methods. Glutaminyl and asparaginyl residues are often deamidated to the corresponding glutamyl and aspartyl residues, respectively. Alternatively, these residues are deamidated under mildly acidic conditions. Both forms of these residues are within the scope of the present invention. Other modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl group of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine, and histidine side chains (TECreighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, 1983, pp. 79-86), acetylation of the N-terminal amine, and amidation of either C-terminal carboxyl group.

[0218] Another type of covalent modification of constructs within the scope of the present invention involves altering the glycosylation pattern of a protein. As is known in the art, glycosylation patterns can depend both on the sequence of the protein (e.g., the presence or absence of particular glycosylated amino acid residues, discussed below) and the host cell or organism in which the protein is produced. Specific expression systems are discussed below. Glycosylation of polypeptides is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Therefore, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used.

[0219] Addition of glycosylation sites to the construct is conveniently accomplished by altering the amino acid sequence to contain one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). Alterations may also be made by adding or substituting one or more serine or threonine residues to the starting sequence (for O-linked glycosylation sites). For ease of use, the amino acid sequence of the construct may be altered through changes at the DNA level, particularly by mutating the DNA encoding the polypeptide at preselected bases, thereby resulting in a codon that will be translated into the desired amino acid.

[0220] Another means of increasing the number of carbohydrate moieties on a construct is by chemical or enzymatic coupling of glycosides to the protein. These procedures are advantageous in that they do not require production of the protein in a host cell with glycosylation capabilities for N- and O-linked glycosylation. Depending on the coupling mode used, sugars can be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyl groups such as those of serine, threonine, or hydroxyproline, (e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan, or (f) the amide group of glutamine. These methods are described in WO 87 / 05330 and Aplin and Wriston, 1981, CRC Crit. Rev. Biochem., pp. 259-306.

[0221] Removal of carbohydrate moieties present on the starting construct can be accomplished chemically or enzymatically. Chemical deglycosylation requires exposure of the protein to the compound trifluoromethanesulfonic acid or an equivalent compound. This treatment results in the cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or N-acetylgalactosamine), while leaving the polypeptide intact. Chemical deglycosylation is described by Hakimuddin et al., 1987, Arch. Biochem. Biophys. 259:52 and Edge et al., 1981, Anal. Biochem. 118:131. Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved using a variety of endo- and exoglycosidases as described by Thotakura et al., 1987, Meth. Enzymol. 138:350. Glycosylation at potential glycosylation sites can be prevented using the compound tunicamycin described by Duskin et al., 1982, J. Biol. Chem. 257:3105. Tunicamycin blocks the formation of protein-N-glycosidic bonds.

[0222] Other modifications of the construct are also contemplated herein. For example, another type of covalent modification of the construct involves linking the construct to various non-proteinaceous polymers, including polyols, in the manner shown in U.S. Patent Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192, or 4,179,337. In addition, as is known in the art, amino acid substitutions can be made at various positions within the construct to facilitate the addition of polymers such as polyethylene glycol (PEG).

[0223] In some embodiments, covalent modification of the constructs of the invention involves the addition of one or more labels. To reduce potential steric hindrance, labeling groups can be linked to the constructs via spacer arms of various lengths. Various methods for labeling proteins are known in the art and can be used in practicing the present invention. The term "label" or "labeling group" refers to any detectable label. Generally, labels are divided into various classes depending on the assay in which they will be detected, examples of which include, but are not limited to: a) Radioisotopes or radionuclides (e.g., 3 H, 14 C. 15 N, 35 S, 89 Zr, 90 Y, 99 Tc, 111 In, 125 I, 131 Isotopic labels, which may be radioactive or heavy isotopes such as I) b) Magnetic labels (e.g., magnetic particles) c) redox-active moieties d) optical dyes (including but not limited to chromophores, phosphors and fluorophores) such as fluorescent labels or fluorophores, which can be either fluorescent groups (e.g., FITC, rhodamine, lanthanide phosphors), chemiluminescent groups and "small molecule" fluorophores or proteinaceous fluorophores; e) Enzyme groups (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase) f) Biotinylation group g) A predetermined polypeptide epitope recognized by a secondary reporter (e.g., a leucine zipper pair sequence, a binding site for a secondary antibody, a metal binding domain, an epitope tag, etc.).

[0224] "Fluorescent label" refers to any molecule that can be detected through its inherent fluorescent properties. Suitable fluorescent labels include fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methyl-coumarin, pyrene, malacite green, stilbene, Lucifer Yellow, Cascade Blue J, Texas Red, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy 5, Cy 5.5, LC Red 705, Oregon Green, Alexa-Fluor dyes (Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660, Alexa Fluor 680), Cascade Blue, Cascade Yellow, and R-phycoerythrin (PE) (Molecular Probes, Eugene, OR), FITC, rhodamine, and Texas Red. Suitable optical dyes, including fluorophores, include, but are not limited to, Cy5, Cy5.5, Cy7 (Amersham Life Science, Pittsburgh, PA). Suitable optical dyes, including fluorophores, are described in Molecular Probes Handbook by Richard P. Haugland.

[0225] Suitable proteinaceous fluorescent labels include green fluorescent proteins, e.g., GFPs of Renilla, Ptilosarcus, or Aequorea species (Chalfie et al., 1994, Science 263:802-805), EGFP (Clontech Laboratories, Inc., Genbank® accession number U55762), blue fluorescent protein (BFP, Quantum Biotechnologies, Inc. 1801 de Maisonneuve Blvd. West, 8th Floor, Montreal, Quebec, Canada H3H 1J9; Stauber, 1998, Biotechniques 24:462-471; Heim et al., 1996, Curr. Biol. 6:178-182), enhanced yellow fluorescent protein (EYFP, Clontech Laboratories, Inc.), luciferase (Ichiki et al., 1994, Science 263:802-805), and the like. al., 1993, J. Immunol. 150:5408-5417), β-galactosidase (Nolan et al. al., 1988, Proc. Natl. Acad. Sci. USA 85:2603-2607) and Renilla (WO 92 / 15673, WO 95 / 07463, WO 98 / 14605, WO 98 / 26277, WO 99 / 49019, U.S. Pat. Nos. 5,292,658; 5,418,155; 5,683,888; 5,741,668; 5,777,079; 5,804,387; 5,874,304; 5,876,995; and 5,925,558).

[0226] Leucine zipper domains are peptides that promote oligomerization of proteins in which they are found. Leucine zippers were originally discovered in a variety of different proteins and have since been identified in several DNA-binding proteins (Landschulz et al., 1988, Science 240:1759). Among the known leucine zippers are naturally occurring peptides and their dimerizing or trimerizing derivatives. Examples of leucine zipper domains suitable for producing soluble oligomeric proteins are described in PCT Application WO 94 / 10308, and a leucine zipper derived from pulmonary surfactant protein D (SPD) is described in Hoppe et al., 1994, FEBS Letters 344:191. The use of a modified leucine zipper that allows stable trimerization of a heterologous protein fused thereto is described in Fanslow et al., 1994, Semin. Immunol. 6:267-78.

[0227] The polypeptide constructs of the present invention may also contain additional domains, for example, to aid in the isolation of the molecule or to improve the pharmacokinetic properties of the molecule. Domains useful for construct isolation can be selected from peptide motifs or secondary introduction moieties that can be captured by isolation methods, such as isolation columns. Non-limiting examples of such additional domains include peptide motifs known as Myc-tags, HAT-tags, HA-tags, TAP-tags, GST-tags, chitin-binding domains (CBD-tags), maltose-binding protein (MBP-tags), Flag-tags, Strep-tags and their variants (e.g., Strep II-tags), and His-tags. All of the constructs disclosed herein featuring identified CDRs may contain a His-tag domain, commonly known as a repeat of consecutive His residues in the amino acid sequence of the molecule, for example, five His residues or six His residues (hexa-histidine). The His-tag can be located, for example, at the N- or C-terminus of the construct. In one embodiment, a hexahistidine tag (HHHHHH) is linked via a peptide bond to the C-terminus of the construct according to the invention. A histidine tag, particularly a 6xHis tag, is preferred.

[0228] The present invention also relates to polynucleotides encoding the polypeptides or polypeptide constructs of the present invention. Nucleic acid molecules are biopolymers composed of nucleotides. Polynucleotides are biopolymers composed of 13 or more nucleotide monomers covalently linked in a chain. DNA (e.g., cDNA) and RNA (e.g., mRNA) are examples of polynucleotides / nucleic acid molecules with different biological functions. Nucleotides are organic molecules that function as monomers or subunits of nucleic acid molecules such as DNA or RNA. Nucleic acid molecules or polynucleotides of the present invention can be double-stranded or single-stranded, linear or circular. It is contemplated that the nucleic acid molecules or polynucleotides are contained in vectors. Furthermore, it is contemplated that such vectors are contained in host cells. The host cells are capable of expressing the constructs, for example, after transformation or transfection with the vectors or polynucleotides / nucleic acid molecules of the present invention. For this purpose, the polynucleotides or nucleic acid molecules are operably linked to regulatory sequences.

[0229] The genetic code is a set of rules by which information encoded in genetic material (nucleic acids) is translated into proteins. Biological decoding in living cells is accomplished by ribosomes, which carry amino acids and use tRNA molecules, which read the mRNA three nucleotides at a time, to link amino acids in the order specified by the mRNA. This code defines how triplet nucleotide sequences, called codons, specify the amino acid to be added next during protein synthesis. With some exceptions, a triplet codon in a nucleic acid sequence specifies one amino acid. Because the majority of genes are encoded using the exact same code, this particular code is often referred to as the canonical or standard genetic code.

[0230] Codon degeneracy is the redundancy of the genetic code, manifested as multiple combinations of three-base-pair codons that specify an amino acid. Degeneracy arises because there are a large number of codons compared to the number of codable amino acids. Codons encoding an amino acid can differ in any of their three positions, but often this difference is in the second or third position. For example, the codons GAA and GAG both specify glutamic acid, demonstrating redundancy, but neither specifies any other amino acid and therefore does not exhibit ambiguity. The genetic code of different organisms may be biased toward using one of several codons that encode the same amino acid over others; i.e., the frequency of one is higher than expected by chance. For example, leucine is specified by six distinct codons, some of which are rarely used. Codon usage tables detailing genomic codon usage for most organisms are available. In recombinant genetic technology, this effect is often exploited by implementing a technique called codon optimization, in which polynucleotides are designed using codons preferred by the respective host cells (e.g., cells of human or hamster origin, Escherichia coli cells, or Saccharomyces cerevisiae cells) to increase, for example, protein expression. Thus, it is envisioned that the polynucleotides / nucleic acid molecules of the present disclosure are codon-optimized. Nevertheless, polynucleotides / nucleic acid molecules encoding the constructs of the present invention may be designed using any codons that encode the desired amino acids.

[0231] According to one embodiment, the polynucleotide / nucleic acid molecule of the present invention encoding the polypeptide construct of the present invention is in the form of one single molecule or in the form of two or more separate molecules. If the construct of the present invention is a single-chain construct, the polynucleotide / nucleic acid molecule encoding such a construct will most likely also be in the form of a single molecule. However, it is also envisaged that different components of the polypeptide construct (e.g., different domains, e.g., a paratope (antigen-binding (epitope-binding) structure)-containing domain that binds to a cell surface antigen, a paratope (antigen-binding (epitope-binding) structure)-containing domain that binds to CD3, and / or further domains such as antibody constant domains) are located on separate polypeptide chains, in which case the polynucleotide / nucleic acid molecule will most likely be in the form of two or more separate molecules.

[0232] The same applies to vectors containing the polynucleotides / nucleic acid molecules of the present invention. When the construct of the present invention is a single-stranded construct, one vector may contain the polynucleotides encoding the construct in one location (as one open reading frame, ORF). One vector may also contain two or more polynucleotides / nucleic acid molecules in separate locations (with individual ORFs), each of which encodes a different component of the construct of the present invention. It is envisioned that vectors containing the polynucleotides / nucleic acid molecules of the present invention may be in the form of a single vector or two or more separate vectors. In one embodiment, for the purpose of expressing the construct in a host cell, the host cell of the present invention should contain the polynucleotides / nucleic acid molecules encoding the construct or a vector containing such polynucleotides / nucleic acid molecules in their entirety, meaning that all components of the construct (whether encoded as a single molecule or in separate molecules / locations) assemble after translation to form together the biologically active construct of the present invention.

[0233] The present invention further relates to vectors comprising the polynucleotide / nucleic acid molecules of the present invention. A vector is a nucleic acid molecule typically used as a vehicle for transferring (exogenous) genetic material into cells to ensure its replication and / or expression. The term "vector" includes, but is not limited to, plasmids, viruses, cosmids, and artificial chromosomes. Some vectors are specifically designed for cloning (cloning vectors), while others are designed for protein expression (expression vectors). So-called transcription vectors are primarily used to amplify their inserts. DNA manipulations are typically performed on E. coli vectors, which contain elements necessary for their maintenance in E. coli. However, vectors can also have elements that allow them to be maintained in other organisms, such as yeast, plant, or mammalian cells; these vectors are called shuttle vectors. Insertion of a vector into a target or host cell is typically referred to as transformation for bacterial cells and transfection for eukaryotic cells, while insertion of a viral vector is often referred to as transduction.

[0234] Generally, engineered vectors contain an origin of replication, a multiple cloning site, and a selectable marker. The vector itself is a nucleotide sequence, usually a DNA sequence, that generally contains an insert (transgene) and a larger sequence that serves as the "backbone" of the vector. While the genetic code determines the polypeptide sequence of a given coding region, other genomic regions can influence when and where these polypeptides are produced. Therefore, in addition to the transgene insert and backbone, modern vectors may include the following additional features: promoters, genetic markers, antibiotic resistance, reporter genes, targeting sequences, and protein purification tags. Vectors called expression vectors (expression constructs) are specifically intended for the expression of transgenes in target cells and generally contain regulatory sequences.

[0235] The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. Control sequences that are suitable for prokaryotes include, for example, a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, Kozak sequences, and enhancers.

[0236] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned to facilitate translation. Generally, "operably linked" means that the nucleotide sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. Enhancers, however, need not be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0237] "Transfection" is the process of intentionally introducing nucleic acid molecules or polynucleotides (including vectors) into target cells. The term is primarily used for non-viral methods in eukaryotic cells. Transduction is often used to describe viral-mediated transfer of nucleic acid molecules or polynucleotides. Transfection of animal cells typically involves creating transient pores or "holes" in the cell membrane to allow uptake of material. Transfection can be performed using biological particles (e.g., viral transfection, also known as viral transduction), chemical-based methods (e.g., using calcium phosphate, lipofection, Fugene, cationic polymers, nanoparticles, etc.), or physical treatments (e.g., electroporation, microinjection, gene guns, cell squeezing, magnetofection, hydrostatic pressure, impalement, sonication, optical transfection, heat shock).

[0238] The term "transformation" is used to describe the non-viral transfer of nucleic acid molecules or polynucleotides (including vectors) into non-animal eukaryotic cells, including bacteria and plant cells. Transformation is thus the genetic modification of bacteria or non-animal eukaryotic cells resulting from the direct uptake and subsequent integration of exogenous genetic material (nucleic acid molecules) from their surroundings through the cell membrane. Transformation can be achieved by artificial means. For transformation to occur, cells or bacteria must be in a state of competence, which can occur as a time-limited response to environmental conditions such as starvation and cell density, and can also be induced artificially.

[0239] Moreover, the present invention provides host cells transformed or transfected with a polynucleotide / nucleic acid molecule of the invention or a vector of the invention.

[0240] As used herein, the terms "host cell" or "recipient cell" are intended to include any individual cell or cell culture that can be or has been a recipient of a vector, exogenous nucleic acid molecule, and / or polynucleotide encoding a construct of the present invention, and / or the recipient of the construct itself. Introduction of individual materials into cells is accomplished by transformation, transfection, and the like (see above). The term "host cell" is also intended to include the passage or potential succession of a single cell. Because certain modifications may occur in successive generations due to either spontaneous, accidental, or deliberate mutations or environmental influences, such passages may not, in fact, be completely identical to the parent cell (in morphology or genomic or total DNA complement), yet still fall within the scope of the term as used herein. Suitable host cells include prokaryotic or eukaryotic cells, including, but not limited to, bacteria (such as E. coli), yeast cells, fungal cells, plant cells, and animal cells (e.g., insect cells and mammalian cells, e.g., hamster, mouse, rat, macaque, or human).

[0241] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for the constructs of the present invention. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, several other genera, species, and strains are commonly available and useful in the present invention, such as Schizosaccharomyces pombe, K. lactis, K. fragilis (ATCC 12424), K. bulgaricus (ATCC 16045), K. wickeramii (ATCC 24178), K. waltii (ATCC 56500), K. drosophilarum (ATCC 16045), and others. Kluyveromyces hosts such as Kluyveromyces spp. (European Patent No. 36906), K. thermotolerans, and K. marxianus; Yarrowia spp. (European Patent No. 402226); Pichia pastoris (European Patent No. 183070); Candida spp.; Trichoderma reesia (European Patent No. 244234); Neurospora crassa; Schwanniomyces occidentalis; Schwanniomyces such as A. occidentalis; and filamentous fungi such as Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.

[0242] Suitable host cells for the expression of glycosylated constructs are derived from multicellular organisms. Examples of invertebrate cells include plant cells and insect cells. Many baculovirus strains and variants have been identified, as well as corresponding permissive insect host cells derived from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori (domestic silkworm). Various virus strains for transfection (e.g., the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV) are publicly available, and such viruses may be used herein as viruses according to the present invention, particularly for transfection of Spodoptera frugiperda cells.

[0243] Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, Arabidopsis, and tobacco can also be used as hosts. Cloning and expression vectors useful for producing proteins in plant cell culture are known to those skilled in the art. See, for example, Hiatt et al., Nature (1989) 342:76-78; Owen et al. (1992) Bio / Technology 10:790-794; Artsaenko et al. (1995) The Plant J 8:745-750; and Fecker et al. (1996) Plant Mol Biol 32:979-986.

[0244] However, most interest has been in vertebrate cells, and propagation of vertebrate cells in culture (cell culture) has become a routine procedure. Examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 lines (e.g., COS-7, ATCC CRL 1651), human embryonic kidney lines (e.g., 293 cells or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)), baby hamster kidney cells (e.g., BHK, ATCC CCL 10), Chinese hamster ovary cells / -DHFR (e.g., CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)), mouse Sertoli cells (e.g., TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (e.g., CVI ATCC CCL 70), African green monkey kidney cells (VERO-76, ATCC CRL1587, etc.), human cervical carcinoma cells (HELA, ATCC CCL 2, etc.), canine kidney cells (MDCK, ATCC CCL 34, etc.), buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442, etc.), human lung cells (W138, ATCC CCL 75, etc.), human hepatocytes (Hep G2, 1413 8065, etc.), mouse mammary tumor cells (MMT 060562, ATCC CCL-51, etc.), TRI cells (Mather et al., Annals NY Acad. Sci. (1982) 383:44-68), MRC 5 cells, FS4 cells, and human hepatoma lines (Hep G2, etc.).

[0245] In a further embodiment, the present invention provides a process for producing a polypeptide or polypeptide construct of the invention, comprising culturing a host cell of the invention under conditions that allow expression of the construct of the invention, and recovering the produced construct from the culture.

[0246] As used herein, the term "culturing" refers to the in vitro maintenance, differentiation, growth, proliferation, and / or propagation of cells in a medium under appropriate conditions. Cells are grown and maintained in a cell growth medium at an appropriate temperature and gas mixture. Culture conditions vary widely for each cell type. Typical growth conditions are a temperature of about 37°C, a CO2 concentration of about 5%, and a humidity of about 95%. Growth medium recipes can vary, for example, in pH, the concentration of the carbon source (e.g., glucose), the nature and concentration of growth factors, and the presence of other nutrients (e.g., amino acids or vitamins). Growth factors used in supplemented media are often derived from serum from animal blood (e.g., fetal bovine serum (FBS), calf serum (FCS), horse serum, and porcine serum). Cells can be grown either in suspension culture or as adherent culture. Some cell lines have been modified to survive in suspension culture and can therefore grow to higher densities than in adherent conditions.

[0247] The term "expression" includes any step involved in producing the construct of the present invention, including, but not limited to, transcription, post-transcriptional modification, translation, folding, post-translational modification, targeting to a specific intracellular or extracellular location, and secretion. The term "recovering" refers to a series of processes intended to isolate the construct from cell culture. The "recovery" or "purification" process separates the protein and non-protein portions of the cell culture, ultimately separating the desired construct from all other polypeptides and proteins. Separation steps typically exploit differences in protein size, physicochemical properties, binding affinity, and biological activity. Preparative purification aims to produce relatively large amounts of purified protein for subsequent use, while analytical purification produces relatively small amounts of protein for various research or analytical purposes.

[0248] When using recombinant techniques, constructs can be produced intracellularly in the periplasmic space or directly secreted into the culture medium. If the construct is produced intracellularly, the first step is to remove particulate debris, such as host cells or lysed fragments, for example, by centrifugation or ultrafiltration. Constructs of the present invention can be produced in bacteria, such as E. coli. After expression, the construct can be isolated in a soluble fraction from the bacterial cell paste and purified, for example, via affinity chromatography and / or size exclusion. Final purification can be carried out similarly to the process for purifying constructs expressed in mammalian cells and secreted into the culture medium. Carter et al. (Biotechnology (NY) 1992 Feb;10(2):163-7) describe a procedure for isolating antibodies secreted into the periplasmic space of E. coli.

[0249] Where the antibody is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, such as an ultrafiltration unit.

[0250] Constructs of the invention prepared from host cells can be recovered or purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography. Other techniques for protein purification, such as fractionation on an ion exchange column, mixed-mode ion exchange, HIC, ethanol precipitation, size exclusion chromatography, reverse-phase HPLC, chromatography on silica, chromatography on heparin Sepharose, chromatography on anion or cation exchange resins (such as polyaspartic acid columns), immunoaffinity (such as protein A / G / L) chromatography, chromatofocusing, SDS-PAGE, ultracentrifugation, and ammonium sulfate precipitation, are also available depending on the construct being recovered.

[0251] In any of the above steps, protease inhibitors may be included to inhibit proteolysis and antibiotics may be included to prevent the growth of contaminants.

[0252] Moreover, the present invention provides pharmaceutical compositions or formulations comprising a polypeptide or polypeptide construct of the invention or a polypeptide or polypeptide construct produced by a process of the invention.

[0253] As used herein, the term "pharmaceutical composition" relates to a composition suitable for administration to a patient, preferably a human patient. Particularly preferred pharmaceutical compositions of the present invention comprise one or more constructs of the present invention, preferably in a therapeutically effective amount. Preferably, the pharmaceutical composition further comprises a suitable formulation of one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, preservatives and / or adjuvants. Acceptable components of the composition are preferably non-toxic to the recipient at the dosages and concentrations used. Pharmaceutical compositions of the present invention include, but are not limited to, liquid, frozen and lyophilized compositions.

[0254] The composition may contain a pharmaceutically acceptable carrier. Generally, as used herein, "pharmaceutically acceptable carrier" refers to all aqueous and non-aqueous solutions, sterile solutions, solvents, buffer solutions, such as phosphate buffered saline (PBS) solutions, water, suspensions, emulsions such as oil / water emulsions, various types of wetting agents, liposomes, dispersion media and coatings that are suitable for pharmaceutical administration, especially parenteral administration. The use of such media and agents in pharmaceutical compositions is well known in the art, and compositions containing such carriers can be formulated by well-known conventional methods.

[0255] Certain embodiments provide pharmaceutical compositions comprising a construct of the invention and one or more additional excipients, such as those illustratively described in this section and elsewhere herein. Excipients may be used in the invention for a variety of purposes, such as adjusting the physical, chemical, or biological properties of the formulation (e.g., adjusting viscosity), and / or may be used in the processes of the invention to improve efficacy and / or stabilize such formulations and processes against degradation and deterioration due to, for example, manufacturing, shipping, storage, preparation prior to use, administration, and subsequent stresses. Excipients are generally intended to be used at their lowest effective concentration.

[0256] In certain embodiments, pharmaceutical compositions may contain formulation materials to modify, maintain, or preserve certain properties of the composition, such as pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption, or penetration (see Remington's Pharmaceutical Sciences, 18th Edition, 1990, Mack Publishing Company). In such embodiments, suitable formulation materials may include, but are not limited to, the following: ·amino acid Antimicrobials, such as antibacterial and antifungal drugs Antioxidants Buffers, buffer systems, and buffering agents used to maintain the composition at or slightly below physiological pH, typically within a pH range of about 5 to about 8 or 9 Non-aqueous solvents, vegetable oils and injectable organic esters Aqueous carriers such as water, alcoholic / aqueous solutions, emulsions or suspensions, e.g. physiological saline solutions and buffered media Biodegradable polymers such as polyester Bulking agent Chelating agents Isotonic and absorption retarding agents Complexing agents Fillers ·carbohydrates (low molecular weight) proteins, polypeptides or proteinaceous carriers, preferably of human origin Coloring and flavoring agents Sulfur-containing reducing agent Diluents ·emulsifier Hydrophilic polymer Salt-forming counterions Preservatives Metal complexes Solvents and co-solvents Sugars and sugar alcohols Suspension agents Surfactants or wetting agents Stabilizer Isotonicity enhancer Parenteral delivery vehicles · Intravenous delivery vehicles.

[0257] It is common knowledge that different components of a pharmaceutical composition may have different effects, for example, amino acids may act as buffers, stabilizers and / or antioxidants; mannitol may act as a bulking agent and / or tonicity enhancer; sodium chloride may act as a delivery vehicle and / or tonicity enhancer, etc.

[0258] In the context of the present invention, the pharmaceutical composition comprises: (a) a polypeptide or polypeptide construct described herein; (b) at least one buffer; (c) at least one sugar, and (d) at least one surfactant and the pH of the pharmaceutical composition is in the range of 3.5 to 6.

[0259] In the above-described composition, the first domain preferably has an isoelectric point (pI) in the range of 4 to 9.5, the second domain has a pI in the range of 8 to 10, preferably 8.5 to 9.0, and the construct optionally comprises a third domain comprising two polypeptide monomers each comprising a hinge, a CH2 domain and a CH3 domain, said two polypeptide monomers being fused to each other via a peptide linker.

[0260] It is further contemplated that in the above-described composition, the at least one buffering agent is present at a concentration ranging from 5 to 200 mM, more preferably from 10 to 50 mM. It is also contemplated that the at least one sugar is selected from the group consisting of monosaccharides, disaccharides, cyclic polysaccharides, sugar alcohols, linear branched dextran, or linear unbranched dextran. It is also contemplated that the disaccharide is selected from the group consisting of sucrose, trehalose, mannitol, sorbitol, and combinations thereof. It is further contemplated that the sugar alcohol is sorbitol. It is also contemplated that the at least one sugar is present at a concentration ranging from 1 to 15% (m / V), preferably from 9 to 12% (m / V). It is further contemplated that the construct is present at a concentration ranging from 0.1 to 8 mg / ml, preferably from 0.2 to 2.5 mg / ml, and more preferably from 0.25 to 1.0 mg / ml.

[0261] According to one embodiment of the above-described composition, the at least one surfactant is selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, poloxamer 188, pluronic F68, Triton X-100, polyoxyethylene, PEG 3350, PEG 4000, and combinations thereof. It is further contemplated that the at least one surfactant is present in a concentration ranging from 0.004 to 0.5% (m / V), preferably from 0.001 to 0.01% (m / V). It is also contemplated that the pH of the composition is in the range of 4.0 to 5.0, preferably 4.2. It is also contemplated that the pharmaceutical composition has an osmolality ranging from 150 to 500 mOsm. It is further contemplated that the pharmaceutical composition further comprises an excipient selected from the group consisting of one or more polyols and one or more amino acids. In the context of the present invention, it is contemplated that the one or more excipients are present in a concentration ranging from 0.1 to 15% (w / V).

[0262] The present invention also provides a pharmaceutical composition comprising: (a) a construct described herein, preferably in a concentration range of 0.1 to 8 mg / ml, preferably 0.2 to 2.5 mg / ml, more preferably 0.25 to 1.0 mg / ml; (b) 10 mM glutamate or acetate; (c) 9% (m / V) sucrose or 6% (m / V) sucrose and 6% (m / V) hydroxypropyl-β-cyclodextrin; and (d) 0.01% (m / V) polysorbate 80, wherein the pH of the liquid pharmaceutical composition is 4.2.

[0263] It is envisaged that the compositions of the present invention may contain, in addition to the constructs of the present invention as defined herein, further biologically active agents depending on the intended use of the composition. Such agents may be drugs acting on the gastrointestinal system, drugs acting as cytostatics, drugs preventing hyperuricemia, drugs suppressing immune responses, drugs modulating inflammatory responses, drugs acting on the circulatory system and / or cytokines known in the art. It is also envisaged that the polypeptide constructs of the present invention may be applied in combination therapy, i.e. in combination with another anti-cancer drug.

[0264] In this regard, it is envisaged that the pharmaceutical compositions of the invention (including constructs comprising a CD3-binding domain and at least one additional binding domain that binds to a cell surface target antigen, preferably a tumor antigen on the surface of a target cell, as described in more detail herein above) further comprise an agent, preferably an antibody or construct, that binds to a protein in an immune checkpoint pathway (such as PD-1 or CTLA-4) or a costimulatory immune checkpoint receptor (such as 4-1BB). The present invention also refers to the combination of a polypeptide construct according to the invention (including constructs comprising a CD3-binding domain and at least one additional binding domain that binds to a cell surface target antigen, preferably a tumor antigen on the surface of a target cell, as described in more detail herein above) with an agent, preferably an antibody or polypeptide construct, that binds to a protein in an immune checkpoint pathway (such as PD-1 or CTLA-4) or a costimulatory immune checkpoint receptor (such as 4-1BB). Due to the nature of the at least two components of the combination, i.e., their pharmaceutical activity, the combination may also be referred to as a therapeutic combination. In some embodiments, the combination may be in the form of a pharmaceutical composition or a kit. According to one embodiment, the pharmaceutical composition or combination comprises a construct of the invention and an antibody or construct that binds to PD-1. Anti-PD-1 binding proteins useful for this purpose are described in detail, for example, in PCT / US2019 / 013205, which is incorporated herein by reference.

[0265] In certain embodiments, the optimal pharmaceutical composition will depend, for example, on the intended route of administration, delivery format, and desired dosage. See, e.g., Remington's Pharmaceutical Sciences, supra. In certain embodiments, such compositions may influence the physical state, stability, in vivo release rate, and in vivo clearance rate of the constructs of the present invention. In certain embodiments, the primary vehicle or carrier in a pharmaceutical composition may be either aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier may be water for injection or physiological saline solution, optionally supplemented with other materials commonly found in compositions for parenteral administration. In certain embodiments, compositions comprising the constructs of the present invention may be prepared for storage in the form of a lyophilized cake or aqueous solution by mixing selected components having the desired degree of purity with optional formulating agents (Remington's Pharmaceutical Sciences, supra). Furthermore, in certain embodiments, the constructs of the present invention may be formulated as a lyophilizate using appropriate excipients.

[0266] When parenteral administration is intended, therapeutic compositions for use in the present invention may be provided in the form of a pyrogen-free, parenterally acceptable aqueous solution containing the desired construct of the present invention in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water, in which the construct of the present invention is formulated as a properly preserved, sterile, isotonic solution. In certain embodiments, this preparation may involve formulating the desired molecule with an agent that can provide controlled or sustained release of the formulation, or an agent that can promote prolonged effective time in the circulation. In certain embodiments, an implantable drug delivery device may be used to introduce the desired construct.

[0267] Further pharmaceutical compositions will be apparent to those skilled in the art, including formulations comprising the constructs of the present invention in sustained or controlled delivery formulations. Techniques for formulating various sustained or controlled delivery means are known to those skilled in the art. The constructs may also be encapsulated in microcapsules prepared in colloidal drug delivery systems or in macroemulsions, for example, by coacervation techniques or interfacial polymerization. Such techniques are disclosed in Remington's Pharmaceutical Sciences, supra.

[0268] Pharmaceutical compositions for in vivo administration are usually provided as sterile preparations.Sterilization can be achieved by filtration through sterile filtration membranes.If the composition is freeze-dried, sterilization using this method can be carried out before or after freeze-drying and reconstitution.Compositions for parenteral administration can be stored in freeze-dried form or in solution.Parenteral compositions are generally placed into a container with a sterile access port, for example, an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic injection needle.

[0269] Another aspect of the present invention includes self-buffering formulations comprising the constructs of the present invention, which can be used as pharmaceutical compositions, as described in WO 2006 / 138181. Regarding protein stabilization and formulation materials and methods useful in this regard, various publications are available, such as Arawaka T. et al., Pharm Res. 1991 March;8(3):285-91; Kendrick et al., "Physical stabilization of proteins in aqueous solution" in: Rational Design of Stable Protein Formulations: Theory and Practice, Carpenter and Manning, eds. Pharmaceutical Biotechnology. 13:61-84 (2002); and Randolph and Jones, Pharm Biotechnol. 2002;13:159-75, see in particular the sections relating to excipients and processes for self-buffering protein formulations, particularly protein pharmaceuticals and processes for use in veterinary and / or human medicine.

[0270] Salts can be used in certain embodiments of the present invention, for example, to adjust the ionic strength and / or tonicity of a composition or formulation and / or improve the solubility and / or physical stability of constructs or other components of a composition according to the present invention. Ions can stabilize proteins in their native state by binding to charged residues on the surface of the protein and by shielding the protein's charged and polar groups, reducing the strength of their electrostatic, attractive, and repulsive interactions. Ions can also stabilize proteins in their denatured state, particularly by binding to the protein's denatured peptide bond (--CONH). Furthermore, ionic interactions with charged and polar groups in proteins can also reduce intermolecular electrostatic interactions, thereby preventing or reducing protein aggregation and insolubilization.

[0271] Ionic species vary significantly in their effects on proteins. Several categorical rankings of ions and their effects on proteins have been developed that can be used in formulating pharmaceutical compositions according to the present invention. One example is the Hofmeister series, which ranks ionic solutes and polar nonionic solutes by their effect on the conformational stability of proteins in solution. Stabilizing solutes are termed "kosmotropics." Destabilizing solutes are termed "chaotropics." Kosmotropes are commonly used at high concentrations to precipitate proteins from solution ("salting out"). Chaotropes are commonly used to denature and / or solubilize proteins ("salting in"). The relative effectiveness of an ion for "salting in" and "salting out" defines its position in the Hofmeister series.

[0272] Free amino acids can be used in formulations or compositions containing the constructs of the present invention according to various embodiments of the present invention as bulking agents, stabilizers, antioxidants, and for other standard uses. Certain amino acids can be used to stabilize proteins in the formulation, while others are useful during lyophilization to ensure the correct cake structure and properties of the active ingredient. Some amino acids can be useful in inhibiting protein aggregation in both liquid and lyophilized formulations, while others are useful as antioxidants.

[0273] Polyols are kosmotropic and are useful as stabilizers in both liquid and lyophilized formulations to protect proteins from physical and chemical degradation processes. Polyols also adjust the tonicity of the formulation and protect against freeze-thaw stress during transportation or for bulk preparation during manufacturing. Polyols can also function as cryoprotectants in the context of the present invention.

[0274] Certain embodiments of formulations or compositions containing the constructs of the present invention can include surfactants. Proteins are prone to surface adsorption, denaturation, and consequent aggregation at air-liquid, solid-liquid, and liquid-liquid interfaces. These adverse interactions generally scale inversely with protein concentration and are typically exacerbated by physical agitation, such as that encountered during product shipping and handling. Surfactants are routinely used to prevent, minimize, or reduce surface adsorption. Surfactants are also commonly used to control the conformational stability of proteins. In this regard, the use of surfactants is protein-specific, as one particular surfactant will typically stabilize some proteins and destabilize others.

[0275] Certain embodiments of formulations or compositions containing the constructs of the present invention may include one or more antioxidants. Harmful oxidation of proteins in pharmaceutical formulations can be prevented to some extent by maintaining appropriate levels of ambient oxygen and temperature and avoiding exposure to light. Antioxidant excipients can also be used to prevent oxidative degradation of proteins. It is envisioned that antioxidants for use in therapeutic protein formulations according to the present invention are water-soluble and capable of maintaining their activity throughout the shelf life of the product (composition containing the construct). Antioxidants can damage proteins, and therefore should also be selected in a manner that eliminates or sufficiently reduces the possibility of the antioxidant damaging the construct or other proteins in the formulation.

[0276] Certain embodiments of formulations or compositions containing the constructs of the present invention can include one or more preservatives. Preservatives are necessary, for example, when developing multi-dose parenteral formulations that involve multiple withdrawals from the same container. Their primary function is to inhibit microbial growth and ensure product sterility over the shelf life or usage period of the formulation. While preservatives have a long history of use with small molecule parenteral agents, developing protein formulations containing preservatives can be challenging. Preservatives frequently have a destabilizing effect on proteins (aggregation), which is a major factor limiting their use in multi-dose protein formulations. To date, most protein drugs have been formulated for single-use only. However, the possibility of multi-dose formulations offers added benefits of patient convenience and increased marketability. Human growth hormone (hGH) is a prime example, where the development of a preserved formulation led to the commercialization of a more convenient multi-use injection pen. Several aspects must be considered during the formulation and development of preserved dosage forms. The effective preservative concentration in the drug product must be optimized, which requires testing a given preservative in the dosage form for a concentration range that confers antimicrobial efficacy without compromising protein stability.

[0277] As expected, developing a liquid formulation containing a preservative is more challenging than a lyophilized formulation. Lyophilized products can be lyophilized without the preservative and reconstituted with a preservative-containing diluent at the time of use. This reduces the time the preservative is in contact with the construct, significantly minimizing the associated stability risks. In liquid formulations, the preservative's effectiveness and stability should be maintained throughout the product's shelf life. It is important to note that the effectiveness of the preservative must be demonstrated in the final formulation containing the active drug and all excipient components. After the pharmaceutical composition is formulated, it can be stored in a sterile vial as a solution, suspension, gel, emulsion, solid, crystalline, or dehydrated or lyophilized powder. Such formulations can be stored either in a ready-to-use form or in a form that is reconstituted (e.g., lyophilized) prior to administration.

[0278] The biological activity of the pharmaceutical compositions defined herein can be determined by in vitro cytotoxicity assays, for example, as described in the Examples below, in WO 99 / 54440, or by Schlereth et al. (Cancer Immunol. Immunother. 20 (2005), 1-12). As used herein, "efficacy" or "in vivo efficacy" refers to the response to therapy with a pharmaceutical composition of the formulation of the present invention, for example, using standardized NCI response criteria. The success of therapy using a pharmaceutical composition of the present invention, or in vivo efficacy, refers to the effectiveness of the composition for its intended purpose, i.e., its ability to cause its desired effect, i.e., the depletion of pathological cells, e.g., tumor cells. In vivo efficacy can be monitored by standard methods established for each disease entity, including, but not limited to, white blood cell count, differentiation, fluorescent cell separation, and bone marrow aspiration. Additionally, various disease-specific clinical chemistry parameters and other established standard methods can be used. Additionally, computer-assisted tomography, X-ray, nuclear magnetic resonance tomography, positron emission tomography scanning, lymph node biopsy / histological methods and other established standard methods can be used.

[0279] Another major challenge in the development of drugs such as the pharmaceutical compositions of the present invention is the predictable regulation of pharmacokinetic properties.To this end, the pharmacokinetic profile of a drug candidate can be established, i.e., the profile of pharmacokinetic parameters that affect the ability of a specific drug to treat a given condition.The pharmacokinetic parameters of a drug that affect the ability of a drug to treat a specific disease include, but are not limited to, half-life, volume of distribution, first-pass metabolism in the liver, and the degree of serum binding.The efficacy of a given drug can be affected by each of the above parameters.

[0280] "Half-life" refers to the time required for an amount to decrease to half of its initial value. In medical science, it refers to the half-life of a substance or drug in the human body. In a medical context, half-life can refer to the time it takes for a substance / drug to lose half of its activity, e.g., pharmacological, physiological, or radiological activity. Half-life can also refer to the time it takes for the concentration of a drug or substance (e.g., a construct of the present invention) in plasma / serum to reach half of its steady-state value ("serum half-life"). Typically, the elimination or removal of an administered substance / drug refers to its cleansing from the body through biological processes such as metabolism and excretion, including the function of the kidneys and liver. "First-pass metabolism" is a drug metabolic phenomenon in which a drug's concentration decreases before it reaches the circulation. This is the proportion of drug lost during the absorption process. Therefore, "hepatic first-pass metabolism" refers to the tendency of a drug to be metabolized upon first contact with the liver, i.e., during its first passage through the liver. "Volume of distribution" (VD) refers to the extent to which a drug is distributed to body tissues rather than to the plasma; a higher VD indicates a greater amount of tissue distribution. Drug retention can occur across various compartments of the body, including intracellular and extracellular spaces, tissues and organs, etc. "Extent of serum binding" refers to the tendency of a drug to interact with and bind to serum proteins such as albumin, leading to a reduction or elimination of the drug's biological activity.

[0281] Pharmacokinetic parameters also include the bioavailability, lag time (T lag), Tmax, absorption rate, and / or Cmax of a given administered amount of drug. "Bioavailability" refers to the proportion of an administered dose of a drug / substance that reaches the systemic circulation (blood compartment). When a pharmaceutical is administered intravenously, its bioavailability is considered to be 100%. However, when a pharmaceutical is administered via other routes (such as orally), its bioavailability is generally reduced. "Lag time" refers to the time delay between the administration of a drug and its detection and measurability in the blood or plasma. Cmax is the maximum plasma concentration that a drug achieves after its administration (and before the administration of a second dose). Tmax is the time to reach Cmax. The time to reach the blood or tissue concentration of a drug required for its biological effect is influenced by all parameters. Pharmacokinetic parameters of constructs exhibiting species specificity that can be determined in preclinical animal studies in primates other than chimpanzees as outlined above, see, for example, Schlereth et al. (supra).

[0282] One embodiment provides a construct of the invention (or a construct produced by a process of the invention) for use as a medicament, in particular for use in the prevention, treatment or amelioration (preferably treatment) of a disease, preferably a neoplastic disease, more preferably a neoplasm, cancer or tumor. Another embodiment provides the use of a construct of the invention (or a construct produced by a process of the invention) in the manufacture of a medicament for the prevention, treatment or amelioration of a disease, preferably a neoplastic disease, more preferably a neoplasm, cancer or tumor. It is also envisaged to provide a method for the prevention, treatment or amelioration of a disease, preferably a neoplastic disease, more preferably a neoplasm, cancer or tumor, comprising the step of administering a construct of the invention (or a construct produced according to a process of the invention) to a subject in need thereof. The terms "subject in need," "patient," or "in need of treatment" include those already affected as well as those in whom the disease is to be prevented. The term also includes human and other mammalian subjects receiving either prophylactic or therapeutic treatment.

[0283] The polypeptides / polypeptide constructs of the present invention and the formulations / pharmaceutical compositions described herein are useful in the treatment, amelioration, and / or prevention of the medical conditions described herein in patients in need thereof. The term "treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Treatment includes the application or administration of a polypeptide / polypeptide construct / pharmaceutical composition to the body, isolated tissues, or cells from a patient with or a subject in need of a disease / disorder described herein, a symptom of such disease / disorder, or a predisposition to such disease / disorder, with the intent to cure, heal, alleviate, relieve, alter, correct, ameliorate, improve, or affect the disease, the symptom of the disease, or the predisposition to the disease. The term "amelioration," as used herein, refers to any improvement in the patient's condition upon administration of a polypeptide construct according to the present invention to such patient or subject in need thereof. Such improvement may be a delay or halt in the progression of the patient's disease and / or a decrease in the severity of the disease symptoms, an increase in the frequency or duration of disease symptom-free periods, or prevention of functional impairment or disability due to the disease. The term "prevention" as used herein means the avoidance of the occurrence or recurrence of a disease identified herein by administration of a construct according to the present invention to a subject in need thereof.

[0284] The term "disease" refers to any condition that would benefit from treatment with the constructs or pharmaceutical compositions described herein. This includes chronic and acute disorders or diseases, including pathological conditions that predispose a mammal to the disease in question. The disease is preferably a neoplastic disease, more preferably a neoplasm, cancer, or tumor. The disease, neoplasm, cancer, or tumor is preferably positive for a tumor antigen, preferably such as those defined herein above, i.e., is characterized by expression or overexpression of a tumor antigen, preferably such as those defined herein above. Overexpression of a tumor antigen means that there is an increase of at least 10%, particularly at least 25%, at least 50%, at least 100%, at least 250%, at least 500%, at least 750%, at least 1000%, or more. Expression is preferably found only in diseased tissue, while expression in corresponding healthy tissue is undetectable or significantly undetectable. According to the present invention, diseases associated with cells expressing a tumor antigen, preferably such as those defined herein above, include cancer diseases. Furthermore, according to the present invention, the cancer disease is preferably one in which the cancer cells express a tumor antigen. According to the present invention, the disease, preferably a neoplastic disease, more preferably a neoplasm, tumor or cancer, is preferably characterized by the presence of BCMA-positive, CD123-positive, CD19-positive, CD20-positive, CD22-positive, CD33-positive, CD70-positive, CDH19-positive, CDH3-positive, CLL1-positive, CS1-positive, CLDN6-positive, CLDN18.2-positive, DLL3-positive, EGFRvIII-positive, FLT3-positive, MAGEB2-positive, MART1-positive, MSLN-positive, MUC17-positive, PSMA-positive or STEAP1-positive cells.In other words, the neoplastic disease, more preferably neoplasm, tumor or cancer, is preferably associated with the presence of BCMA-positive, CD123-positive, CD19-positive, CD20-positive, CD22-positive, CD33-positive, CD70-positive, CDH19-positive, CDH3-positive, CLL1-positive, CS1-positive, CLDN6-positive, CLDN18.2-positive, DLL3-positive, EGFRvIII-positive, FLT3-positive, MAGEB2-positive, MART1-positive, MSLN-positive, MUC17-positive, PSMA-positive or STEAP1-positive cells, Thus, neoplastic diseases, more preferably neoplasms, tumors or cancers, may be referred to as BCMA-positive, CD123-positive, CD19-positive, CD20-positive, CD22-positive, CD33-positive, CD70-positive, CDH19-positive, CDH3-positive, CLL1-positive, CS1-positive, CLDN6-positive, CLDN18.2-positive, DLL3-positive, EGFRvIII-positive, FLT3-positive, MAGEB2-positive, MART1-positive, MSLN-positive, MUC17-positive, PSMA-positive or STEAP1-positive neoplasms, tumors or cancers. It is understood herein that each of said tumor antigen-positive neoplasms, tumors or cancers may be prevented, treated or ameliorated using a polypeptide or polypeptide construct according to the invention comprising a binding domain for a tumor antigen expressed by cells with which said neoplasm, tumor or cancer is associated.BCMA-positive, CD123-positive, CD19-positive, CD20-positive, CD22-positive, CD33-positive, CD70-positive, CDH19-positive, CDH3-positive, CLL1-positive, CS1-positive, CLDN6-positive, CLDN18.2-positive, DLL3-positive, EGFRvIII-positive, FLT3-positive, MAGEB2-positive, MART1-positive, MSLN-positive, MUC17-positive, PSMA-positive, or STEAP1-positive neoplasms, tumors, or cancers are classified as BCMA-positive (for BCMA-positive neoplasms, tumors, or cancers), CD12 3 (for CD123-positive neoplasms, tumors, or cancers), CD19 (for CD19-positive neoplasms, tumors, or cancers), CD20 (for CD20-positive neoplasms, tumors, or cancers), CD22 (for CD22-positive neoplasms, tumors, or cancers), CD33 (for CD33-positive neoplasms, tumors, or cancers), CD70 (for CD70-positive neoplasms, tumors, or cancers), CDH19 (for CDH19-positive neoplasms, tumors, or cancers), CDH3 (for CDH3-positive neoplasms, tumors, or cancers), CLL1 ( and STEAP1 (for STEAP1-positive neoplasms, tumors or cancers).

[0285] A "neoplasm" is an abnormal growth of tissue that usually, but not always, forms a mass. When it forms a mass, it is also usually called a "tumor." A neoplasm or tumor can be benign, potentially malignant (precancerous), or malignant (cancerous). Malignant neoplasms / tumors are generally called cancers. They usually invade and destroy surrounding tissue and can form metastases, i.e., they spread to other parts, tissues, or organs of the body. A "primary tumor" is a tumor that grows at the anatomical site where tumor development begins and progresses to produce a cancerous mass. Most cancers begin at their primary site but then progress and metastasize, or spread, to other parts of the body (e.g., tissues and organs). These additional tumors are "secondary tumors." Most cancers continue to be named after their primary site even after they have spread to other parts of the body.

[0286] Lymphomas and leukemias are lymphoid neoplasms. For purposes of the present invention, they are also encompassed by the terms "tumor" and "cancer." For purposes of the present invention, the terms "neoplasm," "tumor," and "cancer" may be used interchangeably and include both primary tumors / cancers and secondary tumors / cancers (or "metastases"), as well as mass-forming neoplasms (tumors) and lymphoid neoplasms (such as lymphomas and leukemias), and minimal residual disease (MRD).

[0287] The term "minimal residual disease" (MRD) refers to evidence of the presence of a small number of residual cancer cells remaining in a patient after cancer treatment, for example, when the patient is in remission (no symptoms or signs of disease). Standard tests used to assess or detect cancer are not sensitive enough to detect MRD, so typically, very few residual cancer cells cannot be detected by routine means. Recently, highly sensitive molecular biology tests for MRD have become available, such as flow cytometry, PCR, and next-generation sequencing. These tests can measure minimal levels of cancer cells in tissue samples, sometimes as low as one cancer cell per million normal cells. In the context of the present invention, the terms "prevention," "treatment," or "amelioration" of cancer are also intended to encompass "prevention, treatment, or amelioration of MRD," regardless of whether MRD is detected or not.

[0288] The constructs of the present invention will generally be designed for a specific route and method of administration, a specific dosage and frequency of administration, and a specific treatment of a specific disease, particularly in terms of bioavailability and duration. The materials of the composition are preferably formulated in a concentration that is acceptable to the administration site. Thus, formulations and compositions according to the present invention can be designed for delivery by any suitable route of administration. In the context of the present invention, routes of administration include, but are not limited to, topical, enteral, and parenteral routes.

[0289] If the pharmaceutical composition is lyophilized, the lyophilized material is first reconstituted with an appropriate liquid before administration. The lyophilized material can be reconstituted, for example, in bacteriostatic water for injection (BWFI), saline, phosphate-buffered saline (PBS), or the same formulation in which the protein was present before lyophilization. The pharmaceutical compositions and constructs of the present invention are particularly useful for parenteral administration, such as intravenous delivery, for example, injection or infusion. The pharmaceutical composition can be administered using a medical device. Examples of medical devices for administering pharmaceutical compositions are described in U.S. Patent Nos. 4,475,196; 4,439,196; 4,447,224; 4,447,233; 4,486,194; 4,487,603; 4,596,556; 4,790,824; 4,941,880; 5,064,413; 5,312,335; 5,312,335; 5,383,851; and 5,399,163.

[0290] The compositions of the present invention can be administered to a subject at an appropriate dose, which can be determined, for example, by a dose-escalation study. As noted above, constructs of the present invention exhibiting cross-species specificity as described herein can also be advantageously used in preclinical studies in non-chimpanzee primates. The administration schedule will be determined by the attending physician based on clinical factors. As is well known in the medical field, the dosage for any one patient depends on many factors, including the patient's size, body surface area, age, the specific compound administered, sex, time and route of administration, general health, and other drugs administered concomitantly.

[0291] An "effective amount" is the amount of a therapeutic agent sufficient to achieve or at least partially achieve a desired effect. A "therapeutically effective amount" is an amount sufficient to cure or at least partially arrest a disease and its complications, signs and symptoms in a patient suffering from the disease. The amount or dose effective for this use will depend on the disease to be treated (target disease), the construct being delivered, the condition and purpose of the treatment, the severity of the disease, previous therapy, the patient's medical history and response to the treatment, the route of administration, the size (weight, body surface) and / or condition (age and general health) of the patient, and the general state of the patient's own immune system. Appropriate doses can be adjusted based on the judgment of the attending physician to obtain optimal therapeutic effects.

[0292] A therapeutically effective amount of a construct of the invention preferably results in a reduction in the severity of disease symptoms, an increase in the frequency or duration of disease-free symptom-free periods, or prevention of functional impairment or disability due to the disease. In the treatment of tumor antigen-expressing tumors, a therapeutically effective amount of a construct of the invention comprising a binding domain for the tumor antigen preferably inhibits tumor cell growth by at least about 20%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% compared to untreated patients. The ability of a compound to inhibit tumor growth can also be assessed in animal models that are predictive of efficacy in human tumors.

[0293] In a further embodiment, the present invention provides a kit comprising a construct of the invention, a construct produced by a process of the invention, a polynucleotide of the invention, a vector of the invention, and / or a host cell of the invention. In the context of the present invention, the term "kit" refers to two or more components packaged together in a container, recipient, or otherwise, one of which corresponds to a construct, pharmaceutical composition, polynucleotide, vector, or host cell of the invention. A kit can thus be described as a set of products and / or implements sufficient to achieve a particular purpose that can be sold as a single item.

[0294] It is envisioned that a further component of the kit of the invention is an agent, preferably an antibody or construct, that binds to a protein of the immune checkpoint pathway (such as PD-1 or CTLA-4) or a costimulatory immune checkpoint receptor (such as 4-1BB). Such agents are described in further detail herein above. According to one embodiment, the kit comprises a construct of the invention and an antibody or construct that binds to PD-1. Anti-PD-1 binding proteins useful for this purpose are described in detail, for example, in PCT / US2019 / 013205. In certain embodiments, the kit allows for simultaneous and / or sequential administration of the components.

[0295] The kit may comprise one or more containers (e.g., vials, ampoules, containers, syringes, bottles, bags) of any suitable shape, size, and material (preferably waterproof, e.g., plastic or glass) containing a suitable dosage (see above) of the construct or pharmaceutical composition of the invention for administration. The kit may further comprise means for administering the construct or pharmaceutical composition of the invention, such as instructions (e.g., in the form of a booklet or instruction manual), a syringe, a pump, an injector, or the like, means for reconstituting the construct of the invention, and / or means for diluting the construct of the invention.

[0296] The present invention also provides a kit for single-dose administration unit.The kit of the present invention can comprise a first container that contains dried / lyophilized construct or pharmaceutical composition and a second container that contains aqueous formulation.In a specific embodiment of the present invention, a kit is provided that contains a single-chamber and multi-chamber pre-filled syringe.

[0297] Whenever the term "construct" is used herein, said term refers to the polypeptide / polypeptide construct used according to the invention as indicated or its counterpart.

[0298] As used herein, the singular forms "a," "an," a...

Claims

1. A polypeptide or polypeptide construct comprising a binding domain that binds to an extracellular epitope of the human CD3 epsilon chain, comprising or consisting of a VH region and a VL region connected by a peptide linker, said peptide linker comprising or consisting of a G 4 S linker or a G 4 Q linker or repeats thereof; i) the VH region is EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYY ADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSS contains an array of ii) the VL region is QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL contains an array of iii) the VH and VL region sequences are an I at position 34, an A at position 65, a V at position 81, an A at position 99, an A at position 101, and an S at position 106 in the VH region sequence; an I at position 20, a K at position 40, an S at position 69, and an S at position 102 in the VL region sequence; M at position 45, A at position 65, L at position 81, A at position 101, and T at position 106 in the VH region sequence; I at position 20, K at position 40, S at position 69, and S at position 102 in the VL region sequence; M at position 45, A at position 65, L at position 81, A at position 101, S at position 106, F at position 112 in the VH region sequence, I at position 20, I at position 38, K at position 40, E at position 69, S at position 102, and Y at position 93 in the VL region sequence; I at position 34, E at position 39, A at position 65, N at position 101, A at position 102, T at position 106, F at position 112 in the VH region sequence, K at position 40, and S at position 102 in the VL region sequence; an I at position 34, an E at position 64, an A at position 65, an A at position 99, an A at position 101, and a T at position 106 in the VH region sequence; a K at position 40, an A at position 91, and an S at position 102 in the VL region sequence; an I at position 34, an A at position 65, a V at position 81, an A at position 99, an A at position 101, an E at position 102, an I at position 104, and a T at position 106 in the VH region sequence; an I at position 20, a K at position 40, an S at position 69, and an S at position 102 in the VL region sequence; an E at position 64, an A at position 65, a G at position 68, a V at position 81, an A at position 101, and an S at position 106 in the VH region sequence; an I at position 20, an S at position 69, and an S at position 102 in the VL region sequence; I at position 34, E at position 64, A at position 65, A at position 101, T at position 106, F at position 112 in the VH region sequence, K at position 40, A at position 91, and S at position 102 in the VL region sequence; I at position 34, E at position 64, A at position 65, A at position 101, S at position 106, F at position 112 in the VH region sequence, K at position 40, A at position 91, and S at position 102 in the VL region sequence; I at position 34, E at position 64, A at position 65, V at position 81, A at position 99, A at position 101, S at position 106 in the VH region sequence, I at position 20, K at position 40, S at position 69, S at position 102, and Y at position 93 in the VL region sequence; an I at position 34, an A at position 65, an A at position 101, an S at position 106, and an F at position 112 in the VH domain sequence; a K at position 40, an A at position 91, and an S at position 102 in the VL domain sequence; M at position 45, E at position 64, A at position 65, L at position 81, A at position 101, T at position 106 in the VH region sequence, I at position 20, I at position 38, K at position 40, E at position 69, and S at position 102 in the VL region sequence; S at position 30, M at position 45, A at position 65, G at position 68, V at position 81, A at position 101, T at position 106, F at position 112 in the VH region sequence, K at position 40, S at position 69, and Y at position 93 in the VL region sequence; S at position 30, M at position 45, E at position 64, A at position 65, G at position 68, V at position 81, A at position 101, S at position 106 in the VH region sequence, K at position 40, and S at position 69 in the VL region sequence; E at position 64, A at position 65, G at position 68, V at position 81, N at position 101, E at position 102, I at position 104, T at position 106 in the VH region sequence, I at position 20, S at position 69, and S at position 102 in the VL region sequence; A at position 65, G at position 68, V at position 81, N at position 101, E at position 102, I at position 104, S at position 106 in the VH region sequence, and I at position 20, S at position 69, and S at position 102 in the VL region sequence; an I at position 34, an A at position 65, an A at position 99, an A at position 101, and a T at position 106 in the VH domain sequence, a K at position 40, an A at position 91, and an S at position 102 in the VL domain sequence; and S at position 30, E at position 39, E at position 64, A at position 65, V at position 81, A at position 101, E at position 102, I at position 104, and T at position 106 in the VH region sequence; M at position 20, K at position 40, and E at position 69 in the VL region sequence A polypeptide or polypeptide construct comprising a combination of amino acid substitutions selected from:

2. 2. A polypeptide or polypeptide construct according to claim 1, comprising at least one further binding domain.

3. 3. The polypeptide or polypeptide construct of claim 2, wherein the at least one further binding domain binds to a cell surface antigen.

4. The polypeptide or polypeptide construct of claim 3 , wherein the cell surface antigen is a tumor antigen.

5. 5. The polypeptide or polypeptide construct of claim 4, wherein the tumor antigen is selected from the group consisting of BCMA, CD123, CD19, CD20, CD22, CD33, CD70, CDH19, CDH3, CLL1, CS1, CLDN6, CLDN18.2, DLL3, EGFRvIII, FLT3, MAGEB2, MART1, MSLN, MUC17, PSMA, and STEAP1.

6. A polypeptide or polypeptide construct according to any one of claims 1 to 5, which is a single chain polypeptide that is at least bispecific.

7. A polynucleotide encoding a polypeptide or polypeptide construct according to any one of claims 1 to 6.

8. A vector comprising the polynucleotide of claim 7.

9. A host cell transformed or transfected with the polynucleotide of claim 7 or the vector of claim 8.

10. 10. A process for producing a polypeptide or polypeptide construct according to any one of claims 1 to 6, comprising culturing a host cell according to claim 9 under conditions allowing expression of the polypeptide or polypeptide construct according to any one of claims 1 to 6, and recovering the produced polypeptide or polypeptide construct from the culture.

11. A pharmaceutical composition comprising a polypeptide or polypeptide construct according to any one of claims 1 to 6.

12. A polypeptide or polypeptide construct according to any one of claims 4 to 6 for use in a method for the prevention, treatment or amelioration of a disease selected from tumor diseases.

13. A kit comprising a polypeptide or polypeptide construct according to any one of claims 1 to 6, a polynucleotide according to claim 7, a vector according to claim 8 and / or a host cell according to claim 9.

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