Anti-CD3 antibodies, anti-CD123 antibodies, and bispecific antibodies that bind specifically to CD3 and / or CD123

Abstract

The present invention relates to an antibody-like binding protein that binds specifically to CD3 and at least one other antigen, for example, CD123. The present invention also relates to an antibody-like binding protein that binds specifically to CD123 and at least one other antigen. The invention further relates to anti-CD3 antibodies and anti-CD123 antibodies. The invention further relates to pharmaceutical compositions comprising the antibody-like binding protein, the anti-CD3 antibodies, or the anti-CD123 antibodies of the invention, and their use for the treatment of cancer. The invention also relates to isolated nucleic acids, vectors, and host cells comprising a sequence encoding said antibody-like binding protein, anti-CD3 antibody, or anti-CD123 antibody, and to the use of said anti-CD123 antibody as a diagnostic tool.

Description

[0001] The present invention concerns antibody-like binding protein specifically binding to CD3 and binding specifically to at least one further antigen, for example CD123. The invention further concerns anti-CD3 antibodies and anti-CD123 antibodies. The present invention also concerns antibody-like binding protein specifically binding to CD123 and binding specifically to at least one further antigen. The invention also relates to pharmaceutical compositions comprising the antibody-like binding protein, anti-CD3 antibodies or anti-CD123 antibodies of the invention, and their use to treat cancer. The invention further relates to isolated nucleic acids, vectors and host cells comprising a sequence encoding said antibody-like binding protein, anti-CD3 or anti-CD123 antibody and the use of said anti-CD123 antibody as a diagnostic tool.

[0002] The first generation of bispecific antibodies was developed over 20 years ago. Since then a number of clinical studies have tested bispecific antibodies engineered to target cancer cell surface antigens. This group of anti-cancer fusion proteins contains two or more functional domains that localize immunological effector cells in the proximity of targeted cancer cells to achieve anti-cancer activity.

[0003] As bispecific antibody technology developed, a different group of fusion proteins named bispecific T-cell engagers (BiTE) were generated by connecting two antibody single chain variable regions (scFv) only (no Fc amino acid segments were included) with a flexible linker, one scFv binds targeted cells and the other binds CD3 on T cell surface. One BiTE, blinatumomab, with CD19xCD3 bi-specific binding activities showed promising results in Phase II clinical trials for patients with minimal residual disease in B-lineage acute lymphoblastic.

[0004] CD123 (the interleukin-3 receptor alpha chain IL-3Rα) is a tumor antigen over-expressed in a variety of hematological neoplasms. The majority of AML blasts express surface CD123 and this expression does not vary by subtype of AML. Higher expression of CD123 on AML at diagnosis has been reported to be associated with poorer prognosis. It has been reported that CD123 is expressed on leukemic stem cells (LSCs). There is growing evidence to suggest that AML arises from these leukemic stem cells (LSCs) which have been shown to be quiescent and relatively resistant to DNA damaging chemotherapy.

[0005] The increased expression of CD123 on LSCs compared with hematopoietic stem cells (HSCs) presents thus an opportunity for therapeutic targeting of AML-LSCs.

[0006] The monoclonal antibody (MAb) 7G3, raised against CD123, has previously been shown to inhibit IL-3 mediated proliferation and activation of both leukemic cell lines and primary cells (US Patent No. 6,177,078). However, it has remained unclear whether targeting CD123 can functionally impair AML-LSCs.

[0007] The use of CD123xCD3 antibody-like binding protein leads to tumor cell killing, as herein shown by the inventors.

[0008] The idea of producing a bispecific antibody-like binding protein with CD123xCD3 bi-specific binding activities has already been proposed and described in the international patent application WO2013 / 173820.

[0009] Furthermore, a CD123 x CD3 Dual Affinity Re-Targeting (DART) Bi-Specific Antibody Based Molecule from MacroGenics entered phase I clinical trials in 2014.

[0010] However, as shown by the inventors, the CD123xCD3 Dual Affinity Re-Targeting (DART) Bi-Specific Antibody Based Molecule from MacroGenics, for example, has an activation of 82% of CD4+ expressing T-cells and 83% of CD8+ expressing T-cells in the absence of target cells. The inappropriate activation of T-cells may lead to severe side effects, such as the cytokine release syndrome. The cytokine release syndrome refers to the release of cytokines by the activated T cells producing a type of systemic inflammatory response similar to that found in severe infections and characterized by hypotension, pyrexia and rigors. Deaths due to cytokine release syndrome have been reported for example for OKT3.

[0011] Therefore, in spite of these advancements in bispecific antibody technology, there remains a need for additional cancer therapeutics, particularly those that efficiently target and kill cancer cells, either directly or indirectly.

[0012] The inventors have succeeded in generating, screening and selecting specific rat anti-CD3 antibodies displaying high affinity for both human and Macaca fascicularis CD3 protein.

[0013] The inventors developed antibody-like binding proteins having biological and immunological specificity to the antigen CD3 and at least one further target antigen. In one example, to demonstrate the use of these anti-CD3 antibodies in the generation of bispecific antibody-like binding proteins the inventors generated anti-CD3 / anti-CD123 antibody-like binding proteins and demonstrated the therapeutic use thereof. Those bispecific anti-CD3 / anti-CD123 antibody-like binding proteins have a low T-cell activation, as it has been observed for the anti-CD3 antibody alone. However, once the CD123 expressing target cells, such as THP-1 cells, are present the bispecific anti-CD3 / anti-CD123 antibody-like binding protein shows a high activation of T-cells. Accordingly, the anti-CD3 antibody of the invention defined above is particularly useful for the preparation of antibody-like binding proteins of the invention.Definitions

[0014] Throughout the instant application, the term "and / or" is a grammatical conjunction that is to be interpreted as encompassing that one or more of the cases it connects may occur. For example, the wording "such native sequence proteins can be prepared using standard recombinant and / or synthetic methods" indicates that native sequence proteins can be prepared using standard recombinant and synthetic methods or native sequence proteins can be prepared using standard recombinant methods or native sequence proteins can be prepared using synthetic methods.

[0015] Furthermore, throughout the instant application, the term "comprising" is to be interpreted as encompassing all specifically mentioned features as well optional, additional, unspecified ones. As used herein, the use of the term "comprising" also discloses the embodiment wherein no features other than the specifically mentioned features are present (i.e. "consisting of"). Furthermore the indefinite article "a" or "an" does not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage.

[0016] The term "gene" means a DNA sequence that codes for, or corresponds to, a particular sequence of amino acids which comprises all or part of one or more proteins or enzymes, and may or may not include regulatory DNA sequences, such as promoter sequences, which determine for example the conditions under which the gene is expressed. Some genes, which are not structural genes, may be transcribed from DNA to RNA, but are not translated into an amino acid sequence. Other genes may function as regulators of structural genes or as regulators of DNA transcription. In particular, the term gene may be intended for the genomic sequence encoding a protein, i.e. a sequence comprising regulator, promoter, intron and exon sequences.

[0017] A sequence "at least 85% identical to a reference sequence" is a sequence having, on its entire length, 85%, or more, in particular 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the entire length of the reference sequence.

[0018] In the context of the present application, the "percentage of identity" is calculated using a global pairwise alignment (i.e. the two sequences are compared over their entire length). Methods for comparing the identity of two or more sequences are well known in the art. The « needle » program, which uses the Needleman-Wunsch global alignment algorithm (Needleman and Wunsch, 1970 J. Mol. Biol. 48:443-453) to find the optimum alignment (including gaps) of two sequences when considering their entire length, may for example be used. The needle program is for example available on the ebi.ac.uk World Wide Web site. The percentage of identity between two polypeptides, in accordance with the invention, is calculated using the EMBOSS: needle (global) program with a "Gap Open" parameter equal to 10.0, a "Gap Extend" parameter equal to 0.5, and a Blosum62 matrix.

[0019] Proteins consisting of an amino acid sequence "at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical" to a reference sequence may comprise mutations such as deletions, insertions and / or substitutions compared to the reference sequence. In case of substitutions, the protein consisting of an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a reference sequence may correspond to a homologous sequence derived from another species than the reference sequence.

[0020] "Amino acid substitutions" may be conservative or non-conservative. Preferably, substitutions are conservative substitutions, in which one amino acid is substituted for another amino acid with similar structural and / or chemical properties. The substitution preferably corresponds to a conservative substitution as indicated in the table below. Conservative substitutions Type of Amino Acid Ala, Val, Leu, Ile, Met, Pro, Phe, TrpAmino acids with aliphatic hydrophobic side chainsSer, Tyr, Asn, Gln, CysAmino acids with uncharged but polar side chainsAsp, GluAmino acids with acidic side chainsLys, Arg, HisAmino acids with basic side chainsGlyNeutral side chain

[0021] An "antibody" also called "immunoalobulin" may be a natural or conventional antibody in which two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chain, lambda (l) and kappa (k). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each chain contains distinct sequence domains. The light chain includes two domains or regions, a variable domain (VL) and a constant domain (CL). The heavy chain includes four domains, a variable domain (VH) and three constant domains (CH1, CH2 and CH3, collectively referred to as CH). The variable regions of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen. The constant region domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, trans-placental mobility, complement binding, and binding to Fc receptors (FcR). The Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain. The specificity of the antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant. Antibody combining sites are made up of residues that are primarily from the hypervariable or complementarity determining regions (CDRs). Occasionally, residues from nonhypervariable or framework regions (FR) influence the overall domain structure and hence the combining site. Complementarity Determining Regions or CDRs refer to amino acid sequences that together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site. The light and heavy chains of an immunoglobulin each have three CDRs, designated CDR1-L, CDR2-L, CDR3-L and CDR1-H, CDR2-H, CDR3-H, respectively. A conventional antibody antigen-binding site, therefore, includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region.

[0022] In context of the invention, the antibody or immunoglobulin is an IgM, IgD, IgG, IgA and IgE.

[0023] "Framework Regions" (FRs) refer to amino acid sequences interposed between CDRs, i.e. to those portions of immunoglobulin light and heavy chain variable regions that are relatively conserved among different immunoglobulins in a single species. The light and heavy chains of an immunoglobulin each have four FRs, designated FR1-L, FR2-L, FR3-L, FR4-L, and FR1-H, FR2-H, FR3-H, FR4-H, respectively. Accordingly, the light chain variable domain may thus be designated as (FR1-L)-(CDR1-L)-(FR2-L)-(CDR2-L)-(FR3-L)-(CDR3-L)-(FR4-L) and the heavy chain variable domain may thus be designated as (FR1-H)-(CDR1-H)-(FR2-H)-(CDR2-H)-(FR3-H)-(CDR3-H)-(FR4-H).

[0024] Knowing the amino acid sequence of the CDRs one skilled in the art can easily determine the framework regions FR1-L, FR2-L, FR3-L, FR4-L and / or FR1-H, FR2-H, FR3-H, FR4-H.

[0025] As used herein, a "human framework region" is a framework region that is substantially identical (about 85%, or more, in particular 90%, 95%, 97%, 99% or 100%) to the framework region of a naturally occurring human antibody.

[0026] In the context of the invention, CDR / FR definition in an immunoglobulin light or heavy chain is to be determined based on IMGT definition (Lefranc et al. Dev. Comp. Immunol., 2003, 27(1):55-77; www.imgt.org).

[0027] As used herein, the term "antibody" denotes conventional antibodies and fragments thereof, as well as single domain antibodies and fragments thereof, in particular variable heavy chain of single domain antibodies, and chimeric, humanized, bispecific or multispecific antibodies.

[0028] As used herein, antibody or immunoglobulin also includes "single domain antibodies" which have been more recently described and which are antibodies whose complementary determining regions are part of a single domain polypeptide. Examples of single domain antibodies include heavy chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from conventional four-chain antibodies, engineered single domain antibodies. Single domain antibodies may be derived from any species including, but not limited to mouse, human, camel, llama, goat, rabbit, bovine. Single domain antibodies may be naturally occurring single domain antibodies known as heavy chain antibody devoid of light chains. In particular, Camelidae species, for example camel, dromedary, llama, alpaca and guanaco, produce heavy chain antibodies naturally devoid of light chain. Camelid heavy chain antibodies also lack the CH1 domain.

[0029] The variable heavy chain of these single domain antibodies devoid of light chains are known in the art as "VHH" or "nanobody". Similar to conventional VH domains, VHHs contain four FRs and three CDRs. Nanobodies have advantages over conventional antibodies: they are about ten times smaller than IgG molecules, and as a consequence properly folded functional nanobodies can be produced by in vitro expression while achieving high yield. Furthermore, nanobodies are very stable, and resistant to the action of proteases. The properties and production of nanobodies have been reviewed by Harmsen and De Haard HJ (Appl. Microbiol. Biotechnol. 2007 Nov;77(1):13-22).

[0030] The term "monoclonal antibody" or "mAb" as used herein refers to an antibody molecule of a single amino acid composition that is directed against a specific antigen, and is not to be construed as requiring production of the antibody by any particular method. A monoclonal antibody may be produced by a single clone of B cells or hybridoma, but may also be recombinant, i.e. produced by protein engineering.

[0031] The term "chimeric antibody" refers to an engineered antibody which in its broadest sense contains one or more regions from one antibody and one or more regions from on or more other antibody(ies). In particular a chimeric antibody comprises a VH domain and a VL domain of an antibody derived from a non-human animal, in association with a CH domain and a CL domain of another antibody, in particular a human antibody. As the non-human animal, any animal such as mouse, rat, hamster, rabbit or the like can be used. A chimeric antibody may also denote a multispecific antibody having specificity for at least two different antigens.

[0032] The term "humanized antibody" refers to an antibody which is wholly or partially of non-human origin and which has been modified to replace certain amino acids, in particular in the framework regions of the heavy and light chains, in order to avoid or minimize an immune response in humans. The constant domains of a humanized antibody are most of the time human CH and CL domains.

[0033] Numerous methods for humanization of an antibody sequence are known in the art; see e.g. the review by Almagro & Fransson (2008) Front Biosci. 13: 1619-1633. One commonly used method is CDR grafting, or antibody reshaping, which involves grafting of the CDR sequences of a donor antibody, generally a mouse antibody, into the framework scaffold of a human antibody of different specificity. Since CDR grafting may reduce the binding specificity and affinity, and thus the biological activity, of a CDR grafted non-human antibody, back mutations may be introduced at selected positions of the CDR grafted antibody in order to retain the binding specificity and affinity of the parent antibody. Identification of positions for possible back mutations can be performed using information available in the literature and in antibody databases. Amino acid residues that are candidates for back mutations are typically those that are located at the surface of an antibody molecule, while residues that are buried or that have a low degree of surface exposure will not normally be altered. An alternative humanization technique to CDR grafting and back mutation is resurfacing, in which non-surface exposed residues of non-human origin are retained, while surface residues are altered to human residues. Another alternative technique is known as "guided selection" (Jespers et al. (1994) Biotechnology 12, 899) and can be used to derive from for example a murine or rat antibody a fully human antibody conserving the epitope and binding characteristics of the parental antibody. A further method of humanization is the so-called 4D humanization. The 4D humanization protocol is described in the patent application US20110027266 A1 (WO2009032661A1 ) and is exemplified in the following applying the 4D humanization to humanize the rat antibody variable light (VL) and heavy (VH) domains. In one example, a rat antibody homology model was done with typically MOE software (v. 2011.10- Chemical Computing Group, Quebec, Canada) using PDB structures (Berman et al., Nucleic Acids Research, 2000, 28:235-242) as templates and was subsequently energy minimized using the standard procedures implemented in MOE. A molecular dynamics (MD) simulation was then performed on the minimized 3D homology model (done with MOE software) of rat antibody and compared to the, for example, 49 human models derived from the seven representative light chains (vk1, vk2, vk3, vk4, vlambda1, vlambda2, vlambda3) and the seven representative heavy chains (vh1a, vh1b, vh2, vh3, vh4, vh5, vh6) designed by LGCR / SDI and available within MOE. For instance, one model of chains couple (Vkx-Vhx) with the best both hydrophobic, electrostatic components and sequence identity outside CDR has been selected for the "4D humanization". For the pairwise association between the rat antibody variable domain and the selected model, the sequences were aligned based typically on the optimal 3D superposition of the alpha carbons of the corresponding homology models. The unwanted motifs were then considered and mutated. Finally, the resulting humanized sequences were blasted for sequence similarity against, for instance, the IEDB database (http: / / www.immuneepitope.org; version 2012 / 01 / 30 accessible locally) to ensure that none of the sequences contain any known B- or T-cell epitope listed in.

[0034] For chimeric antibodies, humanization typically involves modification of the framework regions of the variable region sequences.

[0035] Amino acid residues that are part of a CDR will typically not be altered in connection with humanization, although in certain cases it may be desirable to alter individual CDR amino acid residues, for example to remove a glycosylation site, a deamidation site or an undesired cysteine residue. N-linked glycosylation occurs by attachment of an oligosaccharide chain to an asparagine residue in the tripeptide sequence Asn-X-Ser or Asn-X-Thr, where X may be any amino acid except Pro. Removal of an N-glycosylation site may be achieved by mutating either the Asn or the Ser / Thr residue to a different residue, in particular by way of conservative substitution. Deamidation of asparagine and glutamine residues can occur depending on factors such as pH and surface exposure. Asparagine residues are particularly susceptible to deamidation, primarily when present in the sequence Asn-Gly, and to a lesser extent in other dipeptide sequences such as Asn-Ala. When such a deamidation site, in particular Asn-Gly, is present in a CDR sequence, it may therefore be desirable to remove the site, typically by conservative substitution to remove one of the implicated residues. Substitution in a CDR sequence to remove one of the implicated residues is also intended to be encompassed by the present invention.

[0036] "Fragments" of (conventional) antibodies comprise a portion of an intact antibody, in particular the antigen binding region or variable region of the intact antibody. Examples of antibody fragments include Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, diabodies, bispecific and multispecific antibodies formed from antibody fragments. A fragment of a conventional antibody may also be a single domain antibody, such as a heavy chain antibody or VHH.

[0037] The term "Fab" denotes an antibody fragment having a molecular weight of about 50,000 and antigen binding activity, in which about a half of the N-terminal side of H chain and the entire L chain, among fragments obtained by treating IgG with a protease, papaine, are bound together through a disulfide bond.

[0038] The term "F(ab')2" refers to an antibody fragment having a molecular weight of about 100,000 and antigen binding activity, which is slightly larger than the Fab bound via a disulfide bond of the hinge region, among fragments obtained by treating IgG with a protease, pepsin.

[0039] The term "Fab' " refers to an antibody fragment having a molecular weight of about 50,000 and antigen binding activity, which is obtained by cutting a disulfide bond of the hinge region of the F(ab')2.

[0040] A single chain Fv ("scFv") polypeptide is a covalently linked VH::VL heterodimer which is usually expressed from a gene fusion including VH and VL encoding genes linked by a peptide-encoding linker. The human scFv fragment of the invention includes CDRs that are held in appropriate conformation, in particular by using gene recombination techniques. Divalent and multivalent antibody fragments can form either spontaneously by association of monovalent scFvs, or can be generated by coupling monovalent scFvs by a peptide linker, such as divalent sc(Fv) 2 . "dsFv" is a VH::VL heterodimer stabilised by a disulphide bond. "(dsFv)2" denotes two dsFv coupled by a peptide linker.

[0041] The term "bispecific antibody" or "BsAb" typically denotes an antibody, which combines the antigen-binding sites of two antibodies within a single molecule. Thus, BsAbs are able to bind two different antigens simultaneously. Genetic engineering has been used with increasing frequency to design, modify, and produce antibodies or antibody derivatives with a desired set of binding properties and effector functions as described for instance in EP 2 050 764 A1.

[0042] The term "multispecific antibody" denotes an antibody that combines the antigen-binding sites of two or more antibodies within a single molecule.

[0043] The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.

[0044] The term "hybridoma" denotes a cell, which is obtained by subjecting a B cell prepared by immunizing a non-human mammal with an antigen to cell fusion with a myeloma cell derived from a mouse or the like which produces a desired monoclonal antibody having an antigen specificity.

[0045] By "purified" and "isolated" it is meant, when referring to a polypeptide (i.e. the antibody of the invention) or a nucleotide sequence, that the indicated molecule is present in the substantial absence of other biological macromolecules of the same type. The term "purified" as used herein in particular means at least 75%, 85%, 95%, or 98% by weight, of biological macromolecules of the same type are present. An "isolated" nucleic acid molecule that encodes a particular polypeptide refers to a nucleic acid molecule that is substantially free of other nucleic acid molecules that do not encode the subject polypeptide; however, the molecule may include some additional bases or moieties, which do not deleteriously affect the basic characteristics of the composition.

[0046] The term "antigen" or "target antigen" as used herein refers to a molecule or a portion of a molecule that is capable of being bound by an antibody or an antibody-like binding protein. The term further refers to a molecule or a portion of a molecule that is capable of being used in an animal to produce antibodies that are capable of binding to an epitope of that antigen. A target antigen may have one or more epitopes. With respect to each target antigen recognized by an antibody or by an antibody-like binding protein, the antibody-like binding protein is capable of competing with an intact antibody that recognizes the target antigen.

[0047] "Affinity" is defined, in theory, by the equilibrium association between the whole antibody and the antigen. Affinity may be expressed for example in half-maximal effective concentration (EC 50 ) or the equilibrium dissociation constant (KD).

[0048] "Half maximal effective concentration" also called "EC 50 " refers to the concentration of a drug, antibody or toxicant which induces a response halfway between the baseline and maximum after a specified exposure time. EC 50 and affinity are inversely related, the lower the EC 50 value the higher the affinity of the antibody.

[0049] "K D " is the equilibrium dissociation constant, a ratio of k off / k on , between the antibody and its antigen. K D and affinity are inversely related. The K D value relates to the concentration of antibody and the lower the K D value and the higher the affinity of the antibody. Affinity can be experimentally assessed by a variety of known methods, such as measuring association and dissociation rates with surface Plasmon resonance or measuring the EC 50 in an immunochemical assay (ELISA, FACS). Enzyme-linked immunosorbent assay (ELISA) is a biochemistry assay that uses a solid-phase enzyme immunoassay to detect the presence of a substance, usually an antigen, in a liquid sample or wet sample. Antigens from the sample are attached to a surface. Then, a further specific antibody is applied over the surface so it can bind to the antigen. This antibody is linked to an enzyme, and, in the final step, a substance containing the enzyme's substrate is added. The subsequent reaction produces a detectable signal, most commonly a color change in the substrate. Fluorescence-activated cell sorting (FACS) provides a method for sorting a heterogeneous mixture of biological cells into two or more containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell. In these assays, the EC 50 is the concentration of the antibody which induces a response halfway between the baseline and maximum after some specified exposure time on a defined concentration of antigen by ELISA (enzyme-linked immuno-sorbent assay) or cell expressing the antigen by FACS (Fluorescence Activated Cell Sorting). Surface plasmon resonance is a label free method wherein the binding of a molecule in the soluble phase (the "analyte") is directly measured to a "ligand" molecule immobilized on a sensor surface. In the sensor device the binding of the ligand is monitored by an optical phenomenon termed surface plasmon. In particular, when the "analyte" molecule dissociates from the "ligand" molecule, a decrease in SPR signal (expressed in resonance units, RU) is observed. Association ('on rate', k a ) and Dissociation rates ('off rate', k d ) are obtained from the signal obtained during the association and dissociation and the equilibrium dissociation constant ('binding constant', K D ) can be calculated therefrom. The signal given in resonance units (RU) depends on the size of the ligand present in the analyte, however in case the experimental conditions are the same, i.e. the ligand is the same molecule at the same condition the obtained RU can indicate affinity, wherein the higher the obtained signal in RU the higher the binding.

[0050] A monoclonal antibody binding to antigen 1(Ag1) is "cross-reactive" to antigen 2 (Ag2) when the EC 50 s are in a similar range for both antigens. In the present application, a monoclonal antibody binding to Ag1 is cross-reactive to Ag2 when the ratio of affinity of Ag2 to affinity of Ag1 is equal or less than 10 (in particular 5, 2, 1 or 0.5), affinities being measured with the same method for both antigens.

[0051] A monoclonal antibody binding to Ag1 is "not significantly cross-reactive" to Ag2 when the affinities are very different for the two antigens. Affinity for Ag2 may not be measurable if the binding response is too low. In the present application, a monoclonal antibody binding to Ag1 is not significantly cross-reactive to Ag2, when the binding response of the monoclonal antibody to Ag2 is less than 5% of the binding response of the same monoclonal antibody to Ag1 in the same experimental setting and at the same antibody concentration. In practice, the antibody concentration used can be the EC 50 or the concentration required to reach the saturation plateau obtained with Ag1.

[0052] As used herein "specificity" denotes the capacity of an antibody to discriminate the target peptide sequence to which its binds ("epitope") from closely related, highly homologous, peptide sequences.

[0053] A monoclonal antibody "binds specifically" to Ag1 when it is not significantly cross-reactive to Ag2.

[0054] A "domain" may be any region of a protein, generally defined on the basis of sequence homologies and often related to a specific structural or functional entity.

[0055] A "recombinant" molecule is one that has been prepared, expressed, created, or isolated by recombinant means.

[0056] As used herein, the term "subject" denotes a mammal, such as a rodent, a feline, a canine, and a primate. In particular, a subject according to the invention is a human.Anti CD3 antibodies

[0057] "CD3" denotes an antigen that is expressed on T-cells as part of the multimolecular T-cell receptor complex and that consists of at least three different chains CD3ε, CD3δ and CD3γ. CD3δ and CD3γ have a low sequence identity and / or similarity to human CD3ε (similarity and identity is less than 20%). CD3ε and CDR3δ can form together a complex, herein called "CD3ε / δ-complex". CD3ε also forms a complex with CDR3y, the so-called "CD3ε / γ-complex" Clustering of CD3 on T-cells, e.g., by immobilized anti-CD3-antibodies, leads to T-cell activation similar to the engagement of the T-cell receptor but independent from its clone typical specificity. "CD3ε" comprises three domains, an intracellular domain, a transmembrane domain and an extracellular domain.

[0058] Most prior art anti-CD3-antibodies recognize the CD3ε-chain. One of such prior art anti-CD3-antibodies is OKT3. Prior art has exemplified T cell activation events employing antibody molecules for example by employing the antibody molecule OKT3. The anti-CD3 antibody and variant thereof have been described in the prior art (US 4,361,549 ; US 4,361 ,549 ; US 5,885,573 ; US 5,929,212 ; and WO 98 / 52975 or US 5,955,358). OKT3 has been further used as potent immunosuppressive agent in clinical transplantation to treat allograft rejection (Thistlethwaite 1984, Transplantation 38, 695-701; Woodle 1991, Transplantation 51, 1207-1212; Choi 2001, Eur. J. Immunol. 31(1), 94-106).

[0059] Major drawbacks of this therapy are T cell activation manifested in cytokine release due to cross-linking between T cells and FcγR-bearing cells and the human antimouse antibody (HAMA) response. Several publications have described alterations such as humanization of OKT3 to reduce these side effects: US 5,929,212; US 5,885,573 and others. On the other hand, OKT3 or other anti-CD3-antibodies can be used as immunopotentiating agents to stimulate T cell activation and proliferation (US 6,406,696 Bluestone; US 6,143,297 Bluestone; US 6,113,901 Bluestone; Yannelly 1990, J. Immunol. Meth. 1 , 91-100). Anti-CD3-anti bodies have also been described as agents used in combination with anti-CD28-antibodies to induce T cell proliferation (US 6,352,694). OKT3 has further been used by itself or as a component of a bispecific antibody to target cytotoxic T cells to tumor cells or virus infected cells (Nitta 1990, Lancet 335, 368-376; Sanna 1995, Bio / Technology 13, 1221-1224; WO 99 / 54440).

[0060] Approaches up to now using antibodies as agents for recruiting T-cells have been hampered by several findings. First, natural or engineered antibodies having a high binding affinity to T-cells often do not activate the T-cells to which they are bound. Second, natural or engineered antibodies having a low binding affinity to T-cells are also often ineffective with respect to their ability to trigger T-cell mediated cell lysis.

[0061] A reference sequence of full-length human CD3ε protein, including the signal peptide, is available from the Uniprot database under accession number P07766 and herein enclosed under SEQ ID NO: 1 (as available on December 12, 2014).

[0062] A reference sequence of full-length Macaca fascicularis CD3ε protein, including the signal peptide, is available from the Uniprot database under accession number Q95LI5 and herein enclosed under SEQ ID NO: 2 (as available on December 12, 2014).

[0063] A sequence of mature human CD3ε His-tagged Fc-fusion proteins, cloned by the inventors from genomic DNA, is disclosed under SEQ ID NO: 3. Said mature human CD3ε His-tagged Fc-fusion protein comprises amino acids 23 to 126 of the full-length human CD3ε protein and thus comprises the extracellular domain of human CD3ε.

[0064] A sequence of mature Macaca fascicularis CD3ε Fc-fusion protein, cloned by the inventors from genomic DNA, is disclosed under SEQ ID NO: 4. Said mature Macaca fascicularis CD3ε Fc-fusion protein comprises amino acids 23 to 117 of the full-length Macaca fascicularis CD3ε protein and thus comprises the extracellular domain of human or Macaca fascicularis CD3ε, containing one Alanine to Valine exchange at the amino acid position 35 in comparison to amino acid position 57 of the wild-type sequence.

[0065] Domain organization of human and Macaca fascicularis CD3ε is as it follows (based on Uniprot P07766 sequence (human) and Uniprot Q95LI5 sequence (Macaca fascicularis)): CD3ε domainsPositions on SEQ ID NO :1 (human)Positions on SEQ ID NO : 2 (Macaca fascicularis)Extracellular23 - 12622 - 117Transmembrane domain127 - 152118 - 138Cytoplasmic153 - 207139 - 198

[0066] Accordingly, the extracellular domain of human CD3ε consists of amino acids at positions 23 - 126 of SEQ ID NO: 1 and the extracellular domain of Macaca fascicularis CD3ε consists of amino acids at positions 22 - 117 of SEQ ID NO: 2.

[0067] The inventors have succeeded in generating, screening and selecting specific mouse and rat anti-CD3 antibodies. These anti-CD3 antibodies display high affinity for both human and Macaca fascicularis CD3 protein, and have however a low T-cell activation in the absence of target cells.

[0068] The inventors have determined the sequence of variable heavy and light chains of such monoclonal antibodies, the so-called anti-CD3 antibodies "20G6-F3", "4B4-D7", "4E7-C9", "18F5-H10", "12D2-E5", "11D7-C3", "11H3-E5", "13H2-C2", "13C1-F6", "18H11-F10", "1E6-C9", "10F4-C10", "10E6-G6", "18G9-H11", "11F3-B9", "12G3-E8", "5B1-G2", "16F8-A7", "11F9-F8", "3G5-E10", "9D7-F3", "8C2-F7", "20E5-F10", "20B5-F10", "6C9-C9", "3E8-G1", "3H6-D2" and "8H2".

[0069] The so-called "20G6-F3" anti-CD3 antibody comprises: a heavy chain variable domain of consisting of sequence EVQLVETGGSLVQPGKSLKLTCATSGFTFTKAW MHWVRQSPEKQLEWVAQIKDKSNS YAT YYAESVKGRFTISRDDSKSTIYLQMNSLKEEDTAIYYCRGVYYALSPFDY WGQGVM VTVSS (SEQ ID NO: 5, with CDRs shown in bold characters and underlined) comprising CDR1-H of sequence SEQ ID NO: 6, a CDR2-H of sequence SEQ ID NO: 7, and a CDR3-H of sequence SEQ ID NO: 8, and a light chain variable domain consisting of sequence DVVMTQTPVSLSVSLGGQVSISCRSSQSLVHNNGNTY LSWYLQKPGQSPQSLIYKVSN RFSGFSDRFSGSGSGTDFTLKISRVDPDDLGVYYCGQGTQYPFT FGSGTKLEIK (SEQ ID NO: 9, with CDRs shown in bold characters and underlined) comprising CDR1-L of sequence SEQ ID NO: 10, a CDR2-L consisting of sequence 'KVS', and a CDR3-L of sequence SEQ ID NO: 11.

[0070] The so-called "4B4-D7" anti-CD3 antibody comprises: a heavy chain variable domain consisting of sequence EVQLVETGGRLVQPGRSLKLTCATSGFTFSNAWMHWVRQSPEKQLEWVAQIKARSNN YAT YYAESVKGRFTISRDDSKSTIYLQMNSLKEEDTAIYYCRGTYYASKPFDY WGQGVM VTVSS (SEQ ID NO: 12, with CDRs shown in bold characters and underlined) comprising CDR1-H of sequence SEQ ID NO: 13, a CDR2-H of sequence SEQ ID NO: 14, and a CDR3-H of sequence SEQ ID NO: 15, and a light chain variable domain consisting of sequence DVVMTQTPVSLSVSLGGQVSISCRSSQSLVHDNGNTY LSWSLQRPGQSPQVLIYKVS N RFSGTSDRFTGSGSGTDFTLKISRVEPDDLGVYYCGQGTQYPFT FGSGTKLEIK (SEQ ID NO: 16, with CDRs shown in bold characters and underlined) comprising CDR1-L of sequence SEQ ID NO: 17, a CDR2-L consisting of sequence 'KVS', and a CDR3-L of sequence SEQ ID NO: 11.

[0071] The so-called "4E7-C9" anti-CD3 antibody comprises: a heavy chain variable domain consisting of sequence EVQVVETGGSL VQPGKSLKL TCA TSGFTFSNA WMHWVRQSPEKQLEWV AQIKDKSNN YAT YYAESLKGRFTISRDDPKRSIYLQMNSLREEDTAIYYCRYVHYGIGYAMDA WGQGT SVTVSS (SEQ ID NO: 18, with CDRs shown in bold characters and underlined) comprising CDR1-H of sequence SEQ ID NO: 13, a CDR2-H of sequence SEQ ID NO: 19, and a CDR3-H of sequence SEQ ID NO: 20, and a light chain variable domain consisting of sequence DVVMTQTPVSLSVSLGGQVSISCRSSQSLEHNNGNTY LSWYLQKPGQSPQPLIYKVS N RFSGISDRFSGSGSGTDFTLKISRVEPDDLGVYYCGQGTQYPFT FGPGTKLELK (SEQ ID NO: 21, with CDRs shown in bold characters and underlined) comprising CDR1-L of sequence SEQ ID NO: 22, a CDR2-L consisting of sequence 'KVS', and a CDR3-L of sequence SEQ ID NO: 11.

[0072] The so-called "18F5-H10" anti-CD3 antibody comprises: a heavy chain variable domain consisting of sequence EVQVVETGGSL VQPGKSLKL TCA TSGFTFTNAWMHWVRRSPEKQLEWV AQIKDKSNN YAT YYAESVKGRFTISRDDSKSSIYLQMNSLKEEDTAIYYCRYVHYRFAYALDA WGRGT SVSVSS (SEQ ID NO: 23, with CDRs shown in bold characters and underlined) comprising CDR1-H of sequence SEQ ID NO: 24, a CDR2-H of sequence SEQ ID NO: 19, and a CDR3-H of sequence SEQ ID NO: 25, and a light chain variable domain consisting of sequence DVLMTQTPVSLSVSLGGQVSISCRSSOSLVHTNGNTYLSWYLQKPGQSPQLLIYKVSNR LSGISDRFSGSGSGTDFTLKISRVEPDDLGVYYCGOGTHYPFTFGAGTKLELK (SEQ ID NO: 26, with CDRs shown in bold characters and underlined) comprising CDR1-L of sequence SEQ ID NO: 27, a CDR2-L consisting of sequence 'KVS', and a CDR3-L of sequence SEQ ID NO:28.

[0073] The so-called "12D2-E5" anti-CD3 antibody comprises: a heavy chain variable domain consisting of sequence EVKLVESGGGLVQPGRSLRLSCAASGFNFYAYW MGWVRQAPGKGLEWIGEIKKDGTTI NYTPSLKDRFTISRDNAQNTL YLQMTKLGSEDT AL YYCAREERDGYFDYWGQGVMVTV SS (SEQ ID NO: 29, with CDRs shown in bold characters and underlined) comprising CDR1-H of sequence SEQ ID NO: 30, a CDR2-H of sequence SEQ ID NO: 31, and a CDR3-H of sequence SEQ ID NO: 32, and a light chain variable domain consisting of sequence QFVLTQPNSVSTNLGSTVKLSCKRSTGNIGSNYVNWYQQHEGRSPTTMIYRDDKRPDG VPDRFSGSIDRSSNSALLTINNVQTEDEADYFCOSYSSGIVFGGGTKLTVL (SEQ ID NO: 33, with CDRs shown in bold characters and underlined) comprising CDR1-L of sequence SEQ ID NO: 34, a CDR2-L consisting of sequence 'RDD', and a CDR3-L of sequence SEQ ID NO: 35.

[0074] The so-called "11D7-C3" anti-CD3 antibody comprises: a heavy chain variable domain consisting of sequence EVQFVETGGSLVQPGKSLKLTCATSGFTFSNAWMHWVRQSPEKQLEWVAQIKAKSNN Y ATYY AESVKGRFTISRDDSKSSVYLQMNSLKEEDTATYYCRGL YYGLSPSDYWGQGV MVTVSS (SEQ ID NO: 36, with CDRs shown in bold characters and underlined) comprising CDR1-H of sequence SEQ ID NO: 13, a CDR2-H of sequence SEQ ID NO: 37, and a CDR3-H of sequence SEQ ID NO: 38, and a light chain variable domain consisting of sequence DVVMTQTPVSLSVSLGGQVSISCRSSQSLVHNNGNTY LSWYLQKPGQSPQLLIYKVS N RFSGISDRFSGSGSGTDFTLKISRVEPDDLGVYYCGQGTHYPFTFGSGTKLEIK (SEQ ID NO: 39, with CDRs shown in bold characters and underlined) comprising CDR1-L of sequence SEQ ID NO: 10, a CDR2-L consisting of sequence 'KVS', and a CDR3-L of sequence SEQ ID NO: 28.

[0075] The so-called "11H3-E5" anti-CD3 antibody comprises: a heavy chain variable domain consisting of sequence EVQLVETGGSLVQPGKSLKLTCATSGFTFSNAW MHWVRQSPEKQLEWVAQIKAKSNN YAT YYAESVKGRFTISRDDSKSSVYLQMNSLKEEDTAIYYCRGTYYAYKPFDY WGQGV MVTVSS (SEQ ID NO: 40, with CDRs shown in bold characters and underlined) comprising CDR1-H of sequence SEQ ID NO: 13, a CDR2-H of sequence SEQ ID NO: 37, and a CDR3-H of sequence SEQ ID NO: 41, and a light chain variable domain consisting of sequence DVVMTQTPVSLSVSLGGQVSISCRSSQSLVHDNGNTY LSWSLQKPGQSPQVLIYKVS N RFSGTSDRFTGSGSGTDFTLKISRVEPDDLGVYYCGQGTQYPFT FGSGTKLEIK (SEQ ID NO: 42, with CDRs shown in bold characters and underlined) comprising CDR1-L of sequence SEQ ID NO: 17, a CDR2-L consisting of sequence 'KVS', and a CDR3-L of sequence SEQ ID NO: 11.

[0076] The so-called "13H2-C2" anti-CD3 antibody comprises: a heavy chain variable domain consisting of sequence EEEL VETGGSL VQPGKSLKL TCA TSGFTFSNAWMHWVRQSPDKQLEWV AQIKAKSNN YAT YYAESVKGRFTISRDDSKSSVYLQMNNLKEEDTAIYYCRYVHYGLAPMDA WGQGT SVTVSS (SEQ ID NO: 43, with CDRs shown in bold characters and underlined) comprising CDR1-H of sequence SEQ ID NO: 13, a CDR2-H of sequence SEQ ID NO: 37, and a CDR3-H of sequence SEQ ID NO: 44, and a light chain variable domain consisting of sequence DVVMTQTPVSLSVSLGGQVSISCRSSQSLVHNNGNTY LSWYLQKAGQSPQLLIYKVS N RFSGISDRFSGSGSGTDFILKISRVEPDDLGVFYCGQGTQYPFTFGAGTKLELK (SEQ ID NO: 45, with CDRs shown in bold characters and underlined) comprising CDR1-L of sequence SEQ ID NO: 10, a CDR2-L consisting of sequence 'KVS', and a CDR3-L of sequence SEQ ID NO: 11.

[0077] The so-called "13C1-F6" anti-CD3 antibody comprises: a heavy chain variable domain consisting of sequence EVQLVETGGTLVQPGKSLKLTCATSGFTFSNAWMHWVRQSPEKQLEWVAQIKAKSNN YAT YYAESVKGRFTISRDDSKTSVYLQLNSLREEDTAIYYCRGTQYGYNPFDY WGQGV MVTVSS (SEQ ID NO: 46, with CDRs shown in bold characters and underlined) comprising CDR1-H of sequence SEQ ID NO: 13, a CDR2-H of sequence SEQ ID NO: 37, and a CDR3-H of sequence SEQ ID NO: 47, and a light chain variable domain consisting of sequence DVVMTQSPVSLSVSLGGQVSISCRSSOSLVHNNGNTYLSWYLQRSGQSPQLLIYKVSN RLSGISDRFSGSGSGTDFTLKISRIEPDDLGVYYCGQGTQYPFTF GSGTRLEIK (SEQ ID NO: 48, with CDRs shown in bold characters and underlined) comprising CDR1-L of sequence SEQ ID NO: 10, a CDR2-L consisting of sequence 'KVS', and a CDR3-L of sequence SEQ ID NO: 11.

[0078] The so-called "18H11-F10" anti-CD3 antibody comprises: a heavy chain variable domain consisting of sequence EVQLVESGGGLVQPGRSLKLSCAASGFIFSDYYMAWVRQAPKKGLEWVATISISGSRTY YPDSVKGRFTVSRDNAKSSLYLQMNSLKSEDTATYYCATNNPGGWFVYWGQGTLVTV SS (SEQ ID NO: 49, with CDRs shown in bold characters and underlined) comprising CDR1-H of sequence SEQ ID NO: 50, a CDR2-H of sequence SEQ ID NO: 51, and a CDR3-H of sequence SEQ ID NO: 52, and a light chain variable domain consisting of sequence NIQMTQSPSLLSASVGDRVTLSCKAGQNINND LAWYQQKLGEAPRLLIYNAN SLQTGIPS RFSGSGSGADFTLTISSLQPEDVATYFCQQYSSGDTFGAGTKLELK (SEQ ID NO: 53, with CDRs shown in bold characters and underlined) comprising CDR1-L of sequence SEQ ID NO: 54, a CDR2-L consisting of sequence 'NAN', and a CDR3-L of sequence SEQ ID NO: 55.

[0079] The so-called "1 E6-C9" anti-CD3 antibody comprises: a heavy chain variable domain consisting of sequence EVQLVETGGSLVQPGKSLKLTCATSGFTFSYAW MHWVRQSPDKQLQWVAQIKAKSNN YAT YYAESVEGRFTISRDDSKSSVYLQMNSLKEEDTAIYYCRGVYYGLLGLDA WGQGT SVTVSS (SEQ ID NO: 56, with CDRs shown in bold characters and underlined) comprising CDR1-H of sequence SEQ ID NO: 57, a CDR2-H of sequence SEQ ID NO: 37, and a CDR3-H of sequence SEQ ID NO: 58, and a light chain variable domain consisting of sequence DVVMTQTPVSLSVRLGGQVSISCRSSQSLVHNNGNTY LSWFLQKPGQSPQLLIYKVS N RFSGISORFSGSASGTOFTLKISRVEPOOLGVYYCGQGTHYPFT FGSGTKLEIK (SEQ ID NO: 59, with CDRs shown in bold characters and underlined) comprising CDR1-L of sequence SEQ ID NO: 10, a CDR2-L consisting of sequence 'KVS', and a CDR3-L of sequence SEQ ID NO: 28.

[0080] The so-called "10F4-C10" anti-CD3 antibody comprises: a heavy chain variable domain consisting of sequence EVQLVETGGSLVQPGKSLKITCATSGFTFSNAWMHWVRQSPEKQLEWVAQIKAKSNNY AT YYAESVKGRFTISRDDSKSSIYLQMNSLKEEDTAIYYCRAVNYGNYPLDY WGQGVMV TVSS (SEQ ID NO: 60, with CDRs shown in bold characters and underlined) comprising CDR1-H of sequence SEQ ID NO: 13, a CDR2-H of sequence SEQ ID NO: 37, and a CDR3-H of sequence SEQ ID NO: 61, and a light chain variable domain consisting of sequence DVVMTQTPVSLSVSLGGQVSISCRSSOSLVHNNGNTYLSWCLQKPGQSPQLLIYKVSN RFSGISDRFSGSGSGTDFTLKISRVEPDDLGVYYCGQGTQYPFTF GAGTKLELK (SEQ ID NO: 62, with CDRs shown in bold characters and underlined) comprising CDR1-L of sequence SEQ ID NO: 10, a CDR2-L consisting of sequence 'KVS', and a CDR3-L of sequence SEQ ID NO: 11.

[0081] The so-called "10E6-G6" anti-CD3 antibody comprises: a heavy chain variable domain consisting of sequence EVQLVETGGGLVQSGKSLKLTCATSGFTVTNAW MHWVRQSPEKQLEWVAQIKAKSNN YET YYAESVKGRFTISRDDSKSSVYLQMNSLKEEDTAIYYCRGTQYGYNPFDY WGQGV MVTVSS (SEQ ID NO: 63, with CDRs shown in bold characters and underlined) comprising CDR1-H of sequence SEQ ID NO: 64, a CDR2-H of sequence SEQ ID NO: 65, and a CDR3-L of sequence SEQ ID NO: 47, and a light chain variable domain consisting of sequence DVVMTQSPVSLSVSLGGQVSISCRSSQSLVHNNGY TYLSWYLQKPGQSPQVFIYKVS N RFSGISDRFSGSGSGTDFTLKISRIEPDDLGVYYCGQGTHYPFT FGSGTKLEIK (SEQ ID NO: 66, with CDRs shown in bold characters and underlined) comprising CDR1-L of sequence SEQ ID NO: 67, a CDR2-L consisting of sequence 'KVS', and a CDR3-L of sequence SEQ ID NO: 28.

[0082] The so-called "18G9-H11" anti-CD3 antibody comprises: a heavy chain variable domain consisting of sequence EVQLVETGGSLVQPGKSLKLTCATSGFTFSNAW IQWVRQSPEKQLEWVAQIKAKSNNY AT YYAESVKGRFTISRDDSKSSVYLQMNSLKEEDTALYYCTWRHYYSSHTMDA WGQG TLVTVSS (SEQ ID NO: 68, with CDRs shown in bold characters and underlined) comprising CDR1-H of sequence SEQ ID NO: 13, a CDR2-H of sequence SEQ ID NO: 37, and a CDR3-H of sequence SEQ ID NO: 69, and a light chain variable domain consisting of sequence DVVMTQTPVSLSVSLGGQVSISCRSSQSLVHNNGNTY LSWYLQKPGQSPQLLIYKVS N RFSGISDRFSGSGSGTDFTLKISRVAPTDLGVYYCGQGSQYPFT FGAGTKLELK (SEQ ID NO: 70, with CDRs shown in bold characters and underlined) comprising CDR1-L of sequence SEQ ID NO: 10, a CDR2-L consisting of sequence 'KVS', and a CDR3-L of sequence SEQ ID NO: 71.

[0083] The so-called "11F3-B9" anti-CD3 antibody comprises: a heavy chain variable domain consisting of sequence EVQLVETGGSLVQPGKSLTLTCATSGFTFSNAW MHWVRQSPEKQLEWVAQIKAKSNN YAT YYAESVKGRFTISRDDSKRSVYLQMNSLKEEDTAIYYCRYVNYGLAPMDV WGQGT SVTVSS (SEQ ID NO: 72, with CDRs shown in bold characters and underlined) comprising CDR1-H of sequence SEQ ID NO: 13, a CDR2-H of sequence SEQ ID NO: 37, and a CDR3-H of sequence SEQ ID NO: 84, and a light chain variable domain consisting of sequence DVVMTQTPVSLSVSLGGQVSISCRSSQSLVHDNGNTY LSWYLQKPGQSPQLLIYKVS N RFSGFSDRFSGSGSGTDFTLKISRVEPDDLGVYYCGQGTQYPFT FGAGTKLELK (SEQ ID NO: 73, with CDRs shown in bold characters and underlined) comprising CDR1-L of sequence SEQ ID NO: 17, a CDR2-L consisting of sequence 'KVS', and a CDR3-L of sequence SEQ ID NO: 11.

[0084] The so-called "12G3-E8" anti-CD3 antibody comprises: a heavy chain variable domain consisting of sequence EVRVVETGGSLVQPGKSLKLTCATSGFTFSLAW MHWVRQSPEKKLEWVAQIKDKANN YAT YYAESVKGRFTISRDDSKRSVYLQMNRLKEEDTAIYYCRGVYYGFSMTPFDY WGQ GVMVTVSS (SEQ ID NO: 74, with CDRs shown in bold characters and underlined) comprising CDR1-H of sequence SEQ ID NO: 75, a CDR2-H of sequence SEQ ID NO: 76, and a CDR3-H of sequence SEQ ID NO: 77, and a light chain variable domain consisting of sequence DVAMTQTPVSLSVSLGGQVSISCRSSQSLVHNNGNTY LSWYLQKPGQSPQVLIYKVS N RFSGISDRFSGSGSGADFTLKISRVEPDDLGVYYCGQGTQYPFT FGAGTKLELK (SEQ ID NO: 78, with CDRs shown in bold characters and underlined) comprising CDR1-L of sequence SEQ ID NO: 10, a CDR2-L consisting of sequence 'KVS', and a CDR3-L of sequence SEQ ID NO: 11.

[0085] The so-called "5B1-G2" anti-CD3 antibody comprises: a heavy chain variable domain consisting of sequence EVQVVETGGSLVQPGKSLKLTCATSGFSFSNAW MHWVRQSPEKQLEWVAQIKDKANN YAT YYAESVKGRFTISRDDSKGSIYLQMNSLKEEDTAVYYCRGLYYGLFPSDY WGQGV MVTVSS (SEQ ID NO: 79, with CDRs shown in bold characters and underlined) comprising CDR1-H of sequence SEQ ID NO: 80, a CDR2-H of sequence SEQ ID NO: 76, and a CDR3-H of sequence SEQ ID NO: 81, and a light chain variable domain consisting of sequence DVVMTQTPVSLSVSLGGQVSISCRSSQSLVHNNGNTY LSWYLLKPGQSPQLLIYKVS NR FSGISDRFSGGGSGTDFTLKISRLEPDDLGIYYCGQGTQYPFT FGSGTKLEIK (SEQ ID NO: 82, with CDRs shown in bold characters and underlined) comprising CDR1-L of sequence SEQ ID NO: 10, a CDR2-L consisting of sequence 'KVS', and a CDR3-L of sequence SEQ ID NO: 11.

[0086] The so-called "16F8-A7" anti-CD3 antibody comprises: a heavy chain variable domain comprising the sequence VETGGNLVQPGKSLKLTCATSGFTFSNAW MHWVRQSPEKQLEWVAQIKAKSNNYAT Y YAESVKGRFTISRDDSKSSVYLQMNSLKEEDTAIYYCRYVNYGLAPMDV WGQGTSVTV SS (SEQ ID NO: 83, with CDRs shown in bold characters and underlined) comprising CDR1-H of sequence SEQ ID NO: 13, a CDR2-H of sequence SEQ ID NO: 37, and a CDR3-H of sequence SEQ ID NO: 84, and a light chain variable domain consisting of sequence DVVMTQTPVSLSVSLGGQVSISCRSSQSLVHNNGNTY LSWYLQKPGQSPQLLIYKVS N RFSGFSDRFSGSGSGTDFTLKISRVEPDDLGVYYCGQGTQYPFT FGAGTKLELK (SEQ ID NO: 85, with CDRs shown in bold characters and underlined) comprising CDR1-L of sequence SEQ ID NO: 10, a CDR2-L consisting of sequence 'KVS', and a CDR3-L of sequence SEQ ID NO: 11.

[0087] The so-called "11F9-F8" anti-CD3 antibody comprises: a heavy chain variable domain consisting of sequence EVQLVETGGTLVQPGKSLKLTCATSGFTFSNAWMHWVRQSPEKQLEWVAQIKAKSNN YAT YYAESVKGRFTISRDDSKTSVYLQLNSLREEDTAIYYCRGTQYGYNPFDY WGQGV MVTVSS (SEQ ID NO: 46, with CDRs shown in bold characters and underlined) comprising CDR1-H of sequence SEQ ID NO: 13, a CDR2-H of sequence SEQ ID NO: 37, and a CDR3-H of sequence SEQ ID NO: 47, and a light chain variable domain consisting of sequence DVVLTQTPVSLSVSLGGQVSISCRSSQSLVHNNGNTY LSWSLQKPGQSPQVLIYKVS NR FSGISNRFSGSGSGTDFTLKISRVEPDDLGVYYCGQGAHYPFT FGSGTKLEIK (SEQ ID NO: 87, with CDRs shown in bold characters and underlined) comprising CDR1-L of sequence SEQ ID NO: 10, a CDR2-L consisting of sequence 'KVS', and a CDR3-L of sequence SEQ ID NO: 88.

[0088] The so-called "3G5-E10" anti-CD3 antibody comprises: a heavy chain variable domain consisting of sequence EVKLVESGGGLVQPGRSLKLSCAASGFNFNVYW MGWVRQAPGKGLEWIGEIKKDSNSI NYTPSLKEKFTISRDNAQNTLYLQVNKLGSEDTAIYYCAREERDGYFDY WGQGVMVTVS S (SEQ ID NO: 89, with CDRs shown in bold characters and underlined) comprising CDR1-H of sequence SEQ ID NO: 90, a CDR2-H of sequence SEQ ID NO: 91, and a CDR3-H of sequence SEQ ID NO: 32, and a light chain variable domain consisting of sequence NIVMTQSPKSMSMSVGERVTLTCKASENVVTY VSWYQQKPEQSPKLLIYGAS NRYTGV PDRFTGSGSATDFTLTISSVQAEDLADYHCGQGYSYPYT FGGGTKLEIK (SEQ ID NO: 92, with CDRs shown in bold characters and underlined) comprising CDR1-L of sequence SEQ ID NO: 93, a CDR2-L consisting of sequence 'GAS', and a CDR3-L of sequence SEQ ID NO: 94.

[0089] The so-called "9D7-F3" anti-CD3 antibody comprises: a heavy chain variable domain consisting of sequence AVQLVESGGGLVQPKESLKISCAASGFTFSNAA MYWVRQAPGKGLEWVARIRTKPNNY AT YYADSVTGRFIISRDDSRSMVYLQMDNLQTEDTAMYYCTALISTAMAA WGQGTSVT VSS (SEQ ID NO: 95, with CDRs shown in bold characters and underlined) comprising CDR1-H of sequence SEQ ID NO: 96, a CDR2-H of sequence SEQ ID NO: 97, and a CDR3-H of sequence SEQ ID NO: 98, and a light chain variable domain consisting of sequence DIQMTQSPSFLSASVGDRVTINCKASQNINKY LNWYHQMLGEAPKLVISNTN NLQAGIPS RFSGSGSGTDFTLTISSLQPEDVATYFCLQHRSGYT FGLGTKLELK (SEQ ID NO: 99, with CDRs shown in bold characters and underlined) comprising CDR1-L of sequence SEQ ID NO: 100, a CDR2-L consisting of sequence 'NTN', and a CDR3-L of sequence SEQ ID NO: 101.

[0090] The so-called "8C2-F7" anti-CD3 antibody comprises: a heavy chain variable domain consisting of sequence QIQLVQSGPELKKPGESVKISCKASGYTFTDFA MNWVKQAPGNGLKWMGWINTQTGK P TYADGFKQRFVFSLETSASTIYLQINNLNIEDTATYFCTRGALAS VGQGVLVTVSS (SEQ ID NO: 102, with CDRs shown in bold characters and underlined) comprising CDR1-H of sequence SEQ ID NO: 103, a CDR2-H of sequence SEQ ID NO: 104, and a CDR3-H of sequence SEQ ID NO: 105, and a light chain variable domain consisting of sequence DVVMTQTPVSLSVSLGSHVSISCRSSQSLVHNNGNTY LSWYLQKPGQSPQPLIYKVSN RFSGISDRFSGSGSGTDFTLEINRVEPDDLGVYYCGQGAQYPFT FGSGTKLEIK (SEQ ID NO: 106, with CDRs shown in bold characters and underlined) comprising CDR1-L of sequence SEQ ID NO: 10, a CDR2-L consisting of sequence 'KVS', and a CDR3-L of sequence SEQ ID NO: 11.

[0091] The so-called "20E5-F10" anti-CD3 antibody comprises: a heavy chain variable domain consisting of sequence EVQL VETGENL VQPGKSLRL TCA TSGFSFSNAWMHWIRQSPEKQLEWV AQIKDKSNNY AT YYAESVNGRFTISRDDSKSSIYLHMDNLKEEDSAIYYCRYVHYGVRFFYTMDV WGQG TSVTVSS (SEQ ID NO: 107, with CDRs shown in bold characters and underlined) comprising CDR1-H of sequence SEQ ID NO: 80, a CDR2-H of sequence SEQ ID NO: 19, and a CDR3-H of sequence SEQ ID NO: 108, and a light chain variable domain consisting of sequence DVVMTQTPVSLSVSLGDQVSISCRPSQSLVHNNGNTY LSWYLQKPGQSPHPLIYKVS N RFSGISDRFSGSGSGTDFTLKISRVEPDDLGVYYCGQGTQYPFT FGSGTKLEIK (SEQ ID NO: 109, with CDRs shown in bold characters and underlined) comprising CDR1-L of sequence SEQ ID NO: 10, a CDR2-L consisting of sequence 'KVS', and a CDR3-L of sequence SEQ ID NO: 11.

[0092] The so-called "20B5-F10" anti-CD3 antibody comprises: a heavy chain variable domain consisting of sequence EVQLVETGGSLVQPGKSLKLTCATSGFTFSNAW MHWVRQSPEKQLEWVAQIKAKSNN YAT YYAESVKGRFTISRDDSISSVYLQMNNLKEEDTAIYYCRGVYYGFLGMDA WGQGT SVTVSS (SEQ ID NO: 110, with CDRs shown in bold characters and underlined) comprising CDR1-H of sequence SEQ ID NO: 13, a CDR2-H of sequence SEQ ID NO: 37, and a CDR3-H of sequence SEQ ID NO: 111, and a light chain variable domain consisting of sequence OVVMTQTPVSLSVSLGGQVSISCRSSQRLVHNNGNTY LSWYLQKPGQSPQLLVYKVS N RFSGISDRFSGSGSGTDFTLKISRVEPDDLGVYYCGQGTEYPFT FGSGTKLEIK (SEQ ID NO: 112, with CDRs shown in bold characters and underlined) comprising CDR1-L of sequence SEQ ID NO: 113, a CDR2-L consisting of sequence 'KVS', and a CDR3-L of sequence SEQ ID NO: 114.

[0093] The so-called "6C9-C9" anti-CD3 antibody comprises: a heavy chain variable domain consisting of sequence AVQLVESGGGLVRPKESLKISCAASGFTFRNAA MYWVRQAPGKGLEWVARIRTQPNNY AK YYADSVKDRFTISRDDSKSMVYLQMDNLKTEDTAMYYCTGLVVTAMDA WGQGTSV TVSS (SEQ ID NO: 115, with CDRs shown in bold characters and underlined) comprising CDR1-H of sequence SEQ ID NO: 116, a CDR2-H of sequence SEQ ID NO: 117, and a CDR3-H of sequence SEQ ID NO: 118, and a light chain variable domain consisting of sequence DIQMTQSPSFLSASVGDRVTINCKASQNINKY LNWYQQKLGEAPKLLIYVTN NLQTGIPS RFSGSGSGTDYTLTISSLQPEDVATYFCLQHRSMYT FGTGTKLELK (SEQ ID NO: 119, with CDRs shown in bold characters and underlined) comprising CDR1-L of sequence SEQ ID NO: 100, a CDR2-L consisting of sequence 'VTN', and a CDR3-L of sequence SEQ ID NO: 120.

[0094] The so-called "3E8-G1" anti-CD3 antibody comprises: a heavy chain variable domain consisting of sequence EVQVVESGGGLVQPGRSLKLSCAASGFTFSNYY MDWVRQAPKKGLEWVATITASGSRI YYPDSVKGRFTISRDNAKSSLYLLMNSLKSEDTATYYCARERTDAYFDY WGQGVMVTV SS (SEQ ID NO: 121, with CDRs shown in bold characters and underlined) comprising CDR1-H of sequence SEQ ID NO: 122, a CDR2-H of sequence SEQ ID NO: 123, and a CDR3-H of sequence SEQ ID NO: 124, and a light chain variable domain consisting of sequence QFILTQPNSVSTILGSTVKLSCKRSTGNIGTNY VSWYQHHEGRSPTTMIYRDD KRPDGV PDRFSGSIDRSSNSALLTINNVQTEDEADYFCQSYISGLNPVFGGGSKLTVL (SEQ ID NO: 125, with CDRs shown in bold characters and underlined) comprising CDR1-L of sequence SEQ ID NO: 126, a CDR2-L consisting of sequence 'RDD', and a CDR3-L of sequence SEQ ID NO: 127.

[0095] The so-called "3H6-D2" anti-CD3 antibody comprises: a heavy chain variable domain consisting of sequence EVQLVETGGRLVQPGKSLKLTCATSGFTFSNAWMHWVRQSPEKQLEWVAQIKDKSNN YAT YYAESVKGRFTISRDDSKSIIYLQMNSLKEEDTAIYYCRALTYYGYKRDAMDG WGH GTSVTVSS (SEQ ID NO: 128, with CDRs shown in bold characters and underlined) comprising CDR1-H of sequence SEQ ID NO: 13, a CDR2-H of sequence SEQ ID NO: 19, and a CDR3-H of sequence SEQ ID NO: 129, and a light chain variable domain consisting of sequence DVVMTQTPVSLSVSLGGQVSISCRSSQSLVHNNGNTY LSWYLQKPGQSPQLLIYKVS N RFSGISDRFSGSGSGTDFTLKISRVEPDDLGVYYCGQGTQYPFTFGAGTKLELK (SEQ ID NO: 130, with CDRs shown in bold characters and underlined) comprising CDR1-L of sequence SEQ ID NO: 10, a CDR2-L consisting of sequence 'KVS', and a CDR3-L of sequence SEQ ID NO: 11.

[0096] The so-called "8H2" anti-CD3 antibody comprises: a heavy chain variable domain consisting of sequence QIQL VQSGPELKKPGESVKISCKASGYTFTDF AMNWVKQAPGNGLKWMGWINTQTGK P TYADGFKQRFVFSLETSASTIYLQINNLNIEDTATYFCTRGALAS VGQGVMVTVSS (SEQ ID NO: 131, with CDRs shown in bold characters and underlined) comprising CDR1-H of sequence SEQ ID NO: 103, a CDR2-H of sequence SEQ ID NO: 104, and a CDR3-H of sequence SEQ ID NO: 105, and a light chain variable domain consisting of sequence DVVMTQTPVSLSVAIGQPASISCKSSQSLVGTSGKTY LNWLLQRPGQSPKRLIYLVS KLD SGIPDRFSGSGSETDFTLKISRVETDDLGVYYCLQGSHFPLT FGSGTKLEIK (SEQ ID NO: 132, with CDRs shown in bold characters and underlined) comprising CDR1-L of sequence SEQ ID NO: 133, a CDR2-L consisting of sequence 'LVS', and a CDR3-L of sequence SEQ ID NO: 134.

[0097] In an embodiment, the anti-CD3 antibody of the invention binds to human CD3. In another embodiment, the anti-CD3 antibody of the invention further binds to Macaca fascicularis CD3. In particular, the anti-CD3 antibody of the invention binds to the extracellular domain of human CD3, or of both human and Macaca fascicularis CD3. More specifically, the antibody binds to CD3ε. More specifically, the anti-CD3 antibody binds to the human and Macaca fascicularis extracellular domain of CD3ε. The anti-CD3 antibody binds to CD3ε when present in the form of a complex, such as a CD3ε / δ complex, or when present as single protein, indifferently whether expressed in isolated form, or present in a soluble extracellular domain or full-length membrane-anchored CD3ε as present in for example in T-cells.

[0098] The anti-CD3 antibody according to the invention is specific for the surface human CD3 protein, or of both human and Macaca fascicularis CD3 proteins, in particular to CD3ε.

[0099] In an embodiment, the anti-CD3 antibody according to the invention has a ratio of affinity for Macaca fascicularis CD3 on affinity for human CD3 (KD(Macaca fascicularis) / KD(human)) which is ≤10, in particular ≤6, ≤5, ≤4, ≤3, for example ≤2, ≤1 or ≤0.5. Such a polypeptide according to the invention may be used in toxicological studies performed in monkeys the toxicity profile observed in monkeys relevant to anticipate potential adverse effects in humans

[0100] In particular, the anti-CD3 antibody of the invention does not bind to, or does not significantly cross-react with CD3γ and / or CD3δ protein(s).

[0101] In particular, the antibody does not bind to, or does not significantly cross-react with the extracellular domain of the aforementioned human and Macaca fascicularis CD3γ and / or CD3δ protein(s).

[0102] The sequence of full-length human CD3δ protein is available in Uniprot database under accession number P04234 (SEQ ID NO: 86, as available on December 14, 2014). The extracellular domain of human CD3δ consists of amino acids at positions 22-105 of SEQ ID NO: 86.

[0103] The sequence of full-length human CD3γ protein is available in Uniprot database under accession number P09693 (SEQ ID NO: 185, as available on December 14, 2014). The extracellular domain of human CD3γ consists of amino acids at positions 23-116 of SEQ ID NO: 185.

[0104] Furthermore, the anti-CD3 antibody according to the invention has an affinity (KD) for human CD3 or Macaca fascicularis CD3, or both, which is ≤90nM, ≤50nM, or ≤30nM, for instance ≤ 20nM, ≤ 10nM, ≤ 8nM, ≤ 6nM, ≤ 4nM or ≤2nM, for instance an affinity of 0.1 nM to 10 nM, in particular of 0.1 nM to 8 nM, or of 0.1 nM to 4 nM.

[0105] Affinity for human CD3 or for Macaca fascicularis CD3 is determined as the KD value with surface plasmon resonance using soluble recombinant CD3ε / δ complex from human and Macaca fascicularis as capture antigen.

[0106] In one example, binding affinities of an anti-CD3 antibody is measured by surface plasmon resonance (SPR) using for instance a Biacore3000 instrument (GE Healthcare). Assay buffer is for example HBS-EP (BR-1001-88, GE Healthcare). As antigen may be used, for example, the human CD3ε and human CD3δ subunit extracellular domain constructs, including the signal peptide, in form of Fc-fusion proteins as described in the examples. Alternatively, use may be made, as antigen, of the Macaca fascicularis CD3ε and Macaca fascicularis CD3δ subunit extracellular domain constructs, including the signal peptide, in form of Fc-fusion proteins as described in the examples. Capture of human or Macaca fascicularis CD3ε / δ-Fc fusion proteins is achieved using, for example, the human antibody capture kit (GE Healthcare). For example, the capture antibody may be coupled to CM5 chips (BR-1001-88, GE Healthcare) to for instance approx. 12.000 RU using for example the amine coupling kit (BR-100-50, GE Healthcare). The CD3ε / δ-Fc fusions proteins are captured at 10 µl / min to approx. 70 RU to yield Rmax values of 30 RU. Binding kinetics for an anti-CD3 antibody may be measured at, for example, 30 µl / min for 240 s and 600 s. for association and dissociation phase, respectively. For example, two fold dilutions of an anti-CD3 antibody from 3 to 400 nM in assay buffer may be used. Regeneration of the capture surface may be performed with for example a 1 min injection of for instance 3M MgCl 2 solution at 30 µl / min. For data analysis for example the BIAevaluation software v.4.1 (GE Healthcare) may be used. Data may be fit globally using a 1:1 Langmuir model with mass transfer.

[0107] In an embodiment, the anti-CD3 antibody of the invention also has an apparent EC50 as, for example, determined by FACS analysis on human T-cells, which is ≤60nM, for instance ≤50nM, ≤40nM, ≤30nM, ≤20nM or ≤15nM. Typically, the apparent EC50 is within the range 1 to 60 nM, in particular 1 to 30nM, for example 1 to 20nM.

[0108] In one embodiment, the anti-CD3 antibody of the invention has a T-cell activation that is less than 10%, less than 8%, less than 6%, less than 4%, less than 2%, less than 1%, for example less than 0.5% in the absence of target cells.

[0109] The term "activation of T-cells" herein refers to triggering CD3 signaling involving cytotoxic granule fusion, transient cytokine release, and proliferation. The antibody-like binding protein and the anti-CD3 antibody of the invention target CD3ε and activate T-cells in the presence of target cells; this activity is also referred to as a "T-cell engaging effect". The T-cell engaging effect induces cytotoxicity in the target cell.

[0110] As known by the skilled in the art, activation of T-cells induces the expression of surface marker such as CD69 and CD25. The activation of T-cells can thus be measured by detecting and measuring the expression of CD4+ / CD25+, CD4 + / CD69 + , CD8+ / CD25+, or CD8 + / CD69 + T cells. Methods to measure T-cell activation are known to the skilled in the art.

[0111] A method to measure T-cell activation is further disclosed in the example section (Example 3.3). Accordingly, in context of the invention T-cell activation is measured either as the percentage of cells expressing CD69 in % of the total number of cells, or as the percentage of cells expressing CD4 andCD69 in % of total number of cells, or as the percentage of cells expressing CD8 and CD69 in % of the total number of cells.

[0112] "Low T-cell activation" in context with the anti-CD3 antibody of the invention refers to less than 10%, less than 8%, less than 6%, less than 4%, less than 2%, less than 1%, for example less than 0.5% T-cell activation in the absence of target cells.

[0113] Alignments of the sequences of the VH and VL regions of the "20G6-F3", "4B4-D7", "4E7-C9", "18F5-H10", "12D2-E5", "11D7-C3", "11H3-E5", "13H2-C2", "13C1-F6", "18H11-F10", "1E6-C9", "10F4-C10", "10E6-G6", "18G9-H11", "11F3-B9", "12G3-E8", "5B1-G2", "16F8-A7", "11F9-F8", "3G5-E10", "9D7-F3", "8C2-F7", "20E5-F10", "20B5-F10", "6C9-C9", "3E8-G1", "3H6-D2" and "8H2" anti-CD3 antibodies were performed. The comparison of the CDR-H and CDR-L sequences tends to indicate that, structurally, "20G6-F3", "4B4-D7", "4E7-C9", "18F5-H10", "11D7-C3", "11H3-E5", "13H2-C2", "13C1-F6", "1E6-C9", "10F4-C10", "10E6-G6", "18G9-H11", "11F3-B9", "12G3-E8", "5B1-G2", "16F8-A7", "11F9-F8", "20E5-F10", "20B5-F10" and "3H6-D2" are closely related, said antibodies probably binding to the same epitope. The comparison of the CDR-H and CDR-L sequences of said related antibodies is presented in Figures 1 and 2, respectively. CDR positions were identified that are strictly conserved between the antibodies and which are thus assumed to be important for specificity, whereas other positions could support substitution.

[0114] Accordingly, the antibody according to the invention comprises: a heavy chain variable domain comprising a CDR1-H consisting of sequence GFX 1 X 2 X 3 X 4 AW (SEQ ID NO: 331) wherein X 1 is T or S, X 2 is F or V, X 3 is S or T and X 4 is N, K, L or Y, or any combination thereof; and a CDR2-H consisting of sequence IKX 1 X 2 X 3 NX 4 YX 5 T (SEQ ID NO: 332) wherein X 1 is A or D, X 2 is K or R, X 3 is S or A, X 4 is N or S and X 5 is A or E, or any combination thereof; and a CDR3-H consisting of sequence TWRHYYSSHTMDA (SEQ ID NO: 69) or RALTYYGYKRDAMDG (SEQ ID NO: 129) or RX 1 X 2 X 3 YX 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 DX 12 (SEQ ID NO: 333) wherein X 1 is Y, G or A, X 2 is V, T or L, X 3 is H, N, Y or Q, X 4 is G, R or A, X 5 is F or V or no amino acid, X 6 is R or no amino acid, X 7 is F, S or I or no amino acid, X 8 is F, L, N, M, Y, S, A or G, X 9 is Y, A, K, S, N, T, F or L, X 10 is A, P, G or T, X 11 is M, L, For S and X 12 is A, V or Y, or any combination thereof, and a light chain variable domain comprising a CDR1-L consisting of sequence QX 1 LX 2 HX 3 NGX 4 TY (SEQ ID NO: 334) wherein X 1 is R or S, X 2 is V or E, X 3 is N, D or T, and X 4 is N or Y, or any combination thereof; and a CDR2-L consisting of sequence 'KVS'; and a CDR3-L consisting of sequence GQGX 1 X 2 YPFT (SEQ ID NO: 335) wherein X 1 is T, A or S and X 2 is H, E or Q, or any combination thereof.

[0115] According to an embodiment, the anti-CD3 antibody according to the invention comprises the CDR sequences of the heavy and / or light chains of one of 28 so-called "20G6-F3", "4B4-D7", "4E7-C9", "18F5-H10", "12D2-E5", "11D7-C3", "11H3-E5", "13H2-C2", "13C1-F6", "18H11-F10", "1E6-C9", "10F4-C10", "10E6-G6", "18G9-H11", "11F3-B9", "12G3-E8", "5B1-G2", "16F8-A7", "11F9-F8", "3G5-E10", "9D7-F3", "8C2-F7", "20E5-F10", "20B5-F10", "6C9-C9", "3E8-G1", "3H6-D2" and "8H2" anti-CD3 antibodies listed above.

[0116] Therefore, the invention relates to an anti-CD3 antibody, which comprises: a) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 6 or a sequence differing from SEQ ID NO: 6 by one amino acid substitution; CDR2-H of sequence SEQ ID NO: 7 or a sequence differing from SEQ ID NO: 7 by one or more amino acid substitution; CDR3-H of sequence SEQ ID NO: 8 or a sequence differing from SEQ ID NO: 8 by one amino acid substitution; and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 10 or SEQ ID NO: 142 or a sequence differing from SEQ ID NO: 10 or SEQ ID NO: 142 by one amino acid substitution substitution; CDR2-L of sequence 'KVS' or a sequence differing from the sequence 'KVS' by one amino acid substitution and CDR3-L of sequence SEQ ID NO: 11 or a sequence differing from SEQ ID NO: 11 by one amino acid substitution; or b) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 13 or a sequence differing from SEQ ID NO: 13 by one amino acid substitution; CDR2-H of sequence SEQ ID NO: 14 or a sequence differing from SEQ ID NO: 14 by one or more amino acid substitutions; CDR3-H of sequence SEQ ID NO: 15 or a sequence differing from SEQ ID NO: 15 by one amino acid substitution; and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 17 or SEQ ID NO: 184 or a sequence differing from SEQ ID NO: 17 or SEQ ID NO: 184 by one amino acid substitution; CDR2-L of sequence 'KVS' or a sequence differing from the sequence 'KVS' by one amino acid substitution and CDR3-L of sequence SEQ ID NO: 11 or a sequence differing from SEQ ID NO: 11 by one amino acid substitution; or c) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 13 or a sequence differing from SEQ ID NO: 13 by one amino acid substitution; CDR2-H of sequence SEQ ID NO: 19 or a sequence differing from SEQ ID NO: 19 by one or more amino acid substitutions; CDR3-H of sequence SEQ ID NO: 20 or a sequence differing from SEQ ID NO: 20 by one amino acid substitution; and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 22 or a sequence differing from SEQ ID NO: 22 by one amino acid substitution; CDR2-L of sequence 'KVS' or a sequence differing from the sequence 'KVS' by one amino acid substitution and CDR3-L of sequence SEQ ID NO: 11 or a sequence differing from SEQ ID NO: 11 by one amino acid substitution; or d) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 24 or a sequence differing from SEQ ID NO: 24 by one amino acid substitution; CDR2-H of sequence SEQ ID NO: 19 or a sequence differing from SEQ ID NO: 19 by one or more amino acid substitutions; CDR3-H of sequence SEQ ID NO: 25 or a sequence differing from SEQ ID NO: 25 by one amino acid substitution; and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 27 or a sequence differing from SEQ ID NO: 27 by one amino acid substitution; CDR2-L of sequence 'KVS' or a sequence differing from the sequence 'KVS' by one amino acid substitution and CDR3-L of sequence SEQ ID NO: 28 or a sequence differing from SEQ ID NO: 28 by one amino acid substitution; or e) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 30 or a sequence differing from SEQ ID NO: 30 by one amino acid substitution; CDR2-H of sequence SEQ ID NO: 31 or a sequence differing from SEQ ID NO: 31 by one or more amino acid substitutions; CDR3-H of sequence SEQ ID NO: 32 or a sequence differing from SEQ ID NO: 32 by one amino acid substitution; and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 34 or a sequence differing from SEQ ID NO: 34 by one amino acid substitution; CDR2-L of sequence 'RDD' or a sequence differing from the sequence 'RDD' by one amino acid substitution and CDR3-L of sequence SEQ ID NO: 35 or a sequence differing from SEQ ID NO: 35 by one amino acid substitution; or f) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 13 or a sequence differing from SEQ ID NO: 13 by one amino acid substitution; CDR2-H of sequence SEQ ID NO: 37 or a sequence differing from SEQ ID NO: 37 by one or more amino acid substitutions; CDR3-H of sequence SEQ ID NO: 38 or a sequence differing from SEQ ID NO: 38 by one amino acid substitution; and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 10 or a sequence differing from SEQ ID NO: 10 by one amino acid substitution; CDR2-L of sequence 'KVS' or a sequence differing from the sequence 'KVS' by one amino acid substitution and CDR3-L of sequence SEQ ID NO: 28 or a sequence differing from SEQ ID NO: 28 by one amino acid substitution; or g) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 13 or a sequence differing from SEQ ID NO: 13 by one amino acid substitution; CDR2-H of sequence SEQ ID NO: 37 or a sequence differing from SEQ ID NO: 37 by one or more amino acid substitutions; CDR3-H of sequence SEQ ID NO: 41 or a sequence differing from SEQ ID NO: 41 by one amino acid substitution; and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 17 or a sequence differing from SEQ ID NO: 17 by one amino acid substitution; CDR2-L of sequence 'KVS' or a sequence differing from the sequence 'KVS' by one amino acid substitution and CDR3-L of sequence SEQ ID NO: 11 or a sequence differing from SEQ ID NO: 11 by one amino acid substitution; or h) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 13 or a sequence differing from SEQ ID NO: 13 by one amino acid substitution; CDR2-H of sequence SEQ ID NO: 37 or a sequence differing from SEQ ID NO: 37 by one or more amino acid substitutions; CDR3-H of sequence SEQ ID NO: 44 or a sequence differing from SEQ ID NO: 44 by one amino acid substitution; and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 10 or a sequence differing from SEQ ID NO: 10 by one amino acid substitution; CDR2-L of sequence 'KVS' or a sequence differing from the sequence 'KVS' by one amino acid substitution and CDR3-L of sequence SEQ ID NO: 11 or a sequence differing from SEQ ID NO: 11 by one amino acid substitution; or i) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 13 or a sequence differing from SEQ ID NO: 13 by one amino acid substitution; CDR2-H of sequence SEQ ID NO: 37 or a sequence differing from SEQ ID NO: 37 by one or more amino acid substitutions; CDR3-H of sequence SEQ ID NO: 47 or a sequence differing from SEQ ID NO: 47 by one amino acid substitution; and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 10 or a sequence differing from SEQ ID NO: 10 by one amino acid substitution; CDR2-L of sequence 'KVS' or a sequence differing from the sequence 'KVS' by one amino acid substitution and CDR3-L of sequence SEQ ID NO: 11 or a sequence differing from SEQ ID NO: 11 by one amino acid substitution; or j) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 50 or a sequence differing from SEQ ID NO: 50 by one amino acid substitution; CDR2-H of sequence SEQ ID NO: 51 or a sequence differing from SEQ ID NO: 51 by one or more amino acid substitutions; CDR3-H of sequence SEQ ID NO: 52 or a sequence differing from SEQ ID NO: 52 by one amino acid substitution; and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 54 or a sequence differing from SEQ ID NO: 54 by one amino acid substitution; CDR2-L of sequence 'NAN' or a sequence differing from the sequence 'NAN' by one amino acid substitution and CDR3-L of sequence SEQ ID NO: 55 or a sequence differing from SEQ ID NO: 55 by one amino acid substitution; or k) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 57 or a sequence differing from SEQ ID NO: 57 by one amino acid substitution; CDR2-H of sequence SEQ ID NO: 37 or a sequence differing from SEQ ID NO: 37 by one or more amino acid substitutions; CDR3-H of sequence SEQ ID NO: 58 or a sequence differing from SEQ ID NO: 58 by one amino acid substitution; and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 10 or a sequence differing from SEQ ID NO: 10 by one amino acid substitution; CDR2-L of sequence 'KVS' or a sequence differing from the sequence 'KVS' by one amino acid substitution and CDR3-L of sequence SEQ ID NO: 28 or a sequence differing from SEQ ID NO: 28 by one amino acid substitution; or l) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 13 or a sequence differing from SEQ ID NO: 13 by one amino acid substitution; CDR2-H of sequence SEQ ID NO: 37 or a sequence differing from SEQ ID NO: 37 by one or more amino acid substitutions; CDR3-H of sequence SEQ ID NO: 61 or a sequence differing from SEQ ID NO: 61 by one amino acid substitution; and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 10 or a sequence differing from SEQ ID NO: 10 by one amino acid substitution; CDR2-L of sequence 'KVS' or a sequence differing from the sequence 'KVS' by one amino acid substitution and CDR3-L of sequence SEQ ID NO: 11 or a sequence differing from SEQ ID NO: 11 by one amino acid substitution; or m) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 64 or a sequence differing from SEQ ID NO: 64 by one amino acid substitution; CDR2-H of sequence SEQ ID NO: 65 or a sequence differing from SEQ ID NO: 65 by one or more amino acid substitutions; CDR3-H of sequence SEQ ID NO: 47 or a sequence differing from SEQ ID NO: 47 by one amino acid substitution; and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 67 or a sequence differing from SEQ ID NO: 67 by one amino acid substitution; CDR2-L of sequence 'KVS' or a sequence differing from the sequence 'KVS' by one amino acid substitution and CDR3-L of sequence SEQ ID NO: 28 or a sequence differing from SEQ ID NO: 28 by one amino acid substitution; or n) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 13 or a sequence differing from SEQ ID NO: 13 by one amino acid substitution; CDR2-H of sequence SEQ ID NO: 37 or a sequence differing from SEQ ID NO: 37 by one or more amino acid substitutions; CDR3-H of sequence SEQ ID NO: 69 or a sequence differing from SEQ ID NO: 69 by one amino acid substitution; and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 10 or a sequence differing from SEQ ID NO: 10 by one amino acid substitution; CDR2-L of sequence 'KVS' or a sequence differing from the sequence 'KVS' by one amino acid substitution and CDR3-L of sequence SEQ ID NO: 71 or a sequence differing from SEQ ID NO: 71 by one amino acid substitution; or o) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 13 or a sequence differing from SEQ ID NO: 13 by one amino acid substitution; CDR2-H of sequence SEQ ID NO: 37 or a sequence differing from SEQ ID NO: 37 by one or more amino acid substitutions; CDR3-H of sequence SEQ ID NO: 84 or a sequence differing from SEQ ID NO: 84 by one amino acid substitution; and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 17 or a sequence differing from SEQ ID NO: 17 by one amino acid substitution; CDR2-L of sequence 'KVS' or a sequence differing from the sequence 'KVS' by one amino acid substitution and CDR3-L of sequence SEQ ID NO: 11 or a sequence differing from SEQ ID NO: 11 by one amino acid substitution; or p) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 75 or a sequence differing from SEQ ID NO: 75 by one amino acid substitution; CDR2-H of sequence SEQ ID NO: 76 or a sequence differing from SEQ ID NO: 76 by one or more amino acid substitutions; CDR3-H of sequence SEQ ID NO: 77 or a sequence differing from SEQ ID NO: 77 by one amino acid substitution; and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 10 or a sequence differing from SEQ ID NO: 10 by one amino acid substitution; CDR2-L of sequence 'KVS' or a sequence differing from the sequence 'KVS' by one amino acid substitution and CDR3-L of sequence SEQ ID NO: 11 or a sequence differing from SEQ ID NO: 11 by one amino acid substitution; or q) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 80 or a sequence differing from SEQ ID NO: 80 by one amino acid substitution; CDR2-H of sequence SEQ ID NO: 76 or a sequence differing from SEQ ID NO: 76 by one or more amino acid substitutions; CDR3-H of sequence SEQ ID NO: 81 or a sequence differing from SEQ ID NO: 81 by one amino acid substitution; and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 10 or a sequence differing from SEQ ID NO: 10 by one amino acid substitution; CDR2-L of sequence 'KVS' or a sequence differing from the sequence 'KVS' by one amino acid substitution and CDR3-L of sequence SEQ ID NO: 11 or a sequence differing from SEQ ID NO: 11 by one amino acid substitution; or r) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 13 or a sequence differing from SEQ ID NO: 13 by one amino acid substitution; CDR2-H of sequence SEQ ID NO: 37 or a sequence differing from SEQ ID NO: 37 by one or more amino acid substitutions; CDR3-H of sequence SEQ ID NO: 84 or a sequence differing from SEQ ID NO: 84 by one amino acid substitution; and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 10 or a sequence differing from SEQ ID NO: 10 by one amino acid substitution; CDR2-L of sequence 'KVS' or a sequence differing from the sequence 'KVS' by one amino acid substitution and CDR3-L of sequence SEQ ID NO: 11 or a sequence differing from SEQ ID NO: 11 by one amino acid substitution; or s) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 13 or a sequence differing from SEQ ID NO: 13 by one amino acid substitution; CDR2-H of sequence SEQ ID NO: 37 or a sequence differing from SEQ ID NO: 37 by one or more amino acid substitutions; CDR3-H of sequence SEQ ID NO: 47 or a sequence differing from SEQ ID NO: 47 by one amino acid substitution; and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 10 or a sequence differing from SEQ ID NO: 10 by one amino acid substitution; CDR2-L of sequence 'KVS' or a sequence differing from the sequence 'KVS' by one amino acid substitution and CDR3-L of sequence SEQ ID NO: 88 or a sequence differing from SEQ ID NO: 88 by one amino acid substitution; or t) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 90 or a sequence differing from SEQ ID NO: 90 by one amino acid substitution; CDR2-H of sequence SEQ ID NO: 91 or a sequence differing from SEQ ID NO: 91 by one or more amino acid substitutions; CDR3-H of sequence SEQ ID NO: 32 or a sequence differing from SEQ ID NO: 32 by one amino acid substitution; and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 93 or a sequence differing from SEQ ID NO: 93 by one amino acid substitution; CDR2-L of sequence 'GAS' or a sequence differing from the sequence 'GAS' by one amino acid substitution and CDR3-L of sequence SEQ ID NO: 94 or a sequence differing from SEQ ID NO: 94 by one amino acid substitution; or u) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 96 or a sequence differing from SEQ ID NO: 96 by one amino acid substitution; CDR2-H of sequence SEQ ID NO: 97 or a sequence differing from SEQ ID NO: 97 by one or more amino acid substitutions; CDR3-H of sequence SEQ ID NO: 98 or a sequence differing from SEQ ID NO: 98 by one amino acid substitution; and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 100 or a sequence differing from SEQ ID NO: 100 by one amino acid substitution; CDR2-L of sequence 'NTN' or a sequence differing from the sequence 'NTN' by one amino acid substitution and CDR3-L of sequence SEQ ID NO: 101 or a sequence differing from SEQ ID NO: 101 by one amino acid substitution; or v) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 103 or a sequence differing from SEQ ID NO: 103 by one amino acid substitution; CDR2-H of sequence SEQ ID NO: 104 or a sequence differing from SEQ ID NO: 104 by one or more amino acid substitutions; CDR3-H of sequence SEQ ID NO: 105 or a sequence differing from SEQ ID NO: 105 by one amino acid substitution; and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 10 or a sequence differing from SEQ ID NO: 10 by one amino acid substitution; CDR2-L of sequence 'KVS' or a sequence differing from the sequence 'KVS' by one amino acid substitution and CDR3-L of sequence SEQ ID NO: 11 or a sequence differing from SEQ ID NO: 11 by one amino acid substitution; or w) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 80 or a sequence differing from SEQ ID NO: 80 by one amino acid substitution; CDR2-H of sequence SEQ ID NO: 19 or a sequence differing from SEQ ID NO: 19 by one or more amino acid substitutions; CDR3-H of sequence SEQ ID NO: 108 or a sequence differing from SEQ ID NO: 108 by one amino acid substitution; and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 10 or a sequence differing from SEQ ID NO: 10 by one amino acid substitution; CDR2-L of sequence 'KVS' or a sequence differing from the sequence 'KVS' by one amino acid substitution and CDR3-L of sequence SEQ ID NO: 11 or a sequence differing from SEQ ID NO: 11 by one amino acid substitution; or x) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 13 or a sequence differing from SEQ ID NO: 13 by one amino acid substitution; CDR2-H of sequence SEQ ID NO: 37 or a sequence differing from SEQ ID NO: 37 by one or more amino acid substitutions; CDR3-H of sequence SEQ ID NO: 111 or a sequence differing from SEQ ID NO: 111 by one amino acid substitution; and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 113 or a sequence differing from SEQ ID NO: 113 by one amino acid substitution; CDR2-L of sequence 'KVS' or a sequence differing from the sequence 'KVS' by one amino acid substitution and CDR3-L of sequence SEQ ID NO: 114 or a sequence differing from SEQ ID NO: 114 by one amino acid substitution; or y) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 116 or a sequence differing from SEQ ID NO: 116 by one amino acid substitution; CDR2-H of sequence SEQ ID NO: 117 or a sequence differing from SEQ ID NO: 117 by one or more amino acid substitutions; CDR3-H of sequence SEQ ID NO: 118 or a sequence differing from SEQ ID NO: 118 by one amino acid substitution; and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 100 or a sequence differing from SEQ ID NO: 100 by one amino acid substitution; CDR2-L of sequence 'VTN' or a sequence differing from the sequence 'VTN' by one amino acid substitution and CDR3-L of sequence SEQ ID NO: 120 or a sequence differing from SEQ ID NO: 120 by one amino acid substitution; or z) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 122 or a sequence differing from SEQ ID NO: 122 by one amino acid substitution; CDR2-H of sequence SEQ ID NO: 123 or a sequence differing from SEQ ID NO: 123 by one or more amino acid substitutions; CDR3-H of sequence SEQ ID NO: 124 or a sequence differing from SEQ ID NO: 124 by one amino acid substitution; and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 126 or a sequence differing from SEQ ID NO: 126 by one amino acid substitution; CDR2-L of sequence 'RDD' or a sequence differing from the sequence 'RDD' by one amino acid substitution and CDR3-L of sequence SEQ ID NO: 127 or a sequence differing from SEQ ID NO: 127 by one amino acid substitution; or aa) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 13 or a sequence differing from SEQ ID NO: 13 by one amino acid substitution; CDR2-H of sequence SEQ ID NO: 19 or a sequence differing from SEQ ID NO: 19 by one or more amino acid substitutions; CDR3-H of sequence SEQ ID NO: 129 or a sequence differing from SEQ ID NO: 129 by one amino acid substitution; and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 10 or a sequence differing from SEQ ID NO: 10 by one amino acid substitution; CDR2-L of sequence 'KVS' or a sequence differing from the sequence 'KVS' by one amino acid substitution and CDR3-L of sequence SEQ ID NO: 11 or a sequence differing from SEQ ID NO: 11 by one amino acid substitution; or bb) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 103 or a sequence differing from SEQ ID NO: 103 by one amino acid substitution; CDR2-H of sequence SEQ ID NO: 104 or a sequence differing from SEQ ID NO: 104 by one or more amino acid substitutions; CDR3-H of sequence SEQ ID NO: 105 or a sequence differing from SEQ ID NO: 105 by one amino acid substitution; and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 133 or a sequence differing from SEQ ID NO: 133 by one amino acid substitution; CDR2-L of sequence 'LVS' or a sequence differing from the sequence 'LVS' by one amino acid substitution and CDR3-L of sequence SEQ ID NO: 134 or a sequence differing from SEQ ID NO: 134 by one amino acid substitution.

[0117] One or more individual amino acids may be altered by substitution, in particular by conservative substitution, in one or more of the above CDR sequences. Such an alteration may be intended for example to remove a glycosylation site or a deamidation site, in connection with humanization of the antibody.

[0118] Based on the alignments of the sequences of the VH and VL regions of the "20G6-F3", "4B4-D7", "4E7-C9", "18F5-H10", "11D7-C3", "11H3-E5", "13H2-C2", "13C1-F6", "1E6-C9", "10F4-C10", "10E6-G6", "18G9-H11", "11F3-B9", "12G3-E8", "5B1-G2", "16F8-A7", "11F9-F8", "20E5-F10", "20B5-F10" and "3H6-D2" different amino acid substitutions were identified. Therefore, in one embodiment, an amino acid is substituted: in CDR1-H at one or more of positions 3 to 6, for instance at position 3, 5 or 6 of CDR1-H of sequence GFTFSNAW (SEQ ID NO: 13) or for instance at position 4 and 5 of CDR1-H of sequence GFTFSNAW (SEQ ID NO: 13); and / or in CDR2-H, at one or more of positions 3 to 5, 7 and 9, for instance at positions 3, 4, 5, 7 or 9 of CDR2-H of sequence IKAKSNNYAT (SEQ ID NO: 37), or for instance at positions 3 and 5 or 3 and 7 of CDR2-H of sequence IKAKSNNYAT (SEQ ID NO: 37); and / or in CDR3-H, at one or more of positions 2 to 4, 7 to 10 and 12, for instance at positions 2, 3, 4, 6, 7, 8, 9 and 10 or at positions 2, 4, 6, 8, 10 and 12 or at positions 2, 3, 7 and 8 of CDR3-H of sequence RGVYYALSPFDY (SEQ ID NO:8), or at position 7 and 8 of CDR3-H of sequence RGLYYGLSPSDY (SEQ ID NO: 38), or at position at positions 2, 3, 4, 6, 7, 8, 9 and 10 of CDR3-H of sequence RGLYYGLSPSDY (SEQ ID NO: 38); and / or in CDR1-L, at one or more of positions 2, 4, 6, and 9, for example at position 6 of CDR1-L of sequence QSLVHDNGNTY (SEQ ID NO: 17) or QSLVHTNGNTY (SEQ ID NO: 27) or at position 2 of CDR1-L of sequence QSLVHNNGNTY (SEQ ID NO : 10) or QRLVHNNGNTY (SEQ ID NO : 113) or at position 4, 6 and 9 of CDR1-L of sequence QSLVHNNGNTY (SEQ ID NO : 10); and / or in CDR3-L, at one or more of positions 4 and 5, for instance at position 4 of CDR3-L of sequence GQGSQYPFT (SEQ ID NO: 71) or GQGTQYPFT (SEQ ID NO: 11), or at position 4 and 5 of CDR3-L of sequence GQGAHYPFT (SEQ ID NO: 88) or position 5 of CDR3-L of sequence GQGTHYPFT (SEQ ID NO: 28) or GQGTEYPFT (SEQ ID NO: 114).

[0119] The anti-CD3 antibody according to the invention is in particular a conventional antibody, in particular a conventional monoclonal antibody, or an antibody fragment, a bispecific or multispecific antibody.

[0120] The anti-CD3 antibody according to the invention in particular comprises or consists of an IgG, or a fragment thereof.

[0121] According to a further embodiment, the invention relates to an anti-CD3 antibody, which comprises: a) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 6, CDR2-H of sequence SEQ ID NO: 7, CDR3-H of sequence SEQ ID NO: 8 and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 10 or SEQ ID NO: 142, CDR2-L of sequence 'KVS' and CDR3-L of sequence SEQ ID NO: 11; or b) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 13, CDR2-H of sequence SEQ ID NO: 14, CDR3-H of sequence SEQ ID NO: 15 and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 17 or SEQ ID NO: 184, CDR2-L of sequence 'KVS' and CDR3-L of sequence SEQ ID NO: 11; or c) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 13, CDR2-H of sequence SEQ ID NO: 19, CDR3-H of sequence SEQ ID NO: 20 and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 22, CDR2-L of sequence 'KVS' and CDR3-L of sequence SEQ ID NO: 11; or d) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 24, CDR2-H of sequence SEQ ID NO: 19, CDR3-H of sequence SEQ ID NO: 25 and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 27, CDR2-L of sequence 'KVS' and CDR3-L of sequence SEQ ID NO: 28; or e) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 30, CDR2-H of sequence SEQ ID NO: 31, CDR3-H of sequence SEQ ID NO: 32 and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 34, CDR2-L of sequence 'RDD' and CDR3-L of sequence SEQ ID NO: 35; or f) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 13, CDR2-H of sequence SEQ ID NO: 37, CDR3-H of sequence SEQ ID NO: 38 and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 10, CDR2-L of sequence 'KVS' and CDR3-L of sequence SEQ ID NO: 28; or g) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 13, CDR2-H of sequence SEQ ID NO: 37, CDR3-H of sequence SEQ ID NO: 41 and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 17, CDR2-L of sequence 'KVS' and CDR3-L of sequence SEQ ID NO: 11; or h) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 13, CDR2-H of sequence SEQ ID NO: 37, CDR3-H of sequence SEQ ID NO: 44 and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 10, CDR2-L of sequence 'KVS' and CDR3-L of sequence SEQ ID NO: 11; or i) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 13, CDR2-H of sequence SEQ ID NO: 37, CDR3-H of sequence SEQ ID NO: 47 and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 10, CDR2-L of sequence 'KVS' and CDR3-L of sequence SEQ ID NO: 11; or j) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 50, CDR2-H of sequence SEQ ID NO: 51, CDR3-H of sequence SEQ ID NO: 52 and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 54, CDR2-L of sequence 'NAN' and CDR3-L of sequence SEQ ID NO: 55; or k) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 57, CDR2-H of sequence SEQ ID NO: 37, CDR3-H of sequence SEQ ID NO: 58 and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 10, CDR2-L of sequence 'KVS' and CDR3-L of sequence SEQ ID NO: 28; or l) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 13, CDR2-H of sequence SEQ ID NO: 37, CDR3-H of sequence SEQ ID NO: 61 and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 10, CDR2-L of sequence 'KVS' and CDR3-L of sequence SEQ ID NO: 11; or m)a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 64, CDR2-H of sequence SEQ ID NO: 65, CDR3-H of sequence SEQ ID NO: 47 and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 67, CDR2-L of sequence 'KVS' and CDR3-L of sequence SEQ ID NO: 28; or n) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 13, CDR2-H of sequence SEQ ID NO: 37, CDR3-H of sequence SEQ ID NO: 69 and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 10, CDR2-L of sequence 'KVS' and CDR3-L of sequence SEQ ID NO: 71; or o) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 13, CDR2-H of sequence SEQ ID NO: 37, CDR3-H of sequence SEQ ID NO: 84 and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 17, CDR2-L of sequence 'KVS' and CDR3-L of sequence SEQ ID NO: 11; or p) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 75, CDR2-H of sequence SEQ ID NO: 76, CDR3-H of sequence SEQ ID NO: 77 and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 10, CDR2-L of sequence 'KVS' and CDR3-L of sequence SEQ ID NO: 11; or q) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 80, CDR2-H of sequence SEQ ID NO: 76, CDR3-H of sequence SEQ ID NO: 81 and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 10, CDR2-L of sequence 'KVS' and CDR3-L of sequence SEQ ID NO: 11; or r) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 13, CDR2-H of sequence SEQ ID NO: 37, CDR3-H of sequence SEQ ID NO: 84 and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 10, CDR2-L of sequence 'KVS' and CDR3-L of sequence SEQ ID NO: 11; or s) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 13, CDR2-H of sequence SEQ ID NO: 37, CDR3-H of sequence SEQ ID NO: 47 and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 10, CDR2-L of sequence 'KVS' and CDR3-L of sequence SEQ ID NO: 88; or t) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 90, CDR2-H of sequence SEQ ID NO: 91, CDR3-H of sequence SEQ ID NO: 32 and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 93, CDR2-L of sequence 'GAS' and CDR3-L of sequence SEQ ID NO: 94; or u) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 96, CDR2-H of sequence SEQ ID NO: 97, CDR3-H of sequence SEQ ID NO: 98 and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 100, CDR2-L of sequence 'NTN' and CDR3-L of sequence SEQ ID NO: 101; or v) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 103, CDR2-H of sequence SEQ ID NO: 104, CDR3-H of sequence SEQ ID NO: 105 and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 10, CDR2-L of sequence 'KVS' and CDR3-L of sequence SEQ ID NO: 11; or w) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 80, CDR2-H of sequence SEQ ID NO: 19, CDR3-H of sequence SEQ ID NO: 108 and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 10, CDR2-L of sequence 'KVS' and CDR3-L of sequence SEQ ID NO: 11; or x) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 13, CDR2-H of sequence SEQ ID NO: 37, CDR3-H of sequence SEQ ID NO: 111 and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 113, CDR2-L of sequence 'KVS' and CDR3-L of sequence SEQ ID NO: 114; or y) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 116, CDR2-H of sequence SEQ ID NO: 117, CDR3-H of sequence SEQ ID NO: 118 and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 100, CDR2-L of sequence 'VTN' and CDR3-L of sequence SEQ ID NO: 120; or z) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 122, CDR2-H of sequence SEQ ID NO: 123, CDR3-H of sequence SEQ ID NO: 124 and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 126, CDR2-L of sequence 'RDD' and CDR3-L of sequence SEQ ID NO: 127; or aa) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 13, CDR2-H of sequence SEQ ID NO: 19, CDR3-H of sequence SEQ ID NO: 129 and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 10, CDR2-L of sequence 'KVS' and CDR3-L of sequence SEQ ID NO: 11; or bb) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 103, CDR2-H of sequence SEQ ID NO: 104, CDR3-H of sequence SEQ ID NO: 105; and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 133, CDR2-L of sequence 'LVS' and CDR3-L of sequence SEQ ID NO: 134.

[0122] The invention also provides an anti-CD3 antibody comprising at least the heavy chain variable domain and / or the light chain variable domain of one of the so-called anti-CD3 antibodies listed above.

[0123] Thus, the invention relates in particular to an anti-CD3 antibody, which comprises: a) a heavy chain variable domain of sequence SEQ ID NO: 5 or a sequence at least 85% identical thereto, and / or a light chain variable domain of sequence SEQ ID NO: 9, or a sequence at least 85% identical thereto; or b) a heavy chain variable domain of sequence SEQ ID NO: 12 or a sequence at least 85% identical thereto, and / or a light chain variable domain of sequence SEQ ID NO: 16, or a sequence at least 85% identical thereto; or c) a heavy chain variable domain of sequence SEQ ID NO: 18 or a sequence at least 85% identical thereto, and / or a light chain variable domain of sequence SEQ ID NO: 21, or a sequence at least 85% identical thereto; or d) a heavy chain variable domain of sequence SEQ ID NO: 23 or a sequence at least 85% identical thereto, and / or a light chain variable domain of sequence SEQ ID NO: 26, or a sequence at least 85% identical thereto; or e) a heavy chain variable domain of sequence SEQ ID NO: 29 or a sequence at least 85% identical thereto, and / or a light chain variable domain of sequence SEQ ID NO: 33, or a sequence at least 85% identical thereto; or f) a heavy chain variable domain of sequence SEQ ID NO: 36 or a sequence at least 85% identical thereto, and / or a light chain variable domain of sequence SEQ ID NO: 39, or a sequence at least 85% identical thereto; or g) a heavy chain variable domain of sequence SEQ ID NO: 40 or a sequence at least 85% identical thereto, and / or a light chain variable domain of sequence SEQ ID NO: 42, or a sequence at least 85% identical thereto; or h) a heavy chain variable domain of sequence SEQ ID NO: 43 or a sequence at least 85% identical thereto, and / or a light chain variable domain of sequence SEQ ID NO: 45, or a sequence at least 85% identical thereto; or i) a heavy chain variable domain of sequence SEQ ID NO: 46 or a sequence at least 85% identical thereto, and / or a light chain variable domain of sequence SEQ ID NO: 48, or a sequence at least 85% identical thereto; or j) a heavy chain variable domain of sequence SEQ ID NO: 49 or a sequence at least 85% identical thereto, and / or a light chain variable domain of sequence SEQ ID NO: 53, or a sequence at least 85% identical thereto; or k) a heavy chain variable domain of sequence SEQ ID NO: 56 or a sequence at least 85% identical thereto, and / or a light chain variable domain of sequence SEQ ID NO: 59, or a sequence at least 85% identical thereto; or l) a heavy chain variable domain of sequence SEQ ID NO: 60 or a sequence at least 85% identical thereto, and / or a light chain variable domain of sequence SEQ ID NO: 62, or a sequence at least 85% identical thereto; or m) a heavy chain variable domain of sequence SEQ ID NO: 63 or a sequence at least 85% identical thereto, and / or a light chain variable domain of sequence SEQ ID NO: 66, or a sequence at least 85% identical thereto; or n) a heavy chain variable domain of sequence SEQ ID NO: 68 or a sequence at least 85% identical thereto, and / or a light chain variable domain of sequence SEQ ID NO: 70, or a sequence at least 85% identical thereto; or o) a heavy chain variable domain of sequence SEQ ID NO: 72 or a sequence at least 85% identical thereto, and / or a light chain variable domain of sequence SEQ ID NO: 73, or a sequence at least 85% identical thereto; or p) a heavy chain variable domain of sequence SEQ ID NO: 74 or a sequence at least 85% identical thereto, and / or a light chain variable domain of sequence SEQ ID NO: 78, or a sequence at least 85% identical thereto; or q) a heavy chain variable domain of sequence SEQ ID NO: 79 or a sequence at least 85% identical thereto, and / or a light chain variable domain of sequence SEQ ID NO: 82, or a sequence at least 85% identical thereto; or r) a heavy chain variable domain comprising sequence SEQ ID NO: 83 or a sequence at least 85% identical thereto, and / or a light chain variable domain of sequence SEQ ID NO: 85, or a sequence at least 85% identical thereto; or s) a heavy chain variable domain of sequence SEQ ID NO: 46 or a sequence at least 85% identical thereto, and / or a light chain variable domain of sequence SEQ ID NO: 87, or a sequence at least 85% identical thereto; or t) a heavy chain variable domain of sequence SEQ ID NO: 89 or a sequence at least 85% identical thereto, and / or a light chain variable domain of sequence SEQ ID NO: 92, or a sequence at least 85% identical thereto; or u) a heavy chain variable domain of sequence SEQ ID NO: 95 or a sequence at least 85% identical thereto, and / or a light chain variable domain of sequence SEQ ID NO: 99, or a sequence at least 85% identical thereto; or v) a heavy chain variable domain of sequence SEQ ID NO: 102 or a sequence at least 85% identical thereto, and / or a light chain variable domain of sequence SEQ ID NO: 106, or a sequence at least 85% identical thereto; or w) a heavy chain variable domain of sequence SEQ ID NO: 107 or a sequence at least 85% identical thereto, and / or a light chain variable domain of sequence SEQ ID NO: 109, or a sequence at least 85% identical thereto; or x) a heavy chain variable domain of sequence SEQ ID NO: 110 or a sequence at least 85% identical thereto, and / or a light chain variable domain of sequence SEQ ID NO: 112, or a sequence at least 85% identical thereto; or y) a heavy chain variable domain of sequence SEQ ID NO: 115 or a sequence at least 85% identical thereto, and / or a light chain variable domain of sequence SEQ ID NO: 119, or a sequence at least 85% identical thereto; or z) a heavy chain variable domain of sequence SEQ ID NO: 121 or a sequence at least 85% identical thereto, and / or a light chain variable domain of sequence SEQ ID NO: 125, or a sequence at least 85% identical thereto; or aa) a heavy chain variable domain of sequence SEQ ID NO: 128 or a sequence at least 85% identical thereto, and / or a light chain variable domain of sequence SEQ ID NO: 130, or a sequence at least 85% identical thereto; or bb) a heavy chain variable domain of sequence SEQ ID NO: 131 or a sequence at least 85% identical thereto, and / or a light chain variable domain of sequence SEQ ID NO: 132, or a sequence at least 85% identical thereto.

[0124] For instance, the sequence of the variable domain of heavy or light chain may differ from the reference sequence SEQ ID NO: 5, 9, 12, 16, 18, 21, 23, 26, 29, 33, 36, 39, 40, 42, 43, 45, 46, 48, 49, 53, 56, 59, 60, 62, 63, 66, 68, 70, 72, 73, 74, 78, 79, 82, 83, 85, 87, 89, 92, 95, 99, 102, 106, 107, 109, 110, 112, 115, 119, 121, 125, 128, 130, 131, 132, as appropriate, by one or more amino acid substitution(s), in particular by one or more conservative amino acid substitution(s) and / or substitution(s) with canonical residues. In particular, the sequence of the variable domain of heavy or light chain may differ from the reference sequence SEQ ID NO: 5, 9, 12, 16, 18, 21, 23, 26, 29, 33, 36, 39, 40, 42, 43, 45, 46, 48, 49, 53, 56, 59, 60, 62, 63, 66, 68, 70, 72, 73, 74, 78, 79, 82, 83, 85, 87, 89, 92, 95, 99, 102, 106, 107, 109, 110, 112, 115, 119, 121, 125, 128, 130, 131, 132, by conservative amino acid substitution(s), only.

[0125] The sequence alterations as compared with sequence SEQ ID NO: 5, 9, 12, 16, 18, 21, 23, 26, 29, 33, 36, 39, 40, 42, 43, 45, 46, 48, 49, 53, 56, 59, 60, 62, 63, 66, 68, 70, 72, 73, 74, 78, 79, 82, 83, 85, 87, 89, 92, 95, 99, 102, 106, 107, 109, 110, 112, 115, 119, 121, 125, 128, 130, 131, 132 will in particular be present essentially in one or more of the framework regions, FR1-L, FR2-L, FR3-L, FR4-L and / or FR1-H, FR2-H, FR3-H, FR4-H.

[0126] However, amino acid substitutions in one or more CDRs are also possible. In particular, the sequence of the light chain variable domain may differ from sequence SEQ ID NO: 9 at least by a S to R substitution at position 28 of SEQ ID NO: 9 (in CDR1-L), and / or at least by a V to E substitution at position 30 of SEQ ID NO: 9 (in CDR1-L), and / or at least by a N to D or T substitution at position 33 of SEQ ID NO: 9 (in CDR1-L), and / or at least by a N to Y substitution at position 35 of SEQ ID NO: 9 (in CDR1-L) and / or the sequence of the light chain variable domain may differ from sequence SEQ ID NO: 9 at least by a T to S or A substitution at position 97 of SEQ ID NO: 9 (in CDR3-L), and / or at least by a Q to H or E substitution at position 98 of SEQ ID NO: 9 (in CDR3-L) and / or the heavy chain variable domain may differ from sequence SEQ ID NO: 5 at least by a T to N or S substitution at position 28 of SEQ ID NO: 5 (in CDR1-H), and / or at least by a F to V substitution at position 29 of SEQ ID NO: 5 (in CDR1-H), or at least by a T to N or Y substitution at position 30 of SEQ ID NO: 5 (in CDR1-H), or at least by a K to L or Y substitution at position 31 of SEQ ID NO: 5 (in CDR1-H) and / or the heavy chain variable domain may differ from sequence SEQ ID NO: 5 at least by a D to A substitution at position 53 of SEQ ID NO: 5 (in CDR2-H), or at least by a K to R substitution at position 54 of SEQ ID NO: 5 (in CDR2-H), or at least by a S to A substitution at position 55 of SEQ ID NO: 5 (in CDR2-H), or at least by a S to N substitution at position 57 of SEQ ID NO: 5 (in CDR2-H), or at least by a A to E substitution at position 59 of SEQ ID NO: 5 (in CDR2-H) and / or the heavy chain variable domain may differ from sequence SEQ ID NO: 5 at least by a G to A substitution at position 100 of SEQ ID NO: 5 (in CDR3-H) and / or at least by a T to V substitution at position 101 of SEQ ID NO: 5 (in CDR3-H) at least by a Q to Y substitution at position 102 of SEQ ID NO: 5 (in CDR3-H).

[0127] In one embodiment, the anti-CD3 antibody of the invention and a fragment thereof is, respectively, a rat antibody and a fragment of a rat antibody.

[0128] In one aspect of the invention, the anti-CD3 antibody of the invention may also be a chimeric antibody, and in particular a rat / human antibody, e.g. an antibody comprising rat variable domains of heavy and light chains and a CH domain and a CL domain from a human antibody.

[0129] In a further aspect of the invention, the anti-CD3 antibody may also be a humanized antibody or a fragment of a humanized antibody obtained, for example, by CDR-grafting or by the 4D method (US20110027266).

[0130] Accordingly, in one embodiment, the anti-CD3 antibody of the invention is a humanized antibody comprising a) a heavy chain variable domain comprising of an amino acid sequence selected from the group consisting of SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 152, SEQ ID NO: 153, and a light chain variable domain comprising of an amino acid sequence selected from the group consisting of SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157 , or b) a heavy chain variable domain comprising of an amino acid sequence selected from the group consisting of SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 183, and a light chain variable domain comprising of an amino acid sequence selected from the group consisting of SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 182.

[0131] According to a further embodiment, the anti-CD3 antibody of the invention is a humanized antibody comprising: a) a heavy chain variable domain of sequence SEQ ID NO: 138 and / or a light chain variable domain of sequence SEQ ID NO: 143; or b) a heavy chain variable domain of sequence SEQ ID NO: 171 and / or a light chain variable domain of sequence SEQ ID NO: 158; or c) a heavy chain variable domain of sequence SEQ ID NO: 176 and / or a light chain variable domain of sequence SEQ ID NO: 164.

[0132] In one embodiment, the anti-CD3 antibody according to the invention comprises the three CDR sequences or the variable domain of the heavy chain, or the six CDR sequences or variable domains of the heavy and light chains of one of so-called anti-CD3 antibodies listed above.

[0133] The invention further refers to a fragment of the humanized anti-CD3 antibody as defined above. In one embodiment, the humanized anti-CD3 antibody described above is a chimeric antibody.

[0134] The anti-CD3 antibody according to the invention may also be a single domain antibody or a fragment thereof. In particular, a single domain antibody fragment may consist of a variable heavy chain (VHH) which comprises the CDR1-H, CDR2-H and CDR3-H of one of the antibodies described above. The anti-CD3 antibody may also be a heavy chain antibody, i.e. an antibody devoid of light chain, which may or may not contain a CH1 domain.

[0135] The single domain antibody or a fragment thereof may also comprise the framework regions of a camelid single domain antibody, and optionally the constant domain of a camelid single domain antibody.

[0136] The anti-CD3 antibody according to the invention may also be an antibody fragment, in particular a humanized antibody fragment, selected from the group consisting of Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, and diabodies.

[0137] Accordingly, the anti-CD3 antibody of the invention is a Fab comprising, or consisting of a) the heavy chain amino acid sequence SEQ ID NO: 186 and / or the light chain amino acid sequence SEQ ID NO: 187; or b) the heavy chain amino acid sequence SEQ ID NO: 188 and / or the light chain amino acid sequence SEQ ID NO: 189; or c) the heavy chain amino acid sequence SEQ ID NO: 190 and / or the light chain amino acid sequence SEQ ID NO: 191; or d) the heavy chain amino acid sequence SEQ ID NO: 192 and / or the light chain amino acid sequence SEQ ID NO: 193.

[0138] In an embodiment, the CD3-antibody is a bispecific or multispecific antibody formed from at least one antibody fragment or at least one variable domain of the anti-CD3 antibodies of the invention. Multispecific antibodies are polyvalent protein complexes as described for instance in EP 2 050 764 A1 or US 2005 / 0003403 A1.

[0139] The bispecific or multispecific CD3-antibodies according to the invention can have specificity for (a) extracellular domain of human or human and Macaca fascicularis CD3, targeted by one of the above described anti-CD3 antibodies and (b) at least one other antigen.

[0140] In one embodiment, the other antigen is CD123 and accordingly the resulting bispecific antibody is a CD3 / CD123 bispecific antibody. Conventional bi-specific antibodies can be produced by techniques that are known to the skilled in the art.

[0141] Antibodies and fragments of anti-CD3 antibodies according to the invention can be used in an isolated (e.g., purified) from or contained in a vector, such as a membrane or lipid vesicle (e.g. a liposome).

[0142] In one further embodiment, the anti-CD3 antibody of the invention is for use for the preparation of antibody-like binding protein of the invention further defined in the section" antibody-like binding protein".

[0143] Any combination of the above embodiments makes part of the invention.Anti CD123 antibodies

[0144] "CD123" (Cluster of Differentiation 123) is also known as "Interleukin 3 receptor, alpha (IL3RA)" or "IL3R", "IL3RX", "IL3RY", "IL3RAY", "hIL-3Ra" and denotes an interleukin 3 specific subunit of a heterodimeric cytokine receptor. The functional interleukin 3 receptor is a heterodimer that comprises a specific alpha chain (IL-3A; CD123) and the IL-3 receptor beta chain (β 0 ; CD 131) that is shared with the receptors for granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin 5 (IL-5). CD123 is a type I integral transmembrane protein with a deduced Molecular Weight of about 43kDa containing an extracellular domain involved in IL-3 binding, a transmembrane domain and a short cytoplasmic tail of about 50 amino acids. The extracellular domain is composed of two regions: an N-terminal region of about 100 amino acids, the sequence of which exhibits similarity to equivalent regions of the GM-CSF and IL-5 receptor alpha-chains; and a region proximal to the transmembrane domain that contains four conserved cysteine residues and a WSXWS motif, common to other members of this cytokine receptor family. The IL-3 binding domain comprises about 200 amino acid residue cytokine receptor motifs (CRMs) made up of two Ig-like folding domains. The extracellular domain of CD123 is highly glycosylated, with N-glycosylation necessary for both ligand binding and receptor signaling. The protein family gathers three members: IL3RA (CD123A), CSF2RA and IL5RA. The overall structure is well conserved between the three members but sequence homologies are very low. One 300 amino-acid long isoform of CD123 has been discovered so far, but only on the RNA level which is accessible on the Getentry database under the accession number ACM24116.1.

[0145] US Patent No. 6,177,078 discloses the anti-IL-3Receptor alpha chain (IL-3Rα, CD123) monoclonal antibody 7G3, and the ability of 7G3 to bind to the N-terminal domain, specifically amino acid residues 19-49, of IL-3Rα. US Patent No. 6,733,743 discloses a method of impairing a hematologic cancer progenitor cell that expresses CD123 but does not significantly express CD131, by contacting the cell with a composition of an antibody and a cytotoxic agent (selected from a chemotherapeutic agent, a toxin or an alpha-emitting radioisotope) whereby the composition binds selectively to CD123 in an amount effective to cause cell death. However, it has remained unclear whether targeting CD123 can functionally impair AML-LSCs.

[0146] A reference sequence of full-length human CD123 protein, including signal peptide, is available from the NCBI database under the accession number NP_002174.1 and under the Uniprot acession number P26951 and is herein disclosed under SEQ ID NO: 194 (as available on December 14, 2014). A reference sequence of full-length Macaca fascicularis CD123 protein, including signal peptide, is available from GenBank database under the accession number EHH61867.1 and under the Uniprot acession number G8F3K3 and is herein disclosed under SEQ ID NO: 195 (as available on December 14, 2014).

[0147] A sequence of a mature human CD123 Strep-II tagged Fc-fusion protein, cloned by the inventors from genomic DNA, is disclosed under SEQ ID NO: 196. Said mature human CD123 Fc-fusion protein comprises amino acids 19 to 305 of the full-length human CD123 protein and thus comprises the extracellular domain of human CD123.

[0148] A sequence of a mature Macaca fascicularis CD123 Strep-II tagged Fc-fusion protein, cloned by the inventors from cDNA, is disclosed under SEQ ID NO: 197. Said mature Macaca fascicularis CD123 Fc-fusion protein comprises amino acids 19 to 305 of the full-length Macaca fascicularis CD123 protein and thus comprises the extracellular domain of Macaca fascicularis CD123.

[0149] Domain organization of human and Macaca fascicularis CD123 is as follows (based on the human CD123 sequence accessible in the NCBI database under accession NP_002174.1 (SEQ ID NO: 194) and based on the Macaca fascicularis CD123 sequence accessible in the Uniprot database under acession number G8F3K3, SEQ ID NO: 195): Human CD123 domainsPositions on SEQ ID NO: 194 (human)Positions on SEQ ID NO: 195 (Macaca fascicularis)Extracellular19 - 30519 - 305Transmembrane domain306 - 325306 - 325Cytoplasmic326 - 378326 - 378

[0150] Accordingly, the extracellular domain of human CD123 consists of amino acids at positions 19 - 305 of SEQ ID NO: 194.

[0151] CD123 (the interleukin-3 receptor alpha chain IL-3Rα) is a tumor antigen over-expressed in a variety of hematological neoplasms. The majority of AML blasts express surface CD123 and this expression does not vary by subtype of AML. Higher expression of CD123 on AML at diagnosis has been reported to be associated with poorer prognosis. CD123 expression has been reported in other hematological malignancies including myelodysplasia, systemic mastocytosis, blastic plasmacytoid dendritic cell neoplasm (BPDCN), ALL and hairy cell leukemia.

[0152] CD123 is expressed on AML leukemic stem cells and growing evidences suggest that AML arises from these LSCs, which have been shown to be quiescent and relatively resistant to DNA damaging chemotherapy. It is hypothesized that the persistence of LSCs underpins relapse after initial remission and thus the eradication of LSCs can be considered a requirement for cure, and an important therapeutic goal.

[0153] "Leukemic stem cells (LSCs)" are cancer cells that possess characteristics associated with normal stem cells, that is, the property of self renewal and the capability to develop multiple lineages. Such cells are proposed to persist in hematological cancers such as AML as distinct populations. The LCS present in AML patients are so called "AML-LCSs".

[0154] "Acute myelogenous leukemia (AML)" is a clonal disorder clinically presenting as increased proliferation of heterogeneous and undifferentiated myeloid blasts. The leukemic hierarchy is maintained by a small population of LSCs (AML-LCSs), which have the distinct ability for self-renewal, and are able to differentiate into leukemic progenitors. These progenitors generate the large numbers of leukemic blasts readily detectable in patients at diagnosis and relapse, leading ultimately to mortality. AML-LSC have been commonly reported as quiescent cells, in contrast to rapidly dividing clonogenic progenitors. This property of AML-LSCs renders conventional chemotherapeutics that target proliferating cells less effective, potentially explaining the current experience in which a high proportion of AML patients enter complete remission, but almost invariably relapse, with <30% of adults surviving for more than 4 years. In addition, minimal residual disease occurrence and poor survival has been attributed to high LSC frequency at diagnosis in AML patients. Consequently, it is imperative for the long-term management of AML (and similarly other above mentioned hematological cancer conditions) that new treatments are developed to specifically eliminate LSCs.

[0155] Over-expression of CD123 has been reported on AML blasts and on CD34 +< / CD38 AML- LSCs relative to normal hematopoietic cells.

[0156] CD123 thus provides an important therapeutic target for cancer therapy, in particular for cancer therapy in patients having poor prognosis.

[0157] The inventors have succeeded in generating, screening and selecting specific mouse and rat anti-CD123 antibodies displaying high affinity for both human and Macaca fascicularis CD123 protein, and which do not significantly cross-react with human CSF2RA and IL5RA proteins, and with Macaca fascicularis CD3 proteins.

[0158] The inventors have determined the sequence of variable heavy and light chains of such monoclonal antibodies, the so-called "3E3-D3", "1E1-G5", "2B8-F3", "2F8-D6", "3B10-E6", "5A5-B4", "6B10-E4", "6C10-C4", "6D6-B8", "8B11-B7", "9B8-G6", "9D7-C8", and "9F6-G3" anti-CD123 antibodies.

[0159] The so-called "3E3-D3" anti-CD123 antibody comprises: a heavy chain variable domain consisting of sequence QVQLQESGPGLVQPSQTLSLTCTVSGFSLTTYD VHWVRQPPGKGLEWMGRIQNGGIT DYNSALKSRLIISRDTSKSQVFLKMNSVQTEDTAMYFCAKTGSYFYAFDH WGQGTLVT VSS (SEQ ID NO: 226, with CDRs shown in bold characters) comprising CDR1-H of sequence SEQ ID NO: 227, a CDR2-H of sequence SEQ ID NO: 228, and a CDR3-H of sequence SEQ ID NO: 229, or a heavy chain variable domain consisting of sequence QVQLQESGPGLVQPSQTLSLTCTVSGFSLTTYDVHWVRQPPGKGLEWMGRIQNAGIT DYNSALKSRLlISRDTSKSQVFLKMNSVQTEDTAMYFCAKTGSYFYAFDHWGQGTLVT VSS (SEQ ID NO: 277, with CDRs shown in bold characters) comprising CDR1-H of sequence SEQ ID NO: 227, a CDR2-H of sequence SEQ ID NO:353, and a CDR3-H of sequence SEQ ID NO: 229, or a heavy chain variable domain consisting of sequence QVQLQESGPGLVQPSQTLSLTCTVSGFSLTTYDVHWVRQPPGKGLEWMGRIQDGGIT DYNSALKSRLIISRDTSKSQVFLKMNSVQTEDTAMYFCAKTGSYFYAFDHWGQGTLVT VSS (SEQ ID NO: 278, with CDRs shown in bold characters) comprising CDR1-H of sequence SEQ ID NO: 227, a CDR2-H of sequence SEQ ID NO: 279, and a CDR3-H of sequence SEQ ID NO: 229, and- a light chain variable domain consisting of sequence QFVLTQPNSVSTNLGSTVKLSCKRNTGNIGSNY VNWYQQHEGRSPTTMIYRDD KRPD GVPDRFSGSIDRSSNSALLTINNVQTEDEADYFCQSYSSGINIIFGGGTKLTVL (SEQ ID NO: 230, with CDRs shown in bold characters) comprising CDR1-L of sequence SEQ ID NO: 231, a CDR2-L consisting of sequence 'RDD', and a CDR3-L of sequence SEQ ID NO: 232.

[0160] The so-called "1E1-G5" anti-CD123 antibody comprises: a heavy chain variable domain consisting of sequence QVQLQESGPTL VKPGOSVKMSCKAFGYTFTDHI I HWVKQSHGKSLEW IGYINPYSGGT NYNEKFKSKATLTVDKSSSTAYMEFSRLTSEDSAICYCALNYGSYYAMDA WGQGTSV TVSS (SEQ ID NO: 198, with CDRs shown in bold characters) comprising CDR1-H of sequence SEQ ID NO: 199, a CDR2-H of sequence SEQ ID NO: 200, and a CDR3-H of sequence SEQ ID NO: 201 and a light chain variable domain consisting of sequence OIQMTQSPASLSASLGQTVTIECRPSEDIYSNLAWFQQKPGSSPQLLIYDANNLAOGVP SRFSGSGSGTQYSLKINSLQSEOVASYFCQQYNKYPYTFGTGTKLELK (SEQ ID NO: 202, with CDRs shown in bold characters) comprising CDR1-L of sequence SEQ ID NO: 203, a CDR2-L consisting of sequence 'DAN', and a CDR3-L of sequence SEQ ID NO: 204.

[0161] The so-called "2B8-F3" anti-CD123 antibody comprises: a heavy chain variable domain consisting of sequence EVQLVESGGGLVQPGRSLKLSCAASGFTFSDYN MAWVRQAPKKGLEWVATILYDGG RT YYRGSVKGRFTISRONAKSTLYLRMOSLRSEOTATYYCATHSRGTDYFDY WGQGV MVTVSS (SEQ ID NO: 205, with CDRs shown in bold characters) comprising CDR1-H of sequence SEQ ID NO: 206, a CDR2-H of sequence SEQ ID NO: 207, and a CDR3-H of sequence SEQ ID NO: 208 and a light chain variable domain consisting of sequence EIVLTQSPTSMTASPGEQVTITCRASSSINY MHWYQQKPGASPRPWIYETS KLASGVP DRFSGSASGTSYSLTINNMEAEDAATYYCQQWNYPSW TFGGGTKLELK (SEQ ID NO: 209, with CDRs shown in bold characters) comprising CDR1-L of sequence SEQ ID NO: 210, a CDR2-L consisting of sequence 'ETS', and a CDR3-L of sequence SEQ ID NO: 211.

[0162] The so-called "2F8-D6" anti-CD123 antibody comprises: a heavy chain variable domain consisting of sequence QVQLKESGPGLVQPSQTLSLTCTVSGFSLTSYG VSWVRQPPGKGLEWIATISSAGST Y YOLVLKSRLSITROTSKSQVFLKVHSLQTEOTAIYLCARDAPVFNYGSYNAMDS WGQG TSVTVSS (SEQ ID NO: 212, with CDRs shown in bold characters) comprising CDR1-H of sequence SEQ ID NO: 213, a CDR2-H of sequence SEQ ID NO: 214, and a CDR3-H of sequence SEQ ID NO: 215 and a light chain variable domain consisting of sequence DIQMTQSPSFLSATVGDRVTINCKASQNINKY LNWYQQKLGEAPKRLIYNTN SLQTGIP SRFSGSGSGTDYTLTISSLQPEDVATYFCLQHKSGL TFGSGTKLEIK (SEQ ID NO: 216, with CDRs shown in bold characters) comprising CDR1-L of sequence SEQ ID NO: 217, a CDR2-L consisting of sequence 'NTN', and a CDR3-L of sequence SEQ ID NO: 218.

[0163] The so-called "3B10-E6" anti-CD123 antibody comprises: a heavy chain variable domain consisting of sequence QVQLQQSGPELRRPGSSVRLSCKASGYRIKDFL IHWIKNRPEHGLEWIGWIDPEDGET KYAQKFQTKATLTADTSSNTAYMQLSSLTSEDTATYFCARWGDVYYGLMRGHVMDA WGQGTSVTVSS (SEQ ID NO: 219, with CDRs shown in bold characters) comprising CDR1-H of sequence SEQ ID NO: 220, a CDR2-H of sequence SEQ ID NO: 221, and a CDR3-H of sequence SEQ ID NO: 222 and a light chain variable domain consisting of sequence DVLMTQTPVSLPVSLGGQVSISCRSSQSLVHSDGDTY LHWYLQKPGQSPQLLIYRVS N RFSGVPDRFSGSGSGTDFTLKISRVEPEDLGLYYCLQTTHFPPWT FGGGTKLEMK (SEQ ID NO: 223, with CDRs shown in bold characters) comprising CDR1-L of sequence SEQ ID NO: 224, a CDR2-L consisting of sequence 'RVS', and a CDR3-L of sequence SEQ ID NO: 225.

[0164] The so-called "5A5-B4" anti-CD123 antibody comprises: a heavy chain variable domain consisting of sequence QIQIVQSGSDVTSTCNGCGTCYFSGFSLSTTGIC VSWIRQPSGKGQEWLADFCWDDG K GYNPSLKNRLSISKOTSNNQVFLKITSVOTAOTATYYCARRRVYYGIYFDY WGQGVM VTVSS (SEQ ID NO: 233, with CDRs shown in bold characters) comprising CDR1-H of sequence SEQ ID NO: 234, a CDR2-H of sequence SEQ ID NO: 235, and a CDR3-H of sequence SEQ ID NO: 236 and a light chain variable domain consisting of sequence DIVMTQSPALAVSPGERVSISCRASNSVSTR MHWYQQKPGQQPKLLIYGAS NLESGVP ARFSGSGSGTDFTLTIDPVEADDIATYFCQQSWNDPLT FGSGTKLEIK (SEQ ID NO: 237, with CDRs shown in bold characters) comprising CDR1-L of sequence SEQ ID NO: 238, a CDR2-L consisting of sequence 'GAS', and a CDR3-L of sequence SEQ ID NO: 239.

[0165] The so-called "6B10-E4" anti-CD123 antibody comprises: a heavy chain variable domain consisting of sequence EVQLVESGGGLVQPGRSLKLSCAASGFTFSHYN MAWVRQAPKKGLEWVATITYDDHS T YYRDSVKGRFTISRDTAKSTLYLQMDSLRSEDTATYYCARLVNYAFAY WGQGTLVTV SS (SEQ ID NO: 240, with CDRs shown in bold characters) comprising CDR1-H of sequence SEQ ID NO: 241, a CDR2-H of sequence SEQ ID NO: 242, and a CDR3-H of sequence SEQ ID NO: 243 and a light chain variable domain consisting of sequence NIVMTQSPKSMSISVGDRVTMNCKASQTVGNN IAWYQQKPGLSPQLLIDYAS NRYTGV PNRFTGGGYGTDFILTINSVQAEDAAFYYCQRMYNSPT FGGGTKLELK (SEQ ID NO: 244, with CDRs shown in bold characters) comprising CDR1-L of sequence SEQ ID NO: 245, a CDR2-L consisting of sequence 'YAS', and a CDR3-L of sequence SEQ ID NO: 246.

[0166] The so-called "6C10-C4" anti-CD123 antibody comprises: a heavy chain variable domain consisting of sequence EVKLQESGPSLVQPSETLSLTCTVSGFSLTSYS VHWVRQHSGKSLEWMGRMWNDGD TSYNSAFTSRLSISRDTSKGQVFLKMNSLQTEDTGTYYCARGHRTPFDY WGQGVMVT VSS (SEQ ID NO: 247, with CDRs shown in bold characters) comprising CDR1-H of sequence SEQ ID NO: 248, a CDR2-H of sequence SEQ ID NO: 249, and a CDR3-H of sequence SEQ ID NO: 250 and a light chain variable domain consisting of sequence DIVMTQSPSSLAVSAGETVTINCKSSQSFLSSGDERNY VAWYQHKPGQSPKLLIYWAS TRHSGVPDRFIGSGSGTDFTLTISSVQAEDLAIYYCQQYYDTPLT FGSGTKLEIK (SEQ ID NO: 251, with CDRs shown in bold characters) comprising CDR1-L of sequence SEQ ID NO: 252, a CDR2-L consisting of sequence 'WAS', and a CDR3-L of sequence SEQ ID NO: 253.

[0167] The so-called "6D6-B8" anti-CD123 antibody comprises: a heavy chain variable domain consisting of sequence QVQLQESGPTLVKPGDSVKMSCKASAYTFTDNI IHWVKQSHGKSLDWIGYINPYSGGT NYNGWFRSKATLTVDKSSSTAYMEFSRLTSDDSAIYYCALNYGSYYAMDA WGQGTS VTVSS (SEQ ID NO: 254, with CDRs shown in bold characters) comprising CDR1-H of sequence SEQ ID NO: 255, a CDR2-H of sequence SEQ ID NO: 200, and a CDR3-H of sequence SEQ ID NO: 201 and a light chain variable domain consisting of sequence DIQMTQSPASLSASLGETVTIDCRPSEDIFNN LAWYQQKPGNSPQLLIYDANSLADGVP SRFSGSGSGTQYSLMIIRLQSEOVASYFCHQYNIYPYT FGAGTKLELK (SEQ ID NO: 256, with CDRs shown in bold characters) comprising CDR1-L of sequence SEQ ID NO: 257, a CDR2-L consisting of sequence 'DAN', and a CDR3-L of sequence SEQ ID NO: 258.

[0168] The so-called "8B11-B7" anti-CD123 antibody comprises: a heavy chain variable domain consisting of sequence QVQLQESGPTLVNPGOSVKMSCKASGYTFTDHI IHWVKQSHGKSLEWIGYINPYSGGA NYNGKFKSKATLTIDKSSSTAYMEFSRLTSGDSAIYYCALNYGSYYAMDA WGQGTSVT VSS (SEQ ID NO: 259, with CDRs shown in bold characters) comprising CDR1-H of sequence SEQ ID NO: 199, a CDR2-H of sequence SEQ ID NO: 260, and a CDR3-H of sequence SEQ ID NO: 201 and a light chain variable domain consisting of sequence DIQMTQSPASLSASLGETVTIECRTSKDIYSN LAWFQQEPGNSPQLLIYDAS NLADGVP SRFSGSGSGTQYSLQINNLQSEDVASYFCHQYNNYPYTFGTGTKLELK (SEQ ID NO: 261, with CDRs shown in bold characters) comprising CDR1-L of sequence SEQ ID NO: 262, a CDR2-L consisting of sequence 'DAS', and a CDR3-L of sequence SEQ ID NO: 263.

[0169] The so-called "9B8-G6" anti-CD123 antibody comprises: a heavy chain variable domain consisting of sequence EVKLQESGPSLVQSSQTLSLTCTVSGFSLTSYHI HWVRQPPGKGLEWMGVMWSDGD T SYSSALKSRLSISROTSQSQVFLKMNSLQTEOTATYYCARGDYSSYIYLWFAY WGQG TLVTVSS (SEQ ID NO: 264, with CDRs shown in bold characters) comprising CDR1-H of sequence SEQ ID NO: 265, a CDR2-H of sequence SEQ ID NO: 266, and a CDR3-H of sequence SEQ ID NO: 267 and a light chain variable domain consisting of sequence DIVMTQSPSSLAVSAGETVTINCKSSQSFLSSGDERNY VAWYQHKPGQSPKLLIYWAS TRHSGVPDRFIGSGSGTDFTLTISSVQAEDLAIYYCQQYYDTPLT FGSGTKLEIK (SEQ ID NO: 251, with CDRs shown in bold characters) comprising CDR1-L of sequence SEQ ID NO: 252, a CDR2-L consisting of sequence 'WAS', and a CDR3-L of sequence SEQ ID NO: 253.

[0170] The so-called "9D7-C8" anti-CD123 antibody comprises: a heavy chain variable domain consisting of sequence EVKLQESGPSLVQSSQTLSLTCTVSGFSLTSYHI HWVRQTPGKGLEWMGVMWSDGD TSYNSALKSRLSISRDTSQSQVFLKMNSLQTEDTATYYCARGYYSSYLYLWFAY WGQ GTLVTVSS (SEQ ID NO: 268, with CDRs shown in bold characters) comprising CDR1-H of sequence SEQ ID NO: 265, a CDR2-H of sequence SEQ ID NO: 266, and a CDR3-H of sequence SEQ ID NO: 269 and a light chain variable domain consisting of sequence DIVMTQSPSSLAVSEGETVTINCKSSQSFLSSGDGKNY VAWYQYKPGQSPKLLIYWAS TRQSGVPDRFIGSGSGTDFTLTISTVQAEDLAIYYCQQYYDTPLT FGSGTKLEIK (SEQ ID NO: 270, with CDRs shown in bold characters) comprising CDR1-L of sequence SEQ ID NO: 271, a CDR2-L consisting of sequence 'WAS', and a CDR3-L of sequence SEQ ID NO: 253.

[0171] The so-called "9F6-G3" anti-CD123 antibody comprises: a heavy chain variable domain consisting of sequence QVQLQESGPTL VKPGOSVKMSCKASGYTFTDYI IHWVKQSHGKSLEW IGYINPYSDGT NYNEKFKSKATLTVDKSTSTAYMEFSRLTSEDSAIYFCALNYGSYYAMDA WGQGTSV TVSS (SEQ ID NO: 272, with CDRs shown in bold characters) comprising CDR1-H of sequence SEQ ID NO: 273, a CDR2-H of sequence SEQ ID NO: 274, and a CDR3-H of sequence SEQ ID NO: 201 and a light chain variable domain consisting of sequence DIQMTQSPASLSASLGETVTIECRPSEDIHSN VAWYQQKPGNSPQLLIYDAS NLADGVP SRFSGSGSGTQYSLKINSLQSEDVASYFCHQYNIYPYT FGSGTKLELK (SEQ ID NO: 275, with CDRs shown in bold characters) comprising CDR1-L of sequence SEQ ID NO: 276, a CDR2-L consisting of sequence 'DAS', and a CDR3-L of sequence SEQ ID NO: 258.

[0172] In one aspect of the invention, the anti-CD123 antibody binds to human CD123. In another embodiment, the anti-CD123 antibody further binds to Macaca fascicularis CD123. In particular, the anti-CD123 antibody of the invention binds to the extracellular domain of human CD123, or of both, human and Macaca fascicularis CD123. More specifically, the anti-CD123 antibody binds to the distal moiety of CD123, for example, to the amino acids at position 19 to 49 of human CD123 of the amino acid sequence SEQ ID NO: 194. The anti-CD123 antibody binds to CD123, indifferently whether expressed in isolated form, or present in a soluble extracellular domain or full-length membrane-anchored CD123 as present in CD123 expressing cells such as AML cells or CD123 transfected cells. The anti-CD123 antibody according to the invention is specific to cells that express human and Macaca fascicularis CD123 proteins on their surface, for example CD123 expressing cancer cells.

[0173] The anti-CD123 antibody according to the invention has a ratio of affinity for Macaca fascicularis CD123 on affinity for human CD123 (KD (Macaca fascicularis) / KD (human) which is ≤10, in particular ≤6, ≤5, ≤4, ≤3, ≤2, ≤1 or ≤0.5. Thus, the polypeptide according to the invention may be used in toxicological studies performed in monkeys the toxicity profile observed in monkeys relevant to anticipate potential adverse effects in humans.

[0174] In particular, the anti-CD123 antibody of the invention does not bind to, or does not significantly cross-react with CSF2RA and IL5RA proteins.

[0175] In particular, the antibody does not bind to, or does not significantly cross-react with the extracellular domain of the aforementioned human and Macaca fascicularis CSF2RA and IL5RA proteins.

[0176] Furthermore, the anti-CD123 antibody according to the invention has an affinity (KD) for human CD123 or Macaca fascicularis CD123, or both, of ≤50nM, ≤40nM, ≤30nM for instance ≤ 20nM, ≤ 15nM, ≤ 10nM, ≤ 8nM, ≤ 6nM, ≤ 4nM, ≤2nM, ≤1 nM or ≤0.5 nM, for instance an affinity of 0.1 nM to 20 nM, for example 0.1 nM to 10 nM, in particular of 0.1 nM to 2 nM, or of 0.1 nM to 1 nM.

[0177] In one example, affinity for human CD3 or for Macaca fascicularis CD3 is determined as the KD value with surface plasmon resonance using recombinant CD123 protein from human and Macaca fascicularis as capture antigen, for example with human and Macaca fascicularis CD123-Fc fusion protein.

[0178] As antigen may be used for example the human or Macaca fascicularis CD123 extracellular domain including the signal sequence corresponding to the amino acid sequence from position M1 to R305 of the wild type protein (SEQ ID NO : 194 (human), SEQ ID NO : 195 (Macaca fascicularis)). The resulting amino acid sequence for human or Macaca fascicularis CD123 mature protein is listed as SEQ ID NO: 196 and SEQ ID NO: 197, respectively. Biacore measurements are known to the skilled in the art. In the present example the Biacore measurement may be performed as described in the section "anti CD3 antibodies" above.

[0179] The anti-CD123 antibody of the invention may also have an apparent affinity constant (apparent KD), as, for example, determined by FACS analysis on CD123 expressing cells such as CD123 transfected HEK293 cells using non purified anti-CD123 antibody in hybridoma supernatant, which is ≤20nM, for instance ≤15nM, ≤10nM, ≤6nM, ≤5nM, ≤4nM, ≤3nM, ≤2nM or ≤1nM. Typically, the apparent KD is within the range 0.1 to 20 nM, in particular 0.1 to 10 nM, for example 0.1 to 5nM.

[0180] Alignments of the sequences of the VH and VL regions of the so-called "3E3-D3", "1E1-G5", "2B8-F3", "2F8-D6", "3B10-E6", "5A5-B4", "6B10-E4", "6C10-C4", "6D6-B8", "8B11-B7", "9B8-G6", "9D7-C8", "9F6-G3" anti-CD123 antibodies.. The comparison of the CDR-H and CDR-L sequences tends to indicate that, structurally, "1E1-G5", "6D6-B8", "8B11-B7" and "9F6-G3" anti-CD123 antibodies, on one hand, and "6C10-C4", 9B8-G6", "9D7-C8" anti-CD123 antibodies, on the other hand, are closely related, said antibodies probably binding to the same epitope. The alignment of the so-called "1E1-G5", "6D6-B8", "8B11-B7" and "9F6-G3" anti-CD123 antibodies and "6C10-C4", 9B8-G6", "9D7-C8" anti-CD123 antibodies are shown in figures 3 and 4, respectively. The comparison of the CDR-H and CDR-L sequences further identifies CDR positions that are strictly conserved between the two groups of antibodies and which are thus assumed to be important for specificity, whereas other positions could support substitution.

[0181] Accordingly, the antibody according to the invention comprises: a) a heavy chain variable domain comprising a CDR1-H consisting of sequence X 1 YTFTDX 2 I (SEQ ID NO: 336) wherein X 1 is G or A and X 2 is H, Y or N, or any combination thereof; and a CDR2-H consisting of sequence INPYSX 1 GX 2 (SEQ ID NO: 337) wherein X 1 is G or D and X 2 is T or A, or any combination thereof; and a CDR3-H consisting of sequence ALNYGSYYAMDA (SEQ ID NO 201), and a light chain variable domain comprising a CDR1-L consisting of sequence X 1 DIX 2 X 3 N (SEQ ID NO: 338) wherein X 1 is E or K, X 2 is F, H or Y and X 3 is N or S, or any combination thereof; and a CDR2-L consisting of sequence 'DAN' or 'DAS'; and a CDR3-L consisting of sequence X 1 QYNX 2 YPYT (SEQ ID NO: 339) wherein X 1 is H or Q and X 2 is I, K or N, or any combination thereof; or b) a heavy chain variable domain comprising a CDR1-H consisting of sequence GFSLTSYX 1 (SEQ ID NO: 340) wherein X 1 is H or S; and a CDR2-H consisting of sequence MWX 1 DGDT (SEQ ID NO: 341) wherein X 1 is S or N; and a CDR3-H consisting of sequence ARGX 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 FX 10 Y (SEQ ID NO: 342) wherein X 1 is D, Y or H, X 2 is Y or R, X 3 is S or T, X 4 is S or P, X 5 is Y or no amino acid, X 6 is L, I or no amino acid, X 7 is Y or no amino acid, X 8 is L or no amino acid, X 9 is W or no amino acid, X 10 is A or D, or any combination thereof, and a light chain variable domain comprising a CDR1-L consisting of sequence QSFLSSGDX 1 X 2 NY (SEQ ID NO: 343) wherein X 1 is E or G and X 2 is R or K, or any combination thereof; and a CDR2-L consisting of sequence 'WAS'; and a CDR3-L consisting of sequence QQYYDTPLT (SEQ ID NO: 253).

[0182] According to an embodiment, the anti-CD123 antibody according to the invention comprises the CDR sequences of the heavy and / or light chains of one of 13 so-called "3E3-D3", "1E1-G5", "2B8-F3", "2F8-D6", "3B10-E6", "5A5-B4", "6B10-E4", "6C10-C4", "6D6-B8", "8B11-B7", "9B8-G6", "9D7-C8", and "9F6-G3" anti-CD123 antibodies listed above.

[0183] Therefore, the invention relates to an anti-CD123 antibody, which comprises: a) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 227 or a sequence differing from SEQ ID NO: 227 by one amino acid substitution; CDR2-H of sequence SEQ ID NO: 228 or SEQ ID NO: 353 or SEQ ID NO: 279 or a sequence differing from SEQ ID NO: 228 or SEQ ID NO: 353 or SEQ ID NO: 279 by one or more amino acid substitutions; CDR3-H of sequence SEQ ID NO: 229 or a sequence differing from SEQ ID NO: 229 by one amino acid substitution; and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 231 or a sequence differing from SEQ ID NO: 231 by one amino acid substitution; CDR2-L of sequence 'RDD'or a sequence differing from 'RDD' by one amino acid substitution and CDR3-L of sequence SEQ ID NO: 232 or a sequence differing from SEQ ID NO: 232 by one amino acid substitution; or b) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 199 or a sequence differing from SEQ ID NO: 199 by one amino acid substitution; CDR2-H of sequence SEQ ID NO: 200 or a sequence differing from SEQ ID NO: 200 by one or more amino acid substitutions; CDR3-H of sequence SEQ ID NO: 201 or a sequence differing from SEQ ID NO: 201 by one amino acid substitution; and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 203 or a sequence differing from SEQ ID NO: 203 by one amino acid substitution; CDR2-L of sequence 'DAN' or a sequence differing from 'DAN' by one amino acid substitution and CDR3-L of sequence SEQ ID NO: 204 or a sequence differing from SEQ ID NO: 204 by one amino acid substitution; or c) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 206 or a sequence differing from SEQ ID NO: 206 by one amino acid substitution; CDR2-H of sequence SEQ ID NO: 207 or a sequence differing from SEQ ID NO: 207 by one or more amino acid substitutions; CDR3-H of sequence SEQ ID NO: 208 or a sequence differing from SEQ ID NO: 208 by one amino acid substitution; and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 210 or a sequence differing from SEQ ID NO: 210 by one amino acid substitution; CDR2-L of sequence 'ETS' or a sequence differing from 'ETS' by one amino acid substitution and CDR3-L of sequence SEQ ID NO: 211 or a sequence differing from SEQ ID NO: 211 by one amino acid substitution; or d) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 213 or a sequence differing from SEQ ID NO: 213 by one amino acid substitution; CDR2-H of sequence SEQ ID NO: 214 or a sequence differing from SEQ ID NO: 214 by one or more amino acid substitutions; CDR3-H of sequence SEQ ID NO: 215 or a sequence differing from SEQ ID NO: 215 by one amino acid substitution; and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 217 or a sequence differing from SEQ ID NO: 217 by one amino acid substitution; CDR2-L of sequence 'NTN' or a sequence differing from 'NTN' by one amino acid substitution and CDR3-L of sequence SEQ ID NO: 218 or a sequence differing from SEQ ID NO: 218 by one amino acid substitution; or e) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 220 or a sequence differing from SEQ ID NO: 220 by one amino acid substitution; CDR2-H of sequence SEQ ID NO: 221 or a sequence differing from SEQ ID NO: 221 by one or more amino acid substitutions; CDR3-H of sequence SEQ ID NO: 222 or a sequence differing from SEQ ID NO: 222 by one amino acid substitution; and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 224 or a sequence differing from SEQ ID NO: 224 by one amino acid substitution; CDR2-L of sequence 'RVS' or a sequence differing from 'RVS' by one amino acid substitution and CDR3-L of sequence SEQ ID NO: 225 or a sequence differing from SEQ ID NO: 225 by one amino acid substitution; or f) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 234 or a sequence differing from SEQ ID NO: 234 by one amino acid substitution; CDR2-H of sequence SEQ ID NO: 235 or a sequence differing from SEQ ID NO: 235 by one or more amino acid substitutions; CDR3-H of sequence SEQ ID NO: 236 or a sequence differing from SEQ ID NO: 236 by one amino acid substitution; and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 238 or a sequence differing from SEQ ID NO: 238 by one amino acid substitution; CDR2-L of sequence 'GAS' or a sequence differing from 'GAS' by one amino acid substitution and CDR3-L of sequence SEQ ID NO: 239 or a sequence differing from SEQ ID NO: 239 by one amino acid substitution; or g) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 241 or a sequence differing from SEQ ID NO: 241 by one amino acid substitution; CDR2-H of sequence SEQ ID NO: 242 or a sequence differing from SEQ ID NO: 242 by one or more amino acid substitutions; CDR3-H of sequence SEQ ID NO: 243 or a sequence differing from SEQ ID NO: 243 by one amino acid substitution; and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 245 or a sequence differing from SEQ ID NO: 245 by one amino acid substitution; CDR2-L of sequence 'VAS' or a sequence differing from 'YAS' by one amino acid substitution and CDR3-L of sequence SEQ ID NO: 246 or a sequence differing from SEQ ID NO: 246 by one amino acid substitution; or h) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 248 or a sequence differing from SEQ ID NO: 248 by one amino acid substitution; CDR2-H of sequence SEQ ID NO: 249 or a sequence differing from SEQ ID NO: 249 by one or more amino acid substitutions; CDR3-H of sequence SEQ ID NO: 250 or a sequence differing from SEQ ID NO: 250 by one amino acid substitution; and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 252 or a sequence differing from SEQ ID NO: 252 by one amino acid substitution; CDR2-L of sequence 'WAS' or a sequence differing from 'WAS' by one amino acid substitution and CDR3-L of sequence SEQ ID NO: 253 or a sequence differing from SEQ ID NO: 253 by one amino acid substitution; or i) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 255 or a sequence differing from SEQ ID NO: 255 by one amino acid substitution; CDR2-H of sequence SEQ ID NO: 200 or a sequence differing from SEQ ID NO: 200 by one or more amino acid substitutions; CDR3-H of sequence SEQ ID NO: 201 or a sequence differing from SEQ ID NO: 201 by one amino acid substitution; and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 257 or a sequence differing from SEQ ID NO: 257 by one amino acid substitution; CDR2-L of sequence 'DAN' or a sequence differing from 'DAN' by one amino acid substitution and CDR3-L of sequence SEQ ID NO: 258 or a sequence differing from SEQ ID NO: 258 by one amino acid substitution; or j) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 199 or a sequence differing from SEQ ID NO: 199 by one amino acid substitution; CDR2-H of sequence SEQ ID NO: 260 or a sequence differing from SEQ ID NO: 260 by one or more amino acid substitutions; CDR3-H of sequence SEQ ID NO: 201 or a sequence differing from SEQ ID NO: 201 by one amino acid substitution; and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 262 or a sequence differing from SEQ ID NO: 262 by one amino acid substitution; CDR2-L of sequence 'DAS' or a sequence differing from 'DAS' by one amino acid substitution and CDR3-L of sequence SEQ ID NO: 263 or a sequence differing from SEQ ID NO: 263 by one amino acid substitution; or k) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 265 or a sequence differing from SEQ ID NO: 265 by one amino acid substitution; CDR2-H of sequence SEQ ID NO: 266 or a sequence differing from SEQ ID NO: 266 by one or more amino acid substitutions; CDR3-H of sequence SEQ ID NO: 267 or a sequence differing from SEQ ID NO: 267 by one amino acid substitution; and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 252 or a sequence differing from SEQ ID NO: 252 by one amino acid substitution; CDR2-L of sequence 'WAS' or a sequence differing from 'WAS' by one amino acid substitution and CDR3-L of sequence SEQ ID NO: 253 or a sequence differing from SEQ ID NO: 253 by one amino acid substitution; or l) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 265 or a sequence differing from SEQ ID NO: 265 by one amino acid substitution; CDR2-H of sequence SEQ ID NO: 266 or a sequence differing from SEQ ID NO: 266 by one or more amino acid substitutions; CDR3-H of sequence SEQ ID NO: 269 or a sequence differing from SEQ ID NO: 269 by one amino acid substitution; and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 271 or a sequence differing from SEQ ID NO: 271 by one amino acid substitution; CDR2-L of sequence 'WAS' or a sequence differing from 'WAS' by one amino acid substitution and CDR3-L of sequence SEQ ID NO: 253 or a sequence differing from SEQ ID NO: 253 by one amino acid substitution; or m) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 273 or a sequence differing from SEQ ID NO: 273 by one amino acid substitution; CDR2-H of sequence SEQ ID NO: 274 or a sequence differing from SEQ ID NO: 274 by one or more amino acid substitutions; CDR3-H of sequence SEQ ID NO: 201 or a sequence differing from SEQ ID NO: 201 by one amino acid substitution; and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 276 or a sequence differing from SEQ ID NO: 276 by one amino acid substitution; CDR2-L of sequence 'DAS' or a sequence differing from 'DAS' by one amino acid substitution and CDR3-L of sequence SEQ ID NO: 258 or a sequence differing from SEQ ID NO: 258 by one amino acid substitution.

[0184] One or more individual amino acids may be altered by substitution, in particular by conservative substitution, in one or more of the above CDR sequences. Such an alteration may be intended for example to remove a glycosylation site or a deamidation site, in connection with humanization of the antibody.

[0185] Based on the alignments of the sequences of the VH and VL regions of "1E1-G5", "6D6-B8", "8B11-B7", "9F6-G3" anti-CD123 antibodies different amino acid substitutions were identified. Therefore, in one embodiment, an amino acid may be substituted: in CDR1-H at one or more of positions 1 and 7, for instance at position 7 of CDR1-H of sequence GYTFTDHI (SEQ ID NO:199) or GYTFTDYI (SEQ ID NO:273) for instance at position 1 and 5 of CDR1-H of sequence AYTFTDNI (SEQ ID NO: 255); and / or in CDR2-H, at one or more of positions 6 and 8, for instance at positions 6 and / or 8 of CDR2-H of sequence INPYSGGT (SEQ ID NO: 200) or INPYSDGT (SEQ ID NO: 274) or INPYSGGA (SEQ ID NO : 260); and / or in CDR1-L, at one or more of positions 1, 4 and 5, for example at position 1 or 1, 4 and 5 of CDR1-L of sequence KDIYSN (SEQ ID NO : 262) or EDIFNN (SEQ ID NO: 257); and / or in CDR2-L, at positions 3 of sequence 'DAN' or 'DAS'; and / or in CDR3-L, at one or more of positions 1 and 5, for instance at position 1 and / or 5 of CDR3-L of sequence HQYNIYPYT (SEQ ID NO: 258) or QQYNKYPYT (SEQ ID NO: 204) or HQYNNYPYT (SEQ ID NO : 263).

[0186] In a further embodiment, an amino acid is substituted: in CDR1-H at position 8, for instance at position 8 of CDR1-H of sequence GFSLTSYH (SEQ ID NO:265) or GFSLTSYS (SEQ ID NO: 248); and / or in CDR2-H at positions 3, for instance at positions 3 of CDR2-H of sequence MWSDGD (SEQ ID NO: 265) or MWNDGD (SEQ ID NO: 248); and / or in CDR3-H at positions 4 and 9, for instance at positions 4 and 9 of CDR3-H of sequence ARGDYSSYIYLWFAY (SEQ ID NO : 267) or ARGYYSSYLYLWFAY(SEQ ID NO : 269); and / or in CDR1-L, at one or more of positions 9 and 10, for example at position 9 and 10 of CDR1-L of sequence QSFLSSGDERNY (SEQ ID NO: 252) or QSFLSSGDGKNY (SEQ ID NO: 271).

[0187] According to an embodiment, the antibody comprises a) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 227, CDR2-H of sequence SEQ ID NO: 228, SEQ ID NO:353, SEQ ID NO:279, CDR3-H of sequence SEQ ID NO: 229 , and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 231, CDR2-L of sequence 'RDD' and CDR3-L of sequence SEQ ID NO: 232; or b) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 199, CDR2-H of sequence SEQ ID NO: 200, CDR3-H of sequence SEQ ID NO: 201 , and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 203, CDR2-L of sequence 'DAN' and CDR3-L of sequence SEQ ID NO: 204; or c) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 206, CDR2-H of sequence SEQ ID NO: 207, CDR3-H of sequence SEQ ID NO: 208 , and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 210, CDR2-L of sequence 'ETS' and CDR3-L of sequence SEQ ID NO: 211; or d) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 213, CDR2-H of sequence SEQ ID NO: 214, CDR3-H of sequence SEQ ID NO: 215, and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 217, CDR2-L of sequence 'NTN' and CDR3-L of sequence SEQ ID NO: 218; or e) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 220, CDR2-H of sequence SEQ ID NO: 221, CDR3-H of sequence SEQ ID NO: 222, and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 224, CDR2-L of sequence 'RVS' and CDR3-L of sequence SEQ ID NO: 225; or f) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 234, CDR2-H of sequence SEQ ID NO: 235, CDR3-H of sequence SEQ ID NO: 236 , and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 238, CDR2-L of sequence 'GAS' and CDR3-L of sequence SEQ ID NO: 239; or g) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 241, CDR2-H of sequence SEQ ID NO: 242, CDR3-H of sequence SEQ ID NO: 243, and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 245, CDR2-L of sequence 'YAS' and CDR3-L of sequence SEQ ID NO: 246; or h) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 248, CDR2-H of sequence SEQ ID NO: 249, CDR3-H of sequence SEQ ID NO: 250, and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 252, CDR2-L of sequence 'WAS' and CDR3-L of sequence SEQ ID NO: 253; or i) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 255, CDR2-H of sequence SEQ ID NO: 200, CDR3-H of sequence SEQ ID NO: 201 , and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 257, CDR2-L of sequence 'DAN' and CDR3-L of sequence SEQ ID NO: 258; or j) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 199, CDR2-H of sequence SEQ ID NO: 260, CDR3-H of sequence SEQ ID NO: 201 , and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 262, CDR2-L of sequence 'DAS' and CDR3-L of sequence SEQ ID NO: 263; or k) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 265, CDR2-H of sequence SEQ ID NO: 266, CDR3-H of sequence SEQ ID NO: 267 , and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 252, CDR2-L of sequence 'WAS' and CDR3-L of sequence SEQ ID NO: 253; or l) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 265, CDR2-H of sequence SEQ ID NO: 266, CDR3-H of sequence SEQ ID NO: 269, and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 271, CDR2-L of sequence 'WAS' and CDR3-L of sequence SEQ ID NO: 253; or m) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 273, CDR2-H of sequence SEQ ID NO: 274, CDR3-H of sequence SEQ ID NO: 201 , and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 276, CDR2-L of sequence 'DAS' and CDR3-L of sequence SEQ ID NO: 258.

[0188] The anti-CD123 antibody according to the invention is in particular a conventional antibody, in particular a conventional monoclonal antibody, or an antibody fragment, a bispecific or multispecific antibody.

[0189] The anti-CD123 antibody according to the invention in particular comprises or consists of an IgG, or a fragment thereof.

[0190] The invention also provides an anti-CD123 antibody as defined above further comprising at least the heavy chain variable domain and / or the light chain variable domain of one of the so-called anti-CD123 antibodies listed above.

[0191] Thus, the invention relates in particular to an anti-CD123 antibody, which comprises: a) a heavy chain variable domain of sequence SEQ ID NO: 226 or SEQ ID NO: 277 or SEQ ID NO: 278 or a sequence at least 85% identical thereto, and / or a light chain variable domain of sequence SEQ ID NO: 230, or a sequence at least 85% identical thereto; or b) a heavy chain variable domain of sequence SEQ ID NO: 198 or a sequence at least 85% identical thereto, and / or a light chain variable domain of sequence SEQ ID NO: 202, or a sequence at least 85% identical thereto; or c) a heavy chain variable domain of sequence SEQ ID NO: 205 or a sequence at least 85% identical thereto, and / or a light chain variable domain of sequence SEQ ID NO: 209, or a sequence at least 85% identical thereto; or d) a heavy chain variable domain of sequence SEQ ID NO: 212 or a sequence at least 85% identical thereto, and / or a light chain variable domain of sequence SEQ ID NO: 216, or a sequence at least 85% identical thereto; or e) a heavy chain variable domain of sequence SEQ ID NO: 219 or a sequence at least 85% identical thereto, and / or a light chain variable domain of sequence SEQ ID NO: 223, or a sequence at least 85% identical thereto; or f) a heavy chain variable domain of sequence SEQ ID NO: 233 or a sequence at least 85% identical thereto, and / or a light chain variable domain of sequence SEQ ID NO: 237, or a sequence at least 85% identical thereto; or g) a heavy chain variable domain of sequence SEQ ID NO: 240 or a sequence at least 85% identical thereto, and / or a light chain variable domain of sequence SEQ ID NO: 244, or a sequence at least 85% identical thereto; or h) a heavy chain variable domain of sequence SEQ ID NO: 247 or a sequence at least 85% identical thereto, and / or a light chain variable domain of sequence SEQ ID NO: 251, or a sequence at least 85% identical thereto; or i) a heavy chain variable domain of sequence SEQ ID NO: 254 or a sequence at least 85% identical thereto, and / or a light chain variable domain of sequence SEQ ID NO: 256, or a sequence at least 85% identical thereto; or j) a heavy chain variable domain of sequence SEQ ID NO: 259 or a sequence at least 85% identical thereto, and / or a light chain variable domain of sequence SEQ ID NO: 261, or a sequence at least 85% identical thereto; or k) a heavy chain variable domain of sequence SEQ ID NO: 264 or a sequence at least 85% identical thereto, and / or a light chain variable domain of sequence SEQ ID NO: 251, or a sequence at least 85% identical thereto; or l) a heavy chain variable domain of sequence SEQ ID NO: 268 or a sequence at least 85% identical thereto, and / or a light chain variable domain of sequence SEQ ID NO: 270, or a sequence at least 85% identical thereto; or m) a heavy chain variable domain of sequence SEQ ID NO: 272 or a sequence at least 85% identical thereto, and / or a light chain variable domain of sequence SEQ ID NO: 275, or a sequence at least 85% identical thereto.

[0192] For instance, the sequence of the variable domain of heavy or light chain may differ from the reference sequence SEQ ID NO: 226, 277, 278, 230, 198, 202, 205, 209, 212, 216, 219, 223, 233, 237, 240, 244, 247, 251, 254, 256, 259, 261, 264, 251, 268, 270, 272 or 275, as appropriate, by one or more amino acid substitution(s), in particular by one or more conservative amino acid substitution(s) and / or substitution(s) with canonical residues. In particular, the sequence of the variable domain of heavy or light chain may differ from the reference sequence SEQ ID NO: 226, 277, 278, 230, 198, 202, 205, 209, 212, 216, 219, 223, 233, 237, 240, 244, 247, 251, 254, 256, 259, 261, 264, 251, 268, 270, 272 or 275 by conservative amino acid substitution(s), only.

[0193] The sequence alterations as compared with sequence SEQ ID NO: 226, 277, 278, 230, 198, 202, 205, 209, 212, 216, 219, 223, 233, 237, 240, 244, 247, 251, 254, 256, 259, 261, 264, 251, 268, 270, 272 or 275 will in particular be present essentially in one or more of the framework regions, FR1-L, FR2-L, FR3-L, FR4-L and / or FR1-H, FR2-H, FR3-H, FR4-H.

[0194] In one embodiment, the anti-CD123 antibody of the invention and a fragment thereof is, respectively, a rat antibody and a fragment of a rat antibody.

[0195] The anti-CD123 antibody of the invention may also be a chimeric antibody, and in particular a rat / human antibody, e.g. an antibody comprising rat variable domains of heavy and light chains and a CH domain and a CL domain from a human antibody. The polypeptide may be a fragment of such an antibody. The anti-CD123 antibody may also be a humanized antibody or a fragment of a humanized antibody obtained by CDR-grafting or by the 4D method (US20110027266).

[0196] Accordingly, in one embodiment, the anti-CD123 antibody of the invention is a humanized antibody comprising : a) a heavy chain variable domain comprising of an amino acid sequence selected from the group consisting of SEQ ID NO: 280, SEQ ID NO: 281, SEQ ID NO: 282, SEQ ID NO: 283, SEQ ID NO: 284, SEQ ID NO: 285, SEQ ID NO: 286, SEQ ID NO: 287, SEQ ID NO: 288, SEQ ID NO: 289, SEQ ID NO: 290, SEQ ID NO: 291, SEQ ID NO: 301 and SEQ ID NO: 302; and b) a light chain variable domain comprising of an amino acid sequence selected from the group consisting of SEQ ID NO: 292, SEQ ID NO: 293, SEQ ID NO: 294, SEQ ID NO: 295, SEQ ID NO: 296, SEQ ID NO: 297, SEQ ID NO: 298, SEQ ID NO: 299, SEQ ID NO: 300, SEQ ID NO: 303, SEQ ID NO: 304 and SEQ ID NO: 305.

[0197] In one embodiment, the anti-CD123 antibody according to the invention comprises the three CDR sequences or the variable domain of the heavy chain, or the six CDR sequences or variable domains of the heavy and light chains of one of so-called anti-CD123 antibodies listed above.

[0198] The invention further refers to a fragment of the humanized anti-CD123 antibody as defined above. In one embodiment, the humanized anti-CD123 antibody described above is a chimeric antibody.

[0199] The anti-CD123 antibody according to the invention may also be a single domain antibody or a fragment thereof. In particular, a single domain antibody fragment may consist of a variable heavy chain (VHH) which comprises the CDR1-H, CDR2-H and CDR3-H of one of the antibodies described above. The CD123-antibody may also be a heavy chain antibody, i.e. an antibody devoid of light chain, which may or may not contain a CH1 domain.

[0200] The single domain antibody or a fragment thereof may also comprise the framework regions of a camelid single domain antibody, and optionally the constant domain of a camelid single domain antibody.

[0201] The anti-CD123 antibody according to the invention may also be an antibody fragment, in particular a humanized antibody fragment, selected from the group consisting of Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, and diabodies.

[0202] The CD123-antibody may also be a bispecific or multispecific antibody formed from at least one antibody fragment or at least one variable domains of the anti-CD123 antibody of the invention. Multispecific antibodies are polyvalent protein complexes as described for instance in EP 2 050 764 A1 or US 2005 / 0003403 A1.

[0203] The bispecific or multispecific CD123-antibodies according to the invention can have specificity for (a) extracellular domain of human or human and Macaca fascicularis CD123 targeted by one of the above described anti-CD123 antibodies and (b) at least one other antigen.

[0204] In a particular embodiment, the other antigen is CD3 and accordingly the resulting bispecific antibody is a CD3 / CD123 bispecific antibody. Conventional bi-specific antibodies can be produced by techniques that are known to the skilled in the art.

[0205] Antibodies and fragments thereof according to the invention can be used in an isolated (e.g., purified) from or contained in a vector, such as a membrane or lipid vesicle (e.g. a liposome).

[0206] In one further embodiment, the anti-CD123 antibody of the invention is used for the preparation of antibody-like binding protein of the invention further defined in the section" antibody-like binding protein".Antibody-like binding proteins

[0207] The inventors have generated several antibody-like binding proteins the so called "7G3x20G6", "7G3x4E7", "7G3x4B4", "7G3x18F5", "hz20G6x7G3", "7G3xhz4B4", "hz4B4x3E3" "hz20G6x7G3-TL4" and "hz20G6xhz7G3" antibody-like binding proteins, wherein the term "hz" denotes humanized antibodies. These antibody-like binding proteins have a CODV design, in particular a CODV-Fab or CODV-Ig design.

[0208] The "CODV format" in context of the present invention refers to the cross-over dual variable (CODV) configuration of bispecific antibodies or multispecific antibodies. The CODV format allows a interchangeability of variable domains with retention of folding and ultimate binding affinity.

[0209] The CODV format has been previously described in the international patent application WO2012 / 135345. Accordingly, in one embodiment, the antibody-like binding protein of the invention is in the CODV format as previously described in the international patent application WO2012 / 135345,

[0210] In one embodiment, the invention refers to an antibody-like binding protein in the CODV-Fab format. Accordingly, in one embodiment, the invention refers to an antibody-like binding protein comprising two polypeptide chains that form two antigen-binding sites, wherein a first polypeptide has a structure represented by the formula [I]:         V D1 -L 1 -V D2 -L 2 -C L      [I] and a second polypeptide has a structure represented by the formula [II]:         V D3 -L 3 -V D4 -L 4 -C H1      [II] wherein: V D1 is a variable domain of heavy or light chain of a first immunoglobulin; V D2 is a variable domain of heavy or light chain of a second immunoglobulin; V D3 is a variable domain of heavy or light chain of said second immunoglobulin; V D4 is a variable domain of heavy or light chain of said first immunoglobulin; C L is a light chain constant domain of an immunoglobulin; C H1 is a C H1 heavy chain constant domain of an immunoglobulin; L 1 , L 2 , L 3 , and L 4 are amino acid linkers; and wherein the first and the second polypeptide form a cross-over light chain-heavy chain pair, and wherein VD1 and VD2 are both either variable domains of light chains, or variable domains of heavy chains, and VD3 and VD4 are both variable domains of heavy chains if VD1 and VD2 are variable domains of light chains, or VD3 and VD4 are both variable domains of light chains if VD1 and VD2 are variable domains of heavy chains.

[0211] Adding a Fc domain to the antibody-like binding protein in the CODV-Fab further stabilizes the antibody-like binding protein. More precisely, adding a Fc domain to the polypeptide of formula (II) of antibody-like binding protein in the CODV-Fab increases the half-life of the antibody-like binding protein and thus improves the pharmacokinetic profile of the antibody-like binding protein. Adding one Fc region to the CODV-Fab results in dimerization of polypeptides containing the Fc domain and the resulting antibody-like binding protein is an antibody-like binding protein in the CODV-Ig format.

[0212] The invention therefore further refers to an antibody like binding protein in the CODV-Ig format.

[0213] Therefore, the invention further refers to an antibody-like binding protein comprising four polypeptide chains that form four antigen-binding sites, wherein two polypeptide chains have a structure represented by the formula [I]:         V D1 -L 1 -V D2 -L 2 -C L      [I] and two polypeptide chains have a structure represented by the formula [III]:         V D3 -L 3 -V D4 -L 4 -C H1 -F c      [III] wherein: V D1 is a variable domain of heavy or light chain of a first immunoglobulin; V D2 is a variable domain of heavy or light chain of a second immunoglobulin; V D3 is a variable domain of heavy or light chain of said second immunoglobulin; V D4 is a variable domain of heavy or light chain of said first immunoglobulin; C L is a light chain constant domain of an immunoglobulin; C H1 is a C H1 heavy chain constant domain of an immunoglobulin; F c is the immunoglobulin hinge region and CH 2 , CH 3 immunoglobulin heavy chain constant domains of an immunoglobulin; L 1 , L 2 , L 3 , and L 4 are amino acid linkers; and wherein the polypeptides of formula I and the polypeptides of formula III form a cross-over light chain-heavy chain pair, and wherein VD1 and VD2 are both either variable domains of light chains, or variable domains of heavy chains, and VD3 and VD4 are both variable domains of heavy chains if VD1 and VD2 are variable domains of light chains, or VD3 and VD4 are both variable domains of light chains if VD1 and VD2 are variable domains of heavy chains.

[0214] In said CODV-Ig format, the two polypeptide chains having a structure represented by the formula [III] dimerize through their F c domains.

[0215] In a further embodiment, a first F c domain is added to the polypeptide of formula [II] of antibody-like binding protein CODV-Fab, and a second F c domain (called F c2 ) is added to the polypeptide of formula [I] of the antibody-like binding protein CODV-Fab. Furthermore, in the same embodiment a linker L 5 is present between C L and the F c2 domain of the polypeptide chains of formula [I] resulting in the polypeptide chains of formula [IV].

[0216] Accordingly, the invention further refers to an antibody-like binding protein comprising two polypeptide chains that form two antigen-binding sites, wherein one polypeptide chain has a structure represented by the formula [IV]:         V D1 -L 1 -V D2 -L 2 -C L -L 5 -F c2      [IV] and one polypeptide chain has a structure represented by the formula [III]:         V D3 -L 3 -V D4 -L 4 -C H1 -F c      [III] wherein: V D1 is a variable domain of heavy or light chain of a first immunoglobulin; V D2 is a variable domain of heavy or light chain of a second immunoglobulin; V D3 is a variable domain of heavy or light chain of said second immunoglobulin; V D4 is a variable domain of heavy or light chain of said first immunoglobulin; C L is a light chain constant domain of an immunoglobulin; C H1 is a C H1 heavy chain constant domain of an immunoglobulin; F c is the immunoglobulin hinge region and CH 2 , CH 3 immunoglobulin heavy chain constant domains of an immunoglobulin; F c2 is the immunoglobulin hinge region and CH 2 , CH 3 immunoglobulin heavy chain constant domains of an immunoglobulin; L 1 , L 2 , L 3 , L 4 and L 5 are amino acid linkers; and wherein the polypeptide of formula [IV] and the polypeptide of formula [III] form a cross-over light chain-heavy chain pair, and wherein VD1 and VD2 are both either variable domains of light chains, or variable domains of heavy chains, and VD3 and VD4 are both variable domains of heavy chains if VD1 and VD2 are variable domains of light chains, or VD3 and VD4 are both variable domains of light chains if VD1 and VD2 are variable domains of heavy chains.

[0217] This CODV format, in which the polypeptide chains represented by the formulae [III] and [IV] dimerize through their respective F c2 and F c regions, is herein called CODV-Fab-TL.

[0218] In another embodiment of the CODV-Fab, a first F c domain is added to the polypeptide chain represented by formula [II] (resulting in formula [III]), and the antibody-like binding protein comprise a third polypeptide chain comprising, or consisting of a second F c domain (called F c3 ).

[0219] The invention further refers to an antibody-like binding protein which comprises three polypeptide chains that form two antigen-binding sites, wherein a first polypeptide has a structure represented by the formula [I]:         V D1 -L 1 -V D2 -L 2 -C L      [I] a second polypeptide has a structure represented by the formula [III]:         V D3 -L 3 -V D4 -L 4 -C H1 -F c      [III] a third polypeptide F c3 (also called Fc stump") which is the immunoglobulin hinge region and CH 2 , CH 3 immunoglobulin heavy chain constant domains of an immunoglobulin; wherein V D1 is a variable domain of heavy or light chain of a first immunoglobulin; V D2 is a variable domain of heavy or light chain of a second immunoglobulin; V D3 is a variable domain of heavy or light chain of said second immunoglobulin; V D4 is a variable domain of heavy or light chain of said first immunoglobulin; C L is a light chain constant domain of an immunoglobulin; C H1 is a C H1 heavy chain constant domain of an immunoglobulin; F c is the immunoglobulin hinge region and CH 2 , CH 3 immunoglobulin heavy chain constant domains of an immunoglobulin; L 1 , L 2 , L 3 , and L 4 are amino acid linkers; and wherein the polypeptide of formula [I] and the polypeptide of formula [III] form a cross-over light chain-heavy chain pair, and wherein V D1 and V D2 are both either variable domains of light chains, or variable domains of heavy chains, and V D3 and V D4 are both variable domains of heavy chains if V D1 and V D2 are variable domains of light chains, or V D3 and V D4 are both variable domains of light chains if V D1 and V D2 are variable domains of heavy chains; and wherein the polypeptide of formula [III] heterodimerizes with the third polypeptide through its F c domain.

[0220] Accordingly, in said embodiment, the so-called "Fc stump" (F c3 ) heterodimerizes with the Fc region of the polypeptide according to formula III. This CODV format is herein called CODV-Fab-OL. This construct avoids that the CODV-Fab form aggregates.

[0221] In one embodiment of the CODV-Fab-OL, both F c and F c3 are immunoglobulin variants in which the CH3 domain has been modified: each of F c and F c3 has been genetically engineered at the CH3-CH3 interface to promote heteromultimer formation according to the so-called "Knob-into-Hole" technology which has been described in patents US5731168 and US8216805, notably,

[0222] Accordingly, in an embodiment the CH3 domain of one of F c and F c3 contains the mutations Y349C, T366S, L368A, and Y407V, while the CH3 domain of the other of F c and F c3 contains the mutations S354C and T366W (amino acid position being indicated by reference to an IgG1 sequence).

[0223] Examples of suitable F c and F c3 pairs include the pair SEQ ID NO: 396 (F c ) and SEQ ID NO: 397 (F c3 ), and the pair SEQ ID NO: 394 (F c ) and SEQ ID NO: 398 (F c3 ).

[0224] In one embodiment of the invention, the first immunoglobulin or the second immunoglobulin is one anti-CD3 antibody as defined in the section «anti-CD3 antibodies» above.

[0225] In another embodiment of the invention, the first immunoglobulin or the second immunoglobulin is one anti-CD123 antibody as defined in the section «anti-CD123 antibodies» above.

[0226] According to one embodiment of the invention, V D1 and V D2 of polypeptide of formula I or formula [IV] are both either variable domains of light chains, or variable domains of heavy chains, and V D3 and V D4 of polypeptide II or III are both variable domains of heavy chains or of light chains. This interchangeability is also referred to as "swapability" and thus determines the cross-over dual variable (CODV) configuration of the antibody-like binding proteins of the invention.

[0227] According to the above definition, V D1 and V D4 are variable domains of heavy or light chain of a first immunoglobulin and V D2 and V D3 are variable domains of heavy or light chain of a second immunoglobulin, V D1 and V D4 are therefore to be considered as cognate domains as well as V D2 and V D3 .

[0228] Accordingly, the term "cross-over" refers to the swapped alignement of V D1 or V D2 of polypeptide of formula [I] or formula [IV] with respect to its cognate variable domain V D4 or V D3 of polypeptide of formula [II] or formula [III].

[0229] In one particular embodiment, V D1 and V D2 are light chain variable domains and V D3 and V D4 are heavy chain variable domains.

[0230] The antibody-like binding proteins of the invention may be prepared using domains or sequences obtained or derived from any human or non-human antibody, including, for example, human, rat, or humanized antibodies.

[0231] In one embodiment, the immunoglobulin is an IgG immunoglobulin.

[0232] Accordingly, in one embodiment, C L is a light chain constant domain of an IgG immunoglobulin. In a further embodiment, C H1 is a C H1 heavy chain constant domain of an IgG immunoglobulin.

[0233] In one embodiment, the antibody-like binding protein of the invention may be prepared using domains or sequences of the anti-CD3 antibody and anti-CD123 antibody herein described.

[0234] The term "linker" as used herein refers to one or more amino acid residues inserted between immunoglobulin domains to provide sufficient mobility for the domains of the light and heavy chains to fold into cross over dual variable region immunoglobulins. In some embodiments, a linker consists of 0 amino acid meaning that the linker is absent. A linker is inserted at the transition between variable domains or between variable and constant domains, respectively, at the sequence level. The transition between domains can be identified because the approximate size of the immunoglobulin domains is well understood. The precise location of a domain transition can be determined by locating peptide stretches that do not form secondary structural elements such as beta-sheets or alpha-helices as demonstrated by experimental data or as can be assumed by techniques of modeling or secondary structure prediction. The linkers described in context of the invention are the linkers L 1 , L 2 , L 3 , L 4 and L 5 . L 1 is located between the N-terminal V D1 domain and the V D2 domain; L 2 is located between the V D2 and the C-terminal C L domain. The linkers L 3 and L 4 are located on polypeptide as defined according to formula II or III of the antibody-like-proteins. More precisely, L 3 is located between the N-terminal V D3 and the V D4 domains and L 4 is located between the V D4 and the C-terminal C H1 -Fc domains. L 5 is located between C L and the N-terminal F c2 . The linkers L 1 , L 2 , L 3 , L 4 and L 5 are independent, but in some embodiments, they have the same sequence and / or length.

[0235] In some antibody-like binding proteins of the invention, the length of L 3 is at least twice the length of L 1 . In other antibody-like binding proteins of the invention, the length of L 4 is at least twice the length of L 2 . In some antibody-like binding proteins of the invention, the length of L 1 is at least twice the length of L 3 . In other antibody-like binding proteins of the invention, the length of L 2 is at least twice the length of L 4 .

[0236] In one embodiment, the linker L 1 , L 2 , L 3 and L 4 comprise 0 to 20 amino acids. In one embodiment, L 5 comprises 0 to 10 amino acids.

[0237] In some antibody-like binding proteins of the invention, L 1 is 3 to 12 amino acid residues in length, L 2 is 3 to 14 amino acid residues in length, L 3 is 1 to 8 amino acid residues in length, and L 4 is 1 to 3 amino acid residues in length. In other antibody-like binding proteins, L 1 is 5 to 10 amino acid residues in length, L 2 is 5 to 8 amino acid residues in length, L 3 is 1 to 5 amino acid residues in length, and L 4 is 1 to 2 amino acid residues in length. In a preferred antibody-like binding protein, L 1 is 7 amino acid residues in length, L 2 is 5 amino acid residues in length, L 3 is 1 amino acid residues in length, and L 4 is 2 amino acid residues in length.

[0238] In some antibody-like binding proteins of the invention, L 1 is 1 to 3 amino acid residues in length, L 2 is 1 to 4 amino acid residues in length, L 3 is 2 to 15 amino acid residues in length, and L 4 is 2 to 15 amino acid residues in length. In other antibody-like binding proteins, Li is 1 to 2 amino acid residues in length, L 2 is 1 to 2 amino acid residues in length, L 3 is 4 to 12 amino acid residues in length, and L 4 is 2 to 12 amino acid residues in length. In a preferred antibody-like binding protein, L 1 is 1 amino acid residue in length, L 2 is 2 amino acid residues in length, L 3 is 7 amino acid residues in length, and L 4 is 5 amino acid residues in length.

[0239] In some antibody-like binding proteins of the invention, L 1 , L 3 , or L 4 may be equal to zero. However, in antibody-like binding proteins wherein L 3 , or L 4 is equal to zero, the corresponding transition linker between the variable region and constant region or between the dual variable domains on the other chain cannot be zero. In some embodiments, L 1 is equal to zero and L 3 is 2 or more amino acid residues, L 3 is equal to zero and L 1 is equal to 1 or more amino acid residues, or L 4 is equal to 0 and L 2 is 3 or more amino acid residues.

[0240] In some antibody-like binding proteins of the invention, at least one of the linkers selected from the group consisting of L 2 , L 3 , and L 4 contains at least one cysteine residue.

[0241] Examples of suitable linkers include a single glycine, threonine or serine residue; a dipeptide such as a diglycine peptide, histidine-threonine peptide or glycine-serine dipeptide; a tripeptide with three glycines, the tripeptide Thr-His-Thr, the tripeptide Gly-Gly-Ser; a peptide with four glycine residues; a peptide with five glycine residues; a peptide with six glycine residues; a peptide with seven glycine residues; a peptide with eight glycine residues. Other combinations of amino acid residues may be used such as the peptide Gly-Gly-Gly-Ser (SEQ ID NO: 354), the peptide Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 344), the peptide Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 355), the peptide Gly-Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 356), the peptide Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 357), the peptide Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 358), and the peptide Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 345). Other suitable linkers include a single Ser, and Val residue; the dipeptide Arg-Thr, Gin-Pro, Ser-Ser, Thr-Lys, and Ser-Leu; Lys-Thr-His-Thr (SEQ ID NO: 359); Lys-Thr-His-Thr-Ser (SEQ ID NO: 360); Asp-Lys-Thr-His-Thr-Ser (SEQ ID NO: 361); Asp-Lys-Thr-His-Thr-Ser-Pro (SEQ ID NO: 362); Ser-Asp-Lys-Thr-His-Thr-Ser-Pro (SEQ ID NO: 363); Ser-Asp-Lys-Thr-His-Thr-Ser-Pro-Pro (SEQ ID NO: 364); Lys-Ser-Asp-Lys-Thr-His-Thr-Ser-Pro-Pro-Ser (SEQ ID NO: 365); Pro-Lys-Ser-Asp-Lys-Thr-His-Thr-Ser-Pro-Pro-Ser (SEQ ID NO: 366); Pro-Lys-Ser-Asp-Lys-Thr-His-Thr-Ser-Pro-Pro-Ser-Pro (SEQ ID NO: 367); Glu-Pro-Lys-Ser-Asp-Lys-Thr-His-Thr-Ser-Pro-Pro-Ser-Pro (SEQ ID NO: 368); Glu-Pro-Lys-Ser-Asp-Lys-Thr-His-Thr-Ser-Pro-Pro-Ser-Pro-Gly (SEQ ID NO: 369); Gly-Glu-Pro-Lys-Ser-Asp-Lys-Thr-His-Thr-Ser-Pro-Pro-Ser-Pro-Gly (SEQ ID NO: 370); Gly-Glu-Pro-Lys-Ser-Asp-Lys-Thr-His-Thr-Ser-Pro-Pro-Ser-Pro-Gly-Gly (SEQ ID NO: 371); Gly-Gly-Glu-Pro-Lys-Ser-Asp-Lys- Thr-His- Thr-Ser-Pro-Pro-Ser-Pro-Gly-Gly (SEQ ID NO: 372); Gly-Gly-Glu-Pro-Lys-Ser-Asp-Lys-Thr-His-Thr-Ser-Pro-Pro-Ser-Pro-Gly-Gly-Gly (SEQ ID NO: 373); Gly-Gly-Gly-Glu-Pro-Lys-Ser-Asp-Lys-Thr-His-Thr-Ser-Pro-Pro-Ser-Pro-Gly-Gly-Gly (SEQ ID NO: 374); Thr-Val-Ala-Ala-Pro (SEQ ID NO: 346), Gln-Pro-Lys-Ala-Ala (SEQ ID NO: 347), Gln-Arg-Ile-Glu-Gly (SEQ ID NO: 348); Ala-Ser-Thr-Lys-Gly-Pro-Ser (SEQ ID NO: 349), Arg-Thr-Val-Ala-Ala-Pro-Ser (SEQ ID NO: 350), Gly-Gln-Pro-Lys-Ala-Ala-Pro (SEQ ID NO: 307), Thr-Lys-Gly-Pro-Ser (SEQ ID NO: 309), His-Ile-Asp-Ser-Pro-Asn-Lys (SEQ ID NO: 351), and Gly-Gly-Ser-Gly-Ser-Ser-Gly-Ser-Gly-Gly (SEQ ID NO: 389). The examples listed above are not intended to limit the scope of the invention in any way, and linkers comprising randomly selected amino acids selected from the group consisting of valine, leucine, isoleucine, serine, threonine, lysine, arginine, histidine, aspartate, glutamate, asparagine, glutamine, glycine, and proline have been shown to be suitable in the antibody-like binding proteins of the invention.

[0242] The identity and sequence of amino acid residues in the linker may vary depending on the type of secondary structural element necessary to achieve in the linker. For example, glycine, serine, and alanine are best for linkers having maximum flexibility. Some combination of glycine, proline, threonine, and serine are useful if a more rigid and extended linker is necessary. Any amino acid residue may be considered as a linker in combination with other amino acid residues to construct larger peptide linkers as necessary depending on the desired properties.

[0243] In one embodiment, the linker L 1 is of sequence Gly-Gln-Pro-Lys-Ala-Ala-Pro (SEQ ID NO: 307), the linker L 2 is of sequence Thr-Lys-Gly-Pro-Ser (SEQ ID NO: 309), the linker L 3 is of sequence 'S' and the linker L 4 is of sequence 'RT'.

[0244] In a further embodiment, the sequences of linkers L 1 , L 2 , L 3 , and L 4 are selected from the grop consisting of threonine; a dipeptide such as a histidine-threonine peptide; the tripeptide Thr-His-Thr, Lys-Thr-His-Thr (SEQ ID NO: 359); Lys-Thr-His-Thr-Ser (SEQ ID NO: 360); Asp-Lys-Thr-His-Thr-Ser (SEQ ID NO: 361); Asp-Lys-Thr-His-Thr-Ser-Pro (SEQ ID NO: 362); Ser-Asp-Lys-Thr-His-Thr-Ser-Pro (SEQ ID NO: 363); Ser-Asp-Lys-Thr-His-Thr-Ser-Pro-Pro (SEQ ID NO: 364); Lys-Ser-Asp-Lys-Thr-His-Thr-Ser-Pro-Pro-Ser (SEQ ID NO: 365); Pro-Lys-Ser-Asp-Lys-Thr-His-Thr-Ser-Pro-Pro-Ser (SEQ ID NO: 366); Pro-Lys-Ser-Asp-Lys-Thr-His-Thr-Ser-Pro-Pro-Ser-Pro (SEQ ID NO: 367); Glu-Pro-Lys-Ser-Asp-Lys-Thr-His-Thr-Ser-Pro-Pro-Ser-Pro (SEQ ID NO: 368); Glu-Pro-Lys-Ser-Asp-Lys-Thr-His-Thr-Ser-Pro-Pro-Ser-Pro-Gly (SEQ ID NO: 369); Gly-Glu-Pro-Lys-Ser-Asp-Lys-Thr-His-Thr-Ser-Pro-Pro-Ser-Pro-Gly (SEQ ID NO: 370); Gly-Glu-Pro-Lys-Ser-Asp-Lys-Thr-His-Thr-Ser-Pro-Pro-Ser-Pro-Gly-Gly (SEQ ID NO: 371); Gly-Gly-Glu-Pro-Lys-Ser-Asp-Lys- Thr-His- Thr-Ser-Pro-Pro-Ser-Pro-Gly-Gly (SEQ ID NO: 372); Gly-Gly-Glu-Pro-Lys-Ser-Asp- Lys- Thr-His- Thr-Ser-Pro-Pro-Ser-Pro-Gly-Gly-Gly (SEQ ID NO: 373) and Gly-Gly-Gly-Glu-Pro-Lys-Ser-Asp-Lys-Thr-His-Thr-Ser-Pro Pro-Ser-Pro-Gly-Gly-Gly (SEQ ID NO: 374).In one embodiment the sequence of linker L 5 is selected from the group consisting of a single serine residue, a dipeptide such as a glycine-serine dipeptide; a tripeptide Gly-Gly-Ser, the peptide Gly-Gly-Gly- Ser (SEQ ID NO: 354), the peptide Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 344), the peptide Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 355), the peptide Gly-Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 356), the peptide Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 357), the peptide Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 358),the peptide Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 345), and the peptide Gly-Gly-Ser-Gly-Ser-Ser-Gly-Ser-Gly-Gly (SEQ ID NO: 389).

[0245] The term "Fc domain" as used herein encompasses native Fc and Fc variants and sequences as defined above. As with Fc variants and native Fc molecules, the term "Fc domain" includes molecules in monomeric or multimeric form, whether digested from whole antibody or produced by other means.

[0246] The term "native Fc" as used herein refers to a molecule comprising the sequence of a non-antigen-binding fragment resulting from digestion of an antibody or produced by other means, whether in monomeric or multimeric form, and can contain the hinge region. The original immunoglobulin source of the native Fc is, in particular, of human origin and can be any of the immunoglobulins, although IgGI and IgG2 are preferred. Native Fc molecules are made up of monomeric polypeptides that can be linked into dimeric or multimeric forms by covalent (i.e., disulfide bonds) and non-covalent association. The number of intermolecular disulfide bonds between monomeric subunits of native Fc molecules ranges from 1 to 4 depending on class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgGI, IgG2, IgG3, IgAl, and IgGA2). One example of a native Fc is a disulfide-bonded dimer resulting from papain digestion of an IgG. The term "native Fc" as used herein is generic to the monomeric, dimeric, and multimeric forms.

[0247] The term "Fc variant" as used herein refers to a molecule or sequence that is modified from a native Fc but still comprises a binding site for the salvage receptor, FcRn (neonatal Fc receptor). Exemplary Fc variants, and their interaction with the salvage receptor, are known in the art. Thus, the term "Fc variant" can comprise a molecule or sequence that is humanized from a non-human native Fc. Furthermore, a native Fc comprises regions that can be removed because they provide structural features or biological activity that are not required for the antibody-like binding proteins of the invention. Thus, the term "Fc variant" comprises a molecule or sequence that lacks one or more native Fc sites or residues, or in which one or more Fc sites or residues has be modified, that affect or are involved in: (1) disulfide bond formation, (2) incompatibility with a selected host cell, (3) N-terminal heterogeneity upon expression in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to an Fc receptor other than a salvage receptor, or (7) antibody-dependent cellular cytotoxicity (ADCC).

[0248] In some embodiments, when the antibody like binding protein contains two F c domains, i.e. in the CODV-Ig (F c and F c2 ), CODV-Fab-TL (two F c domains), and CODV-Fab-OL (F c and F c3 ), the two F c domains are of the same immunoglobulin isotype or isotype subclass. Accordingly, in some embodiments both F c and F c2 of CODV-Ig, or both F c domains of CODV-Fab-TL, or both F c and F c ; of CODV-Fab-OL are of the IgG1 subclass, or of the IgG2 subclass, or of the IgG3 subclass, or of the IgG4 subclass.

[0249] All antibody like binding proteins as described herein have no effector function. This means that when the antibody like binding protein contains one or more F c domain(s) (i.e. F c in formula [III], F c2 in formula [IV][ and / or F c3 ) of the IgG1 subclass, said on or more F c domain(s) of IgG1 backbone contain(s) a double mutation L234A and L235A (so-called "LALA mutation") which abolishes Fc effector function. The Fc double mutant L234A and L235A does not bind FcyR or C1q, and both ADCC and CDC functions of the Fc domain of IgG1 subclass are abolished (Hezareh, M. et al.,J Virol. 2001 Dec; 75(24): 12161-12168).

[0250] In one example, the F c region comprises the amino acid sequence SEQ ID NO: 330, SEQ ID NO: 394, or SEQ ID NO: 396. In one embodiment, the F C2 region comprises two amino acid exchanges within the CH3 domain H435R and Y436F, as described in Jendeberg, L. et al. (1997, J. Immunological Meth. 201: 25-34). Accordingly, in one embodiment the F C2 region comprises the amino acid sequence SEQ ID NO: 327. In another embodiment, the F C2 region comprises the amino acid sequence SEQ ID NO: 392.

[0251] The so-called CODV-Fab "7G3x20G6" antibody-like binding protein comprises: one polypeptide according to formula I of the amino acid sequence DVVMTQTPVSLSVSLGGQVSISCRSSQSLVHNNGNTYLSWYLQKPGQSPQSLIYKVSN RFSGFSDRFSGSGSGTDFTLKISRVDPDDLGVYYCGQGTQYPFTFGSGTKLEIKGQPK AAP DFVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYLQKPGQPPKLLIY WASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPYTFGGGTKLEIK TKGPSRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 306, linkers are indicated in bold and underlined) comprising V D1 of sequence SEQ ID NO: 9, L 1 of sequence SEQ ID NO: 307, V D2 of sequence SEQ ID NO: 308, L 2 of sequence SEQ ID NO: 309 and C L of sequence SEQ ID NO: 310, and one polypeptide according to formula II of the amino acid sequence EVQLQQSGPELVKPGASVKMSCKASGYTFTOYYMKWVKQSHGKSLEWIGOIIPSNGA TFYNQKFKGKATLTVDRSSSTAYMHLNSLTSEDSAVYYCTRSHLLRASWFAYWGQGT LVTVSASEVQLVETGGSLVQPGKSLKLTCATSGFTFTKAWMHWVRQSPEKQLEWVAQ IKDKSNSYATYYAESVKGRFTISRDDSKSTIYLQMNSLKEEDTAIYYCRGVYYALSPFDY WGQGVMVTVSSRTASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV (SEQ ID NO: 311, linkers are indicated in bold and underlined) comprising V D3 of sequence SEQ ID NO: 312, L 3 of the amino acid sequence 'S', V D4 of sequence SEQ ID NO: 5, L 4 of the amino acid sequence 'RT' and C H1 of sequence SEQ ID NO: 313.

[0252] The so-called CODV-Fab "7G3x4E7" antibody-like binding protein comprises: one polypeptide according to formula I of the amino acid sequence DVVMTQTPVSLSVSLGGQVSISCRSSQSLEHNNGNTYLSWYLQKPGQSPQPLIYKVSN RFSGISDRFSGSGSGTDFTLKISRVEPDDLGVYYCGQGTQYPFTFGPGTKLELKGQPK AAP DFVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYLQKPGQPPKLLIY WASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPYTFGGGTKLEIK TKGPSRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 314, linkers are indicated in bold and underlined) comprising V D1 of sequence SEQ ID NO: 21, L 1 of sequence SEQ ID NO: 307, V D2 of sequence SEQ ID NO: 308, L 2 of sequence SEQ ID NO: 309 and C L of sequence SEQ ID NO: 310, and one polypeptide according to formula II of the amino acid sequence EVQLQQSGPELVKPGASVKMSCKASGYTFTDYYMKWVKQSHGKSLEWIGDIIPSNGA TFYNQKFKGKATLTVDRSSSTAYMHLNSLTSEDSAVYYCTRSHLLRASWFAYWGQGT LVTVSASEVQVVETGGSLVQPGKSLKLTCATSGFTFSNAWMHWVRQSPEKQLEWVA QIKDKSNNYATYYAESLKGRFTISRDDPKRSIYLQMNSLREEDTAIYYCRYVHYGIGYA MOAWGQGTSVTVSSRTASTKGPSVFPLAPSSKSTSGGTAALGCLVKOYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV (SEQ ID NO: 315, linkers are indicated in bold and underlined) comprising V D3 of sequence SEQ ID NO: 312, L 3 of the amino acid sequence 'S', V D4 of sequence SEQ ID NO: 18, L 4 of the amino acid sequence 'RT' and C H1 of sequence SEQ ID NO: 313.

[0253] The so-called CODV-Fab "7G3x4B4" antibody-like binding protein comprises: one polypeptide according to formula I of the amino acid sequence DVVMTQTPVSLSVSLGGQVSISCRSSQSLVHDNGNTYLSWSLQRPGQSPQVLIYKVS NRFSGTSORFTGSGSGTOFTLKISRVEP OOLGVYYCGQGTQYPFTFGSGTKLEIKGQP KAAP DFVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYLQKPGQPPKLL IYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPYTFGGGTKLEI KTKGPSRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 316, linkers are indicated in bold and underlined) comprising V D1 of sequence SEQ ID NO: 16, L 1 of sequence SEQ ID NO: 307, V D2 of sequence SEQ ID NO: 308, L 2 of sequence SEQ ID NO: 309 and C L of sequence SEQ ID NO: 310, and one polypeptide according to formula II of the amino acid sequence EVQLQQSGPELVKPGASVKMSCKASGYTFTDYYMKWVKQSHGKSLEWIGDIIPSNGA TFYNQKFKGKATLTVDRSSSTAYMHLNSLTSEDSAVYYCTRSHLLRASWFAYWGQGT LVTVSASEVQLVETGGRLVQPGRSLKLTCATSGFTFSNAWMHWVRQSPEKQLEWVA QIKARSNNYATYYAESVKGRFTISRDDSKSTIYLQMNSLKEEDTAIYYCRGTYYASKPF DYWGQGVMVTVSSRTASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV (SEQ ID NO: 317, linkers are indicated in bold and underlined) comprising V D3 of sequence SEQ ID NO: 312, L 3 of the amino acid sequence 'S', V D4 of sequence SEQ ID NO: 12, L 4 of the amino acid sequence 'RT' and C H1 of sequence SEQ ID NO: 313.

[0254] The so-called CODV-Fab "7G3x18F5" antibody-like binding protein comprises: one polypeptide according to formula I of the amino acid sequence DVLMTQTPVSLSVSLGGQVSISCRSSQSLVHTNGNTYLSWYLQKPGQSPQLLIYKVSN RLSGISDRFSGSGSGTDFTLKISRVEPDDLGVYYCGQGTHYPFTFGAGTKLELKGQPK AAP DFVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYLQKPGQPPKLLIY WASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPYTFGGGTKLEIK TKGPSRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 318, linkers are indicated in bold and underlined) comprising V D1 of sequence SEQ ID NO: 26, L 1 of sequence SEQ ID NO: 307, V D2 of sequence SEQ ID NO: 308, L 2 of sequence SEQ ID NO: 309 and C L of sequence SEQ ID NO: 310, and one polypeptide according to formula II of the amino acid sequence EVQLQQSGPELVKPGASVKMSCKASGYTFTDYYMKWVKQSHGKSLEWIGDIIPSNGA TFYNQKFKGKATLTVDRSSSTAYMHLNSLTSEDSAVYYCTRSHLLRASWFAYWGQGT LVTVSASEVQVVETGGSLVQPGKSLKLTCATSGFTFTNAWMHWVRRSPEKQLEWVA QIKDKSNNYATYYAESVKGRFTISRDDSKSSIYLQMNSLKEEDTAIYYCRYVHYRFAYAL DAWGRGTSVSVSSRTASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV (SEQ ID NO: 319, linkers are indicated in bold and underlined) comprising V D3 of sequence SEQ ID NO: 312, L 3 of the amino acid sequence 'S', V D4 of sequence SEQ ID NO: 23, L 4 of the amino acid sequence 'RT' and C H1 of sequence SEQ ID NO: 313.

[0255] The so-called CODV-Fab "hz20G6x7G3" antibody-like binding protein comprises: one polypeptide according to formula I of the amino acid sequence DFVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYLQKPGQPPKLLIYWA STRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPYTFGGGTKLEIKGQ PKAAPDIVMTQTPLSLSVTPGQPASISCKSSQSLVHNNGNTYLSWYLQKPGQSPQSLI YKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEI KTKGPSRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 320, linkers are indicated in bold and underlined) comprising V D1 of sequence SEQ ID NO: 308, L 1 of sequence SEQ ID NO: 307, V D2 of sequence SEQ ID NO: 143, L 2 of sequence SEQ ID NO: 309 and C L of sequence SEQ ID NO: 310, and one polypeptide according to formula II of the amino acid sequence QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSN SYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQ GTLVTVSSSEVQLQQSGPELVKPGASVKMSCKASGYTFTDYYMKWVKQSHGKSLEWI GDIIPSNGATFYNQKFKGKATLTVDRSSSTAYMHLNSLTSEDSAVYYCTRSHLLRASWF AYWGQGTLVTVSARTASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV (SEQ ID NO: 321, linkers are indicated in bold and underlined) comprising V D3 of sequence SEQ ID NO: 138, L 3 of the amino acid sequence 'S', V D4 of sequence SEQ ID NO: 312, L 4 of the amino acid sequence 'RT' and C H1 of sequence SEQ ID NO: 313.

[0256] The so-called CODV-Fab "7G3xhz4B4" antibody-like binding protein comprises: one polypeptide according to formula I of the amino acid sequence DVVMTQTPVSLSVSVGGRVSISCRSSQSLVHDNGNTYLSWSLQKPGKSPKVLIYKVSN RFSGVSSRFTGSGSGTDFTLKISSVQPDDLGVYYCGQGTQYPFTFGSGTKLEIKGQPK AAP DFVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYLQKPGQPPKLLIY WASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPYTFGGGTKLEIK TKGPS RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 322, linkers are indicated in bold and underlined) comprising V D1 of sequence SEQ ID NO: 158, L 1 of sequence SEQ ID NO: 307, V D2 of sequence SEQ ID NO: 308, L 2 of sequence SEQ ID NO: 309 and C L of sequence SEQ ID NO: 310, and one polypeptide according to formula II of the amino acid sequence EVQLQQSGPELVKPGASVKMSCKASGYTFTDYYMKWVKQSHGKSLEWIGDIIPSNGA TFYNQKFKGKATLTVDRSSSTAYMHLNSLTSEDSAVYYCTRSHLLRASWFAYWGQGT LVTVSASQVQLVETGGGLVKPGQSLKLTCATSGFTFSNAWMHWVRQSPEKGLEWVA QIKARSNNYATYYAESVKGRFTISRDDSKSTIYLQMNSLTPEDTAIYYCRGTYYASKPFD YWGQGVMVTVSSRTASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV (SEQ ID NO: 323, linkers are indicated in bold and underlined) comprising V D3 of sequence SEQ ID NO: 312, L 3 of the amino acid sequence 'S', V D4 of sequence SEQ ID NO: 171, L 4 of the amino acid sequence 'RT' and C H1 of sequence SEQ ID NO: 313.

[0257] The so-called CODV-Fab "hz4B4x3E3" antibody-like binding protein comprises: one polypeptide according to formula I of the amino acid sequence QFVLTQPNSVSTNLGSTVKLSCKRNTGNIGSNYVNWYQQHEGRSPTTMIYRDDKRPD GVPDRFSGSIDRSSNSALLTINNVQTEDEADYFCQSYSSGINIIFGGGTKLTVLGQPKA APDVVMTQTPVSLSVSVGGRVSISCRSSQSLVHDNGNTYLSWSLQKPGKSPKVLIYKV SNRFSGVSSRFTGSGSGTDFTLKISSVQPDDLGVYYCGQGTQYPFTFGSGTKLEIKTK GPSRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 324, linkers are indicated in bold and underlined) comprising V D1 of sequence SEQ ID NO: 230, L 1 of sequence SEQ ID NO: 307, V D2 of sequence SEQ ID NO: 158, L 2 of sequence SEQ ID NO: 309 and C L of sequence SEQ ID NO: 310, and one polypeptide according to formula II of the amino acid sequence QVQLVETGGGLVKPGQSLKLTCATSGFTFSNAWMHWVRQSPEKGLEWVAQIKARSN NYATYYAESVKGRFTISRDDSKSTIYLQMNSLTPEDTAIYYCRGTYYASKPFDYWGQG VMVTVSSS QVQLQESGPGLVQPSQTLSLTCTVSGFSLTTYDVHWVRQPPGKGLEWM GRIQNGGITDYNSALKSRLIISRDTSKSQVFLKMNSVQTEDTAMYFCAKTGSYFYAFDH WGQGTLVTVSSRT ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV (SEQ ID NO: 325, linkers are indicated in bold and underlined) comprising V D3 of sequence SEQ ID NO: 171, L 3 of the amino acid sequence 'S', V D4 of sequence SEQ ID NO: 226, L 4 of the amino acid sequence 'RT' and C H1 of sequence SEQ ID NO: 313.

[0258] The so-called CODV-Fab "hz20G6xhz7G3" antibody-like binding protein comprises: one polypeptide according to formula [I] consisting essentially of the amino acid sequence: DIVMTQSPDSLAVSLGERATINCESSQSLLNSGNQKNYLTWYQQKPGQPPKPLIYWAS TRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYSYPYTFGQGTKLEIKGGS GSSGSGG DIVMTQTPLSLSVTPGQPASISCKSSQSLVHNNANTYLSWYLQKPGQSPQ SLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTK VEIKGGSGSSGSGG RTVAAPSVFIFPPSOEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC (SEQ ID NO: 388, linkers are indicated in bold and underlined) comprising V D1 of sequence SEQ ID NO: 385, L 1 of sequence SEQ ID NO: 389, V D2 of sequence SEQ ID NO: 141, L 2 of sequence SEQ ID NO: 389 and C L of sequence SEQ ID NO: 310, and one polypeptide according to formula [II] consisting essentially of the amino acid sequence QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSN SYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQ GTLVTVSSEVOLVOSGAEVKKPGESLKISCKGSGYSFTDYYMKWAROMPGKGLEWM GDIIPSSGATFYNQKFKGQVTISADKSISTTYLQWSSLKASDTAMYYCARSHLLRASWF AYWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV (SEQ ID NO: 390) comprising V D3 of sequence SEQ ID NO: 138, L 3 is 0 amino acid, V D4 of sequence SEQ ID NO: 383 (in italic and underlined), L 4 is 0 amino acid and C H1 of sequence SEQ ID NO: 313.

[0259] In one example, the polypeptides according to formula II of the so-called CODV-Fab "7G3x20G6", "7G3x4E7", "7G3x4B4", "7G3x18F5", "hz20G6x7G3", "7G3xhz4B4", "hz4B4x3E3" and "hz20G6xhz7G3" antibody-like binding proteins further comprise the sequence EPKSCDKTHTHHHHHH (SEQ ID NO: 352) corresponding to a hinge sequence and a Histag used, for example, for purification.

[0260] The so-called CODV-Fab "hz20G6x7G3-TL4" (also called CODV-Fab-TL4 "hz20G6x7G3") antibody-like binding protein comprises: one polypeptide according to formula [IV] consisting essentially of the amino acid sequence DFVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYLQKPGQPPKLLIYWA STRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPYTFGGGTKLEIKGQ PKAAP DIVMTQTPLSLSVTPGQPASISCKSSQSLVHNNGNTYLSWYLQKPGQSPQSLI YKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEI KTKGPSRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECESK YGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWY VDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNRFTQKSLSLSLG (SEQ ID NO: 326, linkers are indicated in bold and underlined) comprising V D1 of sequence SEQ ID NO: 308, L 1 of sequence SEQ ID NO: 307, V D2 of sequence SEQ ID NO: 143, L 2 of sequence SEQ ID NO: 309, C L of sequence SEQ ID NO: 310 and F c2 (underlined) of sequence SEQ ID NO: 327, and one polypeptide according to formula [III] of the amino acid sequence QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSN SYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQ GTLVTVSSSEVQLQQSGPELVKPGASVKMSCKASGYTFTDYYMKWVKQSHGKSLEWI GDIIPSNGATFYNQKFKGKATLTVDRSSSTAYMHLNSLTSEDSAVYYCTRSHLLRASWF AYWGQGTLVTVSART ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES KYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNW YVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 328, linkers are indicated in bold and underlined) comprising V D3 of sequence SEQ ID NO: 138, L 3 of the amino acid sequence 'S', V D4 of sequence SEQ ID NO: 312, L 4 of the amino acid sequence 'RT', and C H1 of sequence SEQ ID NO: 329, and F c of sequence SEQ ID NO: 330.

[0261] In the above CODV-Fab-TL4 "hz20G6x7G3" antibody-like binding protein, the Fc of sequence SEQ ID NO: 330 and F c2 of sequence SEQ ID NO: 327 are from an IgG4 backbone. Said antibody-like binding protein is in a CODV-Fab-TL format and contains or consists of one polypeptide of formula III and one polypeptide of formula IV.

[0262] The so-called CODV-Fab-TL1 "hz20G6xhz7G3" antibody-like binding protein comprises: one polypeptide according to formula IV of the amino acid sequence DIVMTQSPDSLAVSLGERATINCESSQSLLNSGNQKNYLTWYQQKPGQPPKPLIYWAS TRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYSYPYTFGQGTKLEIKGGS GSSGSGG DIVMTQTPLSLSVTPGQPASISCKSSQSLVHNNANTYLSWYLQKPGQSPQ SLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTK VEIKGGSGSSGSGG RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGECDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPG (SEQ ID NO: 391, linkers are indicated in bold and underlined) comprising V D1 of sequence SEQ ID NO: 385, L 1 of sequence SEQ ID NO: 389, V D2 of sequence SEQ ID NO: 141, L 2 of sequence SEQ ID NO: 389, C L of sequence SEQ ID NO: 310, L 5 which contains 0 amino acid, and F c2 (underlined) of sequence SEQ ID NO: 392; and one polypeptide according to formula III of the amino acid sequence QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSN SYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQ GTLVTVSSEVQLVQSGAEVKKPGESLKISCKGSGYSFTDYYMKWARQMPGKGLEWM GDIIPSSGATFYNQKFKGQVTISADKSISTTYLQWSSLKASDTAMYYCARSHLLRASWF AYWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO : 393) comprising V D3 of sequence SEQ ID NO: 138, L 3 is 0 amino acid, V D4 of sequence SEQ ID NO: 383 (in italic), L 4 is 0 amino acid, C H1 of sequence SEQ ID NO: 313, and and F c (underlined) of sequence SEQ ID NO: 394.

[0263] In the CODV-Fab-TL1 "hz20G6x7G3" antibody-like binding protein, the Fc of sequence SEQ ID NO: 394 and F c2 of sequence SEQ ID NO: 392 are from an IgG1 backbone. Said antibody-like binding protein is in a CODV-Fab-TL format. It contains or consists of one polypeptide of formula IV and one polypeptide of formula III.

[0264] The so-called CODV-Fab-OL1 "hz20G6xhz7G3" antibody-like binding protein comprises: one polypeptide according to formula I of the amino acid sequence DIVMTQSPDSLAVSLGERATINCESSQSLLNSGNQKNYLTWYQQKPGQPPKPLIYWAS TRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYSYPYTFGQGTKLEIKGGS GSSGSGGDIVMTQTPLSLSVTPGQPASISCKSSQSLVHNNANTYLSWYLQKPGQSPQ SLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTK VEIKGGSGSSGSGG RTVAAPSVFIFPPSOEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC (SEQ ID NO: 388, linkers are indicated in bold and underlined) comprising V D1 of sequence SEQ ID NO: 385, L 1 of sequence SEQ ID NO: 389, V D2 of sequence SEQ ID NO: 141, L 2 of sequence SEQ ID NO: 389, and C L of sequence SEQ ID NO: 310; and one polypeptide according to formula III of the amino acid sequence QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSN SYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQ GTLVTVSSEVOLVOSGAEVKKPGESLKISCKGSGYSFTDYYMKWAROMPGKGLEWM GDIIPSSGATFYNQKFKGQVTISADKSISTTYLQWSSLKASDTAMYYCARSHLLRASWF AYWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPG (SEQ ID NO : 395) comprising V D3 of sequence SEQ ID NO: 138, L 3 is 0 amino acid, V D4 of sequence SEQ ID NO: 383 (in italic and underlined), L 4 is 0 amino acid, C H1 of sequence SEQ ID NO: 313, and F c (underlined) of sequence SEQ ID NO: 396; and wherein the so-called CODV-Fab-OL1 "hz20G6xhz7G3" antibody-like binding protein further comprises a Fc stump (Fc3) of the amino acid sequence: GSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 397) and which heterodimerises with the Fc region of the polypeptide according to formula III.

[0265] Said antibody-like binding protein is in a CODV-Fab-OL format, i.e. it contains or consists of one polypeptide of formula I, one polypeptide of formula III, and one Fc stump. Its F c and F c3 sequences have been engineered according to the "Knob-into-Hole" technology and further contain the double mutation L234A and L235A.

[0266] The F c sequence of sequence SEQ ID NO: 396 has been designed to contain RF residues at positions 200-221 (in bold above), instead of HY residues which would have otherwise been present at these positions of the Fc region. The HY > RF mutation (i.e. H435R and Y436F in CH3 domain as described by Jendeberg, L. et al. 1997, J. Immunological Meth., 201: 25-34) is advantageous for purification purposes as it abolishes binding to protein A. In the case of CODV-Fab-OL1 "hz20G6xhz7G3", the F c stump of sequence SEQ ID NO: 397 comprises HY residues at positions 217-218 (in bold above).

[0267] The so-called CODV-Fab-OL1a "hz20G6xhz7G3" antibody-like binding protein comprise: one polypeptide according to formula I of the amino acid sequence DIVMTQSPDSLAVSLGERATINCESSQSLLNSGNQKNYLTWYQQKPGQPPKPLIYWAS TRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYSYPYTFGQGTKLEIKGGS GSSGSGGDIVMTQTPLSLSVTPGQPASISCKSSQSLVHNNANTYLSWYLQKPGQSPQ SLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTK VEIKGGSGSSGSGGRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC (SEQ ID NO: 388) which comprises V D1 of sequence SEQ ID NO: 385, L 1 of sequence SEQ ID NO: 389, V D2 of sequence SEQ ID NO: 141, L 2 of sequence SEQ ID NO: 389, and C L of sequence SEQ ID NO: 310; one polypeptide according to formula III of the amino acid sequence: QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKOKSN SYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQ GTLVTVSSEVOLVOSGAEVKKPGESLKISCKGSGYSFTDYYMKWAROMPGKGLEWM GDIIPSSGATFYNQKFKGQVTISADKSISTTYLQWSSLKASDTAMYYCARSHLLRASWF AYWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 399) comprising V D3 of sequence SEQ ID NO: 138, L 3 is 0 amino acid, V D4 of sequence SEQ ID NO: 383 (in italic and underlined), L 4 is 0 amino acid, C H1 of sequence SEQ ID NO: 313, and F c (underlined) of sequence SEQ ID NO: 400; and wherein the so-called COOV-Fab-OL1a "hz20G6xhz7G3" antibody-like binding protein further comprises a Fc stump (F c3 ) of the amino acid sequence: GSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPG (SEQ ID NO: 398) which heterodimerises with the Fc region of the polypeptide according to formula III.

[0268] The F c of sequence SEQ ID NO: 400 comprises HY residues at positions 200-221 (in bold above) while the F c stump of sequence SEQ ID NO: 398 comprises RF residues at positions 217-218 (in bold above).

[0269] Said antibody-like binding protein is in a CODV-Fab-OL format, i.e. it contains or consists of one polypeptide of formula I, one polypeptide of formula III and one Fc stump. Its F c and F c3 sequences have been engineered according to the "Knob-into-Hole" technology and contain the double mutation L234A and L235A.

[0270] In one embodiment, the first immunoglobulin or the second immunoglobulin is one anti-CD123 antibody selected from the so-called "3E3-D3", "1E1-G5", "2B8-F3", "2F8-D6", "3B10-E6", "5A5-B4", "6B10-E4", "6C10-C4", "6D6-B8", "8B11-B7", "9B8-G6", "9D7-C8", and "9F6-G3" anti-CD123 antibodies, or a humanized form thereof, or the anti-CD123 antibody "7G3" described herein below, for example the anti-CD123 antibodies "3E3-D3" or "7G3", or a humanized form thereof.

[0271] In one embodiment, the first immunoglobulin or the second immunoglobulin is one anti-CD3 antibody selected from the so-called "20G6-F3", "4B4-D7", "4E7-C9", "18F5-H10", "12D2-E5", "11D7-C3", "11H3-E5", "13H2-C2", "13C1-F6", "18H11-F10", "1E6-C9", "10F4-C10", "10E6-G6", "18G9-H11", "11F3-B9", "12G3-E8", "5B1-G2", "16F8-A7", "11F9-F8", "3G5-E10", "9D7-F3", "8C2-F7", "20E5-F10", "20B5-F10", "6C9-C9", "3E8-G1", "3H6-D2", and "8H2" anti-CD3 antibodies, or a humanized form thereof, for example the so-called "20G6-F3", "4B4-D7", "4E7-C9", "18F5-H10", and "hz20G6" anti-CD3 antibodies, for instance the so-called so-called "20G6-F3", "4B4-D7" anti-CD3 antibodies.

[0272] Accordingly, V D1 and V D4 , or V D2 and V D3 are the variable domains of a heavy or light chain of an anti-CD3 antibody, wherein said anti-CD3 antibody comprises: a) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 6, CDR2-H of sequence SEQ ID NO: 7, CDR3-H of sequence SEQ ID NO: 8 and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 10 or SEQ ID NO: 142, CDR2-L of sequence 'KVS' and CDR3-L of sequence SEQ ID NO: 11; or b) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 13, CDR2-H of sequence SEQ ID NO: 14, CDR3-H of sequence SEQ ID NO: 15 and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 17 or SEQ ID NO: 184, CDR2-L of sequence 'KVS' and CDR3-L of sequence SEQ ID NO: 11; or c) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 13, CDR2-H of sequence SEQ ID NO: 19, CDR3-H of sequence SEQ ID NO: 20 and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 22, CDR2-L of sequence 'KVS' and CDR3-L of sequence SEQ ID NO: 11; or d) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 24, CDR2-H of sequence SEQ ID NO: 19, CDR3-H of sequence SEQ ID NO: 25 and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 27, CDR2-L of sequence 'KVS' and CDR3-L of sequence SEQ ID NO: 28,and wherein V D1 is the variable domain of heavy chain as defined above if V D4 is the variable domain of light chain or V D1 is the variable domain of the light chain as defined above if V D4 is the variable domain of the heavy chain, or V D2 is the variable domain of heavy chain as defined above if V D3 is the variable domain of light chain or V D2 is the variable domain of the light chain as defined above if V D3 is the variable domain of the heavy chain.

[0273] In a further embodiment, V D1 and V D4 or V D2 and V D3 are the variable domains of a heavy or light chain of an anti-CD3 antibody, wherein said anti-CD3 antibody is a humanized antibody and comprises: a) a heavy chain variable domain of sequence SEQ ID NO: 138, or a sequence at least 85% identical thereto and / or a light chain variable domain of sequence SEQ ID NO: 143, or a sequence at least 85% identical thereto; or b) a heavy chain variable domain of sequence SEQ ID NO: 171, or a sequence at least 85% identical thereto and / or a light chain variable domain of sequence SEQ ID NO: 158, or a sequence at least 85% identical thereto; or c) a heavy chain variable domain of sequence SEQ ID NO: 176, or a sequence at least 85% identical thereto and / or a light chain variable domain of sequence SEQ ID NO: 164, or a sequence at least 85% identical thereto; or wherein V D1 is the variable domain of heavy chain as defined above if V D4 is the variable domain of light chain or V D1 is the variable domain of the light chain as defined above if V D4 is the variable domain of the heavy chain, or V D2 is the variable domain of heavy chain as defined above if V D3 is the variable domain of light chain or V D2 is the variable domain of the light chain as defined above if V D3 is the variable domain of the heavy chain.

[0274] In said sequence at least 85% identical to SEQ ID NO: 138, SEQ ID NO: 143, SEQ ID NO: 171, SEQ ID NO: 158, SEQ ID NO: 176, or SEQ ID NO: 164, the sequences of the 6 CDRs are unchanged compared to the 6 CDRs present in the reference sequence SEQ ID NO: 138, SEQ ID NO: 143, SEQ ID NO: 171, SEQ ID NO: 158, SEQ ID NO: 176, or SEQ ID NO: 164.

[0275] In an embodiment, the antibody-like binding protein of the invention binds to human CD3. In another embodiment, the antibody-like binding protein of the invention further binds to Macaca fascicularis CD3. In particular, the antibody-like binding protein of the invention binds to the extracellular domain of human CD3, or of both human and Macaca fascicularis CD3. More specifically, the antibody binds to CD3ε. More specifically, the antibody-like binding protein binds to the human or human and Macaca fascicularis extracellular domain of CD3ε. The antibody-like binding protein binds to CD3ε when present in the form of a complex, such as a CD3ε / δ complex, or when present as single protein, indifferently whether expressed in isolated form, or present in a soluble extracellular domain or full-length membrane-anchored CD3ε as present in for example in T-cells. The antibody-like binding protein according to the invention is specific for the surface human CD3 protein, or of both human and Macaca fascicularis CD3 proteins, in particular to CD3ε.

[0276] The antibody-like binding according to the invention has a ratio of affinity for Macaca fascicularis CD3 on affinity for human CD3 (KD(Macaca fascicularis) / KD(human) which is ≤10, in particular ≤6, ≤5, ≤4, ≤3, ≤2, ≤1 or ≤0.5. Thus, the antibody-like binding protein according to the invention may be used in toxicological studies performed in monkeys the toxicity profile observed in monkeys relevant to anticipate potential adverse effects in humans.

[0277] Furthermore, the antibody-like binding protein according to the invention has an affinity (KD) for human CD3 or Macaca fascicularis CD3, or both, which is ≤50nM, ≤40nM, or ≤30nM, for instance ≤ 20nM,for example an affinity of 0.1 nM to 30 nM, in particular of 0.4 nM to 20 nM, or of 0.4 nM to 15 nM.

[0278] In one embodiment, the antibody-like binding protein of the invention has a T-cell activation that is lower than less than 20%, less than 18%, less than 16%, less than 14%, less than 12%, less than 10% in the absence of target cells.

[0279] In one embodiment, the antibody-like binding protein of the invention has a T-cell activation that is higher higher than 55%, higher than 60%, higher than 62%, higher than 64%, higher than 66%, higher than 68%, higher than 70% in the presence of target cells.

[0280] "Low T-cell activation" in the context of the antibody-like binding proteins of the invention refers to a T-cell activation less than 20%, less than 18%, less than 16%, less than 14%, less than 12%, less than 10%.

[0281] "Target cells" herein refer to cells that express the second antigen, in one example target cells herein refer to CD123 expressing cells such as THP-1 cells.

[0282] "High T-cell activation" herein refers to a T-cell activation higher than 50%, higher than 55%, higher than 60%, higher than 62%, higher than 64%, higher than 66%, higher than 68%, higher than 70%.

[0283] In a further embodiment, the invention relates to an antibody-like binding protein having biological and immunological specificity to at least one further target antigen.

[0284] Therefore, in one aspect of the invention, the antibody-like binding protein of the invention binds further to at least one other target antigen. Accordingly, in one embodiment, the antibody-like binding protein of the invention is bispecific and capable of binding two different antigen targets or epitopes.

[0285] Accordingly, in one embodiment the first immunoglobulin is an immunoglobulin directed against at least one further target if the second immunoglobulin is one anti-CD123 antibody as defined in the section «anti-CD123 antibodies» above, or the second immunoglobulin is an immunoglobulin directed against at least one further target if the first immunoglobulin is one anti-CD123 antibody as defined in the section «anti-CD123 antibodies» above.

[0286] In one further embodiment the first immunoglobulin is an immunoglobulin directed against at least one further target if the second immunoglobulin is one anti-CD3 antibody as defined in the section «anti-CD3 antibodies» above, or the second immunoglobulin is an immunoglobulin directed against at least one further target if the first immunoglobulin is one anti-CD3 antibody as defined in the section «anti-CD3 antibodies» above.

[0287] The antibody-like binding protein of the invention has a T-cell engaging effect. This T-cell engaging effect induces cytotoxicity in the target cell. In one embodiment, the target cell is a CD123 expressing cell, such as a CD123 expressing cancer cell, for example THP-1 or TF-1.

[0288] Accordingly, in one embodiment the antibody-like binding protein according to the invention is able to engage primary T-cells and to lyse target cells in vitro wherein the (EC 50 ) is ≤40pM, ≤35pM, for instance ≤30pM.

[0289] "Cytotoxicity" herein refers to the quality of a compound, such as the antibody-like binding protein or an anti-CD123 antibody of the invention, to be toxic to cells. Cytotoxicity may be induced by different mechanisms of action and can thus be divided into cell-mediated cytotoxicity, apoptosis, antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

[0290] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a mechanism of cell-mediated immune defense whereby an effector cell of the immune system actively lyses a target cell, whose membrane-surface antigens have been bound by specific antibodies.

[0291] "Complement-dependent cytotoxicity" or "CDC", in the context of the invention, refers to lysis of a target cell in the presence of complement system proteins.

[0292] "Cell-mediated cytotoxicity" refers to cytolysis of a target cell by effector lymphocytes, such as cytotoxic T lymphocytes or natural killer cells and can thus be distinguished into T-cell-mediated cytotoxicity and NK-cell cytotoxicity.

[0293] In one embodiment, cytotoxicity herein refers to Cell-mediated cytotoxicity for example T-cell-mediated cytotoxicity.

[0294] Furthermore, in one embodiment the cell-mediated cytotoxicity refers to cell-mediated cytotoxicity by T-cells.

[0295] Accordingly, the antibody-like binding protein of the invention induces cell-mediated cytotoxicity in the target cell mediated by T-cells.

[0296] Methods to measure cytotoxicity are known to the skilled in the art and include using 51-Chromium (Cr) release assay, live / dead cell staining of target cells including propidium iodide, 7-AAD, and other stains that are known to the skilled in the art, detection of lytic molecules released by T cells including granzyme and perforin by flow cytometry or ELISA, detection of lactate dehydrogenase (LDH) released into the media from damaged cells as a biomarker for cellular cytotoxicity and cytolysis, detection of cell surface mobilization of CD107a, Annexin V (calcium-dependent phospholipid-binding proteins) staining of apoptotic target cells and for example detection of activated Caspase-3 (CASP3).. Furthermore, the skilled in the art can distinguish between the different mechanisms of cytotoxicity based on the test selected and based on the experimental set up.

[0297] In one example, cell-mediated cytotoxicity may be for example measured using CFSE to label target cells and 7-AAD to label dead cells as described, for instance, in example 3.2.

[0298] In a further embodiment, the antibody-like binding protein is capable of binding to CD3 and at least one further antigen target, for example CD123.

[0299] In one embodiment, the antibody-like binding protein is capable of inhibiting the function of this further antigen target, for example CD123.

[0300] In one aspect of the invention, the antibody-like binding protein binds to human CD123. In another embodiment, the antibody-like binding protein further binds to Macaca fascicularis CD123. In particular, the antibody-like binding protein of the invention binds to the extracellular domain of human CD123, or of both, human and Macaca fascicularis CD123. More specifically, the antibody-like binding protein binds to the distal moiety of CD123, for example, to the amino acids starting from position 19 to 49 of human CD123 of the amino acid sequence SEQ ID NO: 104. The antibody-like binding protein binds to CD123, indifferently whether expressed in isolated form, or present in a soluble extracellular domain or full-length membrane-anchored CD123 as present in CD123 expressing cells such as AML cells or CD123 transfected cells. The antibody-like binding protein according to the invention is specific to cells that express human or human and Macaca fascicularis CD123 proteins on their surface, for example CD123 expressing cancer cells.

[0301] Accordingly, the antibody-like binding protein according to the invention has an affinity (KD) for human CD123 or Macaca fascicularis CD123, or both, which is ≤20nM, ≤15nM, or ≤10nM, for instance ≤ 5nM,for example an affinity of 0.01 nM to 5 nM, in particular of 0.1 nM to 5 nM.

[0302] Accordingly, in one embodiment, the first immunoglobulin is one anti-CD3 antibody selected from the so-called "20G6-F3", "4B4-D7", "4E7-C9", "18F5-H10", "12D2-E5", "11D7-C3", "11H3-E5", "13H2-C2", "13C1-F6", "18H11-F10", "1E6-C9", "10F4-C10", "10E6-G6", "18G9-H11", "11F3-B9", "12G3-E8", "5B1-G2", "16F8-A7", "11F9-F8", "3G5-E10", "9D7-F3", "8C2-F7", "20E5-F10", "20B5-F10", "6C9-C9", "3E8-G1", "3H6-D2", and "8H2" anti-CD3 antibodies, or a humanized form thereof, for example the so-called "20G6-F3", "4B4-D7", "4E7-C9", "18F5-H10", "hz4B4" and "hz20G6" anti-CD3 antibodies, and the second immunoglobulin is one anti-CD123 antibody selected from the so-called "3E3-D3", "1E1-G5", "2B8-F3", "2F8-D6", "3B10-E6", "5A5-B4", "6B10-E4", "6C10-C4", "6D6-B8", "8B11-B7", "9B8-G6", "9D7-C8", and "9F6-G3" anti-CD123 antibodies.

[0303] Accordingly, in a further embodiment, the second immunoglobulin is one anti-CD3 antibody selected from the so-called "20G6-F3", "4B4-D7", "4E7-C9", "18F5-H10", "12D2-E5", "11D7-C3", "11H3-E5", "13H2-C2", "13C1-F6", "18H11-F10", "1E6-C9", "10F4-C10", "10E6-G6", "18G9-H11", "11F3-B9", "12G3-E8", "5B1-G2", "16F8-A7", "11F9-F8", "3G5-E10", "9D7-F3", "8C2-F7", "20E5-F10", "20B5-F10", "6C9-C9", "3E8-G1", "3H6-D2", and "8H2" anti-CD3 antibodies, or a humanized form thereof, for example the so-called "20G6-F3", "4B4-D7", "4E7-C9", "18F5-H10", "hz4B4" and "hz20G6" anti-CD3 antibodies, and the first immunoglobulin is one anti-CD123 antibody selected from the so-called "3E3-D3", "1E1-G5", "2B8-F3", "2F8-D6", "3B10-E6", "5A5-B4", "6B10-E4", "6C10-C4", "6D6-B8", "8B11-B7", "9B8-G6", "9D7-C8", and "9F6-G3" anti-CD123 antibodies.

[0304] Accordingly, in one embodiment, the V D1 and V D4 or V D2 and V D3 comprise a heavy chain variable domain and a light chain variable domain, each of them either defined by three CDR sequences or by heavy and light chain variable domain sequences of one the 13 so-called "3E3-D3", "1E1-G5", "2B8-F3", "2F8-D6", "3B10-E6", "5A5-B4", "6B10-E4", "6C10-C4", "6D6-B8", "8B11-B7", "9B8-G6", "9D7-C8", and "9F6-G3" anti-CD123 antibodies as defined above, wherein V D1 and V D4 , both comprise the three CDR sequences of the heavy and light chain variable domain sequences of one anti-CD123 antibody as defined above, if V D2 and V D3 , both comprise three CDR sequences of heavy and light chain variable domain sequences of one of the anti-CD3 antibodies as defined above, or wherein V D2 and V D3 , both comprise the three CDRs of the heavy and light chain variable domain sequences of one anti-CD123 antibody as defined above, if V D1 and V D4 comprise CDR sequences of heavy and light chain variable domain sequences of one of the anti-CD3 antibodies as defined above.

[0305] Accordingly, in one embodiment V D1 and V D4 are the variable domain of heavy or light chain of an anti-CD3 antibody, wherein said anti-CD3 antibody comprises a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 6, CDR2-H of sequence SEQ ID NO: 7, CDR3-H of sequence SEQ ID NO: 8 and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 10 or SEQ ID NO: 142, CDR2-L of sequence 'KVS' and CDR3-L of sequence SEQ ID NO: 11 and V D2 and V D3 are the variable domain of heavy or light chain of an anti-CD123 antibody selected from the group consisting of "3E3-D3", "1E1-G5", "2B8-F3", "2F8-D6", "3B10-E6", "5A5-B4", "6B10-E4", "6C10-C4", "6D6-B8", "8B11-B7", "9B8-G6", "9D7-C8", "9F6-G3" anti-CD123 antibodies as described above under the section "anti-CD123 antibodies", wherein V D1 and V D2 are both variable domains of light chains if V D3 and V D4 are both variable domains of heavy chains or V D1 and V D2 are both variable domains of heavy chains if V D3 and V D4 are both variable domains of light chains.

[0306] Accordingly, in a further embodiment V D2 and V D3 are the variable domain of heavy or light chain of an anti-CD3 antibody, wherein said anti-CD3 antibody comprises a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 6, CDR2-H of sequence SEQ ID NO: 7, CDR3-H of sequence SEQ ID NO: 8 and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 10 or SEQ ID NO: 142, CDR2-L of sequence 'KVS' and CDR3-L of sequence SEQ ID NO: 11 and V D1 and V D4 are the variable domain of heavy or light chain of an anti-CD123 antibody selected from the group consisting of "3E3-D3", "1E1-G5", "2B8-F3", "2F8-D6", "3B10-E6", "5A5-B4", "6B10-E4", "6C10-C4", "6D6-B8", "8B11-B7", "9B8-G6", "9D7-C8", "9F6-G3" anti-CD123 antibodies as described above under the section "anti-CD123 antibodies", wherein V D1 and V D2 are both variable domains of light chains if V D3 and V D4 are both variable domains of heavy chains or V D1 and V D2 are both variable domains of heavy chains if V D3 and V D4 are both variable domains of light chains.

[0307] Accordingly, in a further embodiment V D1 and V D4 are the variable domain of heavy or light chain of an anti-CD3 antibody, wherein said anti-CD3 antibody comprises a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 13, CDR2-H of sequence SEQ ID NO: 14, CDR3-H of sequence SEQ ID NO: 15 and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 17 or SEQ ID NO: 184, CDR2-L of sequence 'KVS' and CDR3-L of sequence SEQ ID NO: 11 and V D2 and V D3 are the variable domain of heavy or light chain of an anti-CD123 antibody selected from the group consisting of "3E3-D3", "1E1-G5", "2B8-F3", "2F8-D6", "3B10-E6", "5A5-B4", "6B10-E4", "6C10-C4", "6D6-B8", "8B11-B7", "9B8-G6", "9D7-C8", "9F6-G3" anti-CD123 antibodies as described above under the section "anti-CD123 antibodies", wherein V D1 and V D2 are both variable domains of light chains if V D3 and V D4 are both variable domains of heavy chains or V D1 and V D2 are both variable domains of heavy chains if V D3 and V D4 are both variable domains of light chains.

[0308] Accordingly, in a further embodiment V D1 and V D4 are the variable domain of heavy or light chain of an anti-CD3 antibody, wherein said anti-CD3 antibody comprises a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 13, CDR2-H of sequence SEQ ID NO: 14, CDR3-H of sequence SEQ ID NO: 15 and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 17 or SEQ ID NO: 184, CDR2-L of sequence 'KVS' and CDR3-L of sequence SEQ ID NO: 11 and V D2 and V D3 are the variable domain of heavy or light chain of an anti-CD123 antibody selected from the group consisting of "3E3-D3", "1E1-G5", "2B8-F3", "2F8-D6", "3B10-E6", "5A5-B4", "6B10-E4", "6C10-C4", "6D6-B8", "8B11-B7", "9B8-G6", "9D7-C8", "9F6-G3" anti-CD123 antibodies as described above under the section "anti-CD123 antibodies", wherein V D1 and V D2 are both variable domains of light chains if V D3 and V D4 are both variable domains of heavy chains or V D1 and V D2 are both variable domains of heavy chains if V D3 and V D4 are both variable domains of light chains.

[0309] In a further embodiment V D1 and V D4 are the variable domain of heavy or light chain of a humanized anti-CD3 antibody, wherein said anti-CD3 antibody comprises a heavy chain variable a heavy chain variable domain of sequence SEQ ID NO: 138 and / or a light chain variable domain of sequence SEQ ID NO: 143 and V D2 and V D3 are the variable domain of heavy or light chain of an anti-CD123 antibody selected from the group consisting of "3E3-D3", "1E1-G5", "2B8-F3", "2F8-D6", "3B10-E6", "5A5-B4", "6B10-E4", "6C10-C4", "6D6-B8", "8B11-B7", "9B8-G6", "9D7-C8", "9F6-G3" anti-CD123 antibodies as described above under the section "anti-CD123 antibodies", wherein V D1 and V D2 are both variable domains of light chains if V D3 and V D4 are both variable domains of heavy chains or V D1 and V D2 are both variable domains of heavy chains if V D3 and V D4 are both variable domains of light chains.

[0310] In a further embodiment V D2 and V D3 are the variable domain of heavy or light chain of a humanized anti-CD3 antibody, wherein said anti-CD3 antibody comprises a heavy chain variable a heavy chain variable domain of sequence SEQ ID NO: 138 and / or a light chain variable domain of sequence SEQ ID NO: 143 and V D1 and V D4 are the variable domain of heavy or light chain of an anti-CD123 antibody selected from the group consisting of "3E3-D3", "1E1-G5", "2B8-F3", "2F8-D6", "3B10-E6", "5A5-B4", "6B10-E4", "6C10-C4", "6D6-B8", "8B11-B7", "9B8-G6", "9D7-C8", "9F6-G3" anti-CD123 antibodies as described above under the section "anti-CD123 antibodies", wherein V D1 and V D2 are both variable domains of light chains if V D3 and V D4 are both variable domains of heavy chains or V D1 and V D2 are both variable domains of heavy chains if V D3 and V D4 are both variable domains of light chains.

[0311] In a further embodiment V D1 and V D4 are the variable domain of heavy or light chain of a humanized anti-CD3 antibody, wherein said anti-CD3 antibody comprises a heavy chain a heavy chain variable domain of sequence SEQ ID NO: 171 and / or a light chain variable domain of sequence SEQ ID NO: 158 and V D2 and V D3 are the variable domain of heavy or light chain of an anti-CD123 antibody selected from the group consisting of "3E3-D3", "1E1-G5", "2B8-F3", "2F8-D6", "3B10-E6", "5A5-B4", "6B10-E4", "6C10-C4", "6D6-B8", "8B11-B7", "9B8-G6", "9D7-C8", "9F6-G3" anti-CD123 antibodies as described above under the section "anti-CD123 antibodies", wherein V D1 and V D2 are both variable domains of light chains if V D3 and V D4 are both variable domains of heavy chains or V D1 and V D2 are both variable domains of heavy chains if V D3 and V D4 are both variable domains of light chains.

[0312] In a further embodiment V D2 and V D3 are the variable domain of heavy or light chain of a humanized anti-CD3 antibody, wherein said anti-CD3 antibody comprises a heavy chain a heavy chain variable domain of sequence SEQ ID NO: 171 and / or a light chain variable domain of sequence SEQ ID NO: 158 and V D1 and V D4 are the variable domain of heavy or light chain of an anti-CD123 antibody selected from the group consisting of "3E3-D3", "1E1-G5", "2B8-F3", "2F8-D6", "3B10-E6", "5A5-B4", "6B10-E4", "6C10-C4", "6D6-B8", "8B11-B7", "9B8-G6", "9D7-C8", "9F6-G3" anti-CD123 antibodies as described above under the section "anti-CD123 antibodies", wherein V D1 and V D2 are both variable domains of light chains if V D3 and V D4 are both variable domains of heavy chains or V D1 and V D2 are both variable domains of heavy chains if V D3 and V D4 are both variable domains of light chains.

[0313] In a further embodiment V D1 and V D4 are the variable domain of heavy or light chain of a humanized anti-CD3 antibody, wherein said anti-CD3 antibody comprises a heavy chain a heavy chain variable domain of sequence SEQ ID NO: 176 and / or a light chain variable domain of sequence SEQ ID NO: 164 and V D2 and V D3 are the variable domain of heavy or light chain of an anti-CD123 antibody selected from the group consisting of "3E3-D3", "1E1-G5", "2B8-F3", "2F8-D6", "3B10-E6", "5A5-B4", "6B10-E4", "6C10-C4", "6D6-B8", "8B11-B7", "9B8-G6", "9D7-C8", "9F6-G3" anti-CD123 antibodies as described above under the section "anti-CD123 antibodies", wherein V D1 and V D2 are both variable domains of light chains if V D3 and V D4 are both variable domains of heavy chains or V D1 and V D2 are both variable domains of heavy chains if V D3 and V D4 are both variable domains of light chains.

[0314] In a further embodiment V D2 and V D3 are the variable domain of heavy or light chain of a humanized anti-CD3 antibody, wherein said anti-CD3 antibody comprises a heavy chain a heavy chain variable domain of sequence SEQ ID NO: 176 and / or a light chain variable domain of sequence SEQ ID NO: 164 and V D1 and V D4 are the variable domain of heavy or light chain of an anti-CD123 antibody selected from the group consisting of "3E3-D3", "1E1-G5", "2B8-F3", "2F8-D6", "3B10-E6", "5A5-B4", "6B10-E4", "6C10-C4", "6D6-B8", "8B11-B7", "9B8-G6", "9D7-C8", "9F6-G3" anti-CD123 antibodies as described above under the section "anti-CD123 antibodies", wherein V D1 and V D2 are both variable domains of light chains if V D3 and V D4 are both variable domains of heavy chains or V D1 and V D2 are both variable domains of heavy chains if V D3 and V D4 are both variable domains of light chains.

[0315] According to a further aspect of the invention, the first or second immunoglobulin is the anti-CD123 antibody 7G3. Therefore, in one embodiment V D1 and V D4 or V D2 and V D3 comprise a heavy chain variable domain and a light chain variable domain as defined by CDR sequences of heavy and light chain variable domain sequences of the antibody 7G3 as defeined herein below. In one embodiment V D1 and V D4 , or V D2 and V D3 comprise a heavy chain variable domain and a light chain variable domain of the antibody 7G3 as described in the patent application WO2013 / 173820,

[0316] Accordingly, the so-called "7G3" anti-CD123 antibody as herein used comprises: a heavy chain variable domain consisting of sequence EVQLQQSGPELVKPGASVKMSCKASGYTFTDYY MKWVKQSHGKSLEWIGDIIPSNGA TFYNQKFKGKATLTVDRSSSTAYMHLNSLTSEDSAVYYCTRSHLLRASWFAY WGQGT LVTVSA (SEQ ID NO: 312, with CDRs shown in bold characters) comprising CDR1-H of sequence SEQ ID NO: 375, a CDR2-H of sequence SEQ ID NO: 376, and a CDR3-H of sequence SEQ ID NO: 377 and a light chain variable domain consisting of sequence DFVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNY LTWYLQKPGQPPKLLIYWA STRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPYT FGGGTKLEIK (SEQ ID NO: 308, with CDRs shown in bold characters) comprising CDR1-L of sequence SEQ ID NO: 378, a CDR2-L of sequence 'WAS', and a CDR3-L of sequence SEQ ID NO: 379.

[0317] In a further aspect of the invention, the antibody 7G3 may also be a humanized antibody or a fragment of a humanized antibody. Accordingly, in one embodiment, the antibody 7G3 of the invention is a humanized antibody comprising a heavy chain variable domain consisting of sequence EVQLVQSGAEVKKPGESLKISCKGSGYSFTDYY MKWARQMPGKGLEWMGDIIPSNGA T FYNQKFKGQVTISADKSISTTYLQWSSLKASDTAMYYCARSHLLRASWFAY WGQGT MVTVSS (SEQ ID NO: 380, with CDRs shown in bold characters) comprising CDR1-H of sequence SEQ ID NO: 381, a CDR2-H of sequence SEQ ID NO: 377, and a CDR3-H of sequence SEQ ID NO: 382, or a heavy chain variable domain consisting of sequence EVQLVQSGAEVKKPGESLKISCKGSGYSFTDYYMKWARQMPGKGLEWMGDIIPSSGA TFYNQKFKGQVTISADKSISTTYLQWSSLKASDTAMYYCARSHLLRASWFAYWGQGT MVTVSS (SEQ ID NO: 383, with CDRs shown in bold characters) comprising CDR1-H of sequence SEQ ID NO: 381, a CDR2-H of sequence SEQ ID NO: 384, and a CDR3-H of sequence SEQ ID NO: 382, or a light chain variable domain consisting of sequence DIVMTQSPDSLAVSLGERATINCESSQSLLNSGNQKNY LTWYQQKPGQPPKPLIYWAS TRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYSYPYT FGQGTKLElK (SEQ ID NO: 385, with CDRs shown in bold characters) comprising CDR1-L of sequence SEQ ID NO: 378, a CDR2-L of sequence 'WAS', and a CDR3-L of sequence SEQ ID NO: 379.

[0318] In one embodiment, the antibody-like binding protein that binds specifically to human CD3ε and human CD123 comprises a) a light chain variable domain of a first immunoglobulin (V D1 ) consisting of the amino acid sequence SEQ ID NO: 9 or a sequence at least 85% identical thereto, a light chain variable domain of a second immunoglobulin (V D2 ) consisting of the amino acid sequence SEQ ID NO : 308 or a sequence at least 85% identical thereto, a heavy chain variable domain of the second immunoglobulin (V D3 ) consisting of the amino acid sequence SEQ ID NO : 312 or a sequence at least 85% identical thereto and a heavy chain variable domain of the first immunoglobulin (V D4 ) consisting of the amino acid sequence SEQ ID NO : 5 or a sequence at least 85% identical thereto, or b) a light chain variable domain of a first immunoglobulin (V D1 ) consisting of the amino acid sequence SEQ ID NO: 21 or a sequence at least 85% identical thereto, a light chain variable domain of a second immunoglobulin (V D2 ) consisting of the amino acid sequence SEQ ID NO : 308 or a sequence at least 85% identical thereto, a heavy chain variable domain of the second immunoglobulin (V D3 ) consisting of the amino acid sequence SEQ ID NO : 312 or a sequence at least 85% identical thereto and a heavy chain variable domain of the first immunoglobulin (V D4 ) consisting of the amino acid sequence SEQ ID NO : 18 or a sequence at least 85% identical thereto, or c) a light chain variable domain of a first immunoglobulin (V D1 ) consisting of the amino acid sequence SEQ ID NO: 16 or a sequence at least 85% identical thereto, a light chain variable domain of a second immunoglobulin (V D2 ) consisting of the amino acid sequence SEQ ID NO : 308 or a sequence at least 85% identical thereto, a heavy chain variable domain of the second immunoglobulin (V D3 ) consisting of the amino acid sequence SEQ ID NO : 312 or a sequence at least 85% identical thereto and a heavy chain variable domain of the first immunoglobulin (V D4 ) consisting of the amino acid sequence SEQ ID NO 12 or a sequence at least 85% identical thereto, or d) a light chain variable domain of a first immunoglobulin (V D1 ) consisting of the amino acid sequence SEQ ID NO: 26 or a sequence at least 85% identical thereto, a light chain variable domain of a second immunoglobulin (V D2 ) consisting of the amino acid sequence SEQ ID NO : 308 or a sequence at least 85% identical thereto, a heavy chain variable domain the second immunoglobulin (V D3 ) consisting of the amino acid sequence SEQ ID NO : 312 or a sequence at least 85% identical thereto and a heavy chain variable domain of the first immunoglobulin (V D4 ) consisting of the amino acid sequence SEQ ID NO : 23 or a sequence at least 85% identical thereto, or e) a light chain variable domain of a first immunoglobulin (V D1 ) consisting of the amino acid sequence SEQ ID NO: 308 or a sequence at least 85% identical thereto, a light chain variable domain of a second immunoglobulin (V D2 ) consisting of the amino acid sequence SEQ ID NO : 143 or a sequence at least 85% identical thereto, a heavy chain variable domain of the second immunoglobulin (V D3 ) consisting of the amino acid sequence SEQ ID NO : 138 or a sequence at least 85% identical thereto and a heavy chain variable domain of the first immunoglobulin (V D4 ) consisting of the amino acid sequence SEQ ID NO: 312 or a sequence at least 85% identical thereto, or f) a light chain variable domain of a first immunoglobulin (V D1 ) consisting of the amino acid sequence SEQ ID NO: 158 or a sequence at least 85% identical thereto, a light chain variable domain of a second immunoglobulin (V D2 ) consisting of the amino acid sequence SEQ ID NO : 308 or a sequence at least 85% identical thereto, a heavy chain variable domain of the second immunoglobulin (V D3 ) consisting of the amino acid sequence SEQ ID NO : 312 or a sequence at least 85% identical thereto and a heavy chain variable domain of a first immunoglobulin (V D4 ) consisting of the amino acid sequence SEQ ID NO : 171 or a sequence at least 85% identical thereto, g) a light chain variable domain of a first immunoglobulin (V D1 ) consisting of the amino acid sequence SEQ ID NO: 230 or a sequence at least 85% identical thereto, a light chain variable domain of a second immunoglobulin (V D2 ) consisting of the amino acid sequence SEQ ID NO : 158 or a sequence at least 85% identical thereto, a heavy chain variable domain of the second immunoglobulin (V D3 ) consisting of the amino acid sequence SEQ ID NO : 171 or a sequence at least 85% identical thereto and a heavy chain variable domain of the first immunoglobulin (V D4 ) consisting of the amino acid sequence SEQ ID NO : 226 or a sequence at least 85% identical thereto, h) a light chain variable domain of a first immunoglobulin (V D1 ) consisting of the amino acid sequence SEQ ID NO: 385 or a sequence at least 85% identical thereto, a light chain variable domain of a second immunoglobulin (V D2 ) consisting of the amino acid sequence SEQ ID NO : 141 or a sequence at least 85% identical thereto, a heavy chain variable domain of the second immunoglobulin (V D3 ) consisting of the amino acid sequence SEQ ID NO : 138 or a sequence at least 85% identical thereto and a heavy chain variable domain of the first immunoglobulin (V D4 ) consisting of the amino acid sequence SEQ ID NO : 383 or a sequence at least 85% identical thereto.

[0319] In said above sequence at least 85% identical to a reference sequence (e;g. a sequence at least 85% identical to SEQ ID NO: 383 or SEQ ID NO: 385), the sequences of the 6 CDRs are unchanged compared to the 6 CDRs present in the reference sequence.

[0320] In one embodiment the antibody binding protein according to any of the definitions a) to g) further comprises the linker L 1 of sequence SEQ ID NO: 307, L 2 of sequence SEQ ID NO: 309, L 3 of the amino acid sequence 'S', L 4 of the amino acid sequence 'RT' and C H1 of sequence SEQ ID NO: 313.

[0321] In one embodiment, the antibody binding protein according to any of the definitions a) to g) further comprises F c2 of sequence SEQ ID NO: 327.

[0322] In one embodiment, the antibody binding protein according to definition h) further comprises the linker L 1 of sequence SEQ ID NO: 389, L 2 of sequence SEQ ID NO: 389, L 3 and L 4 consisting of 0 amino acid, and C H1 of sequence SEQ ID NO: 313.

[0323] In one embodiment, the antibody binding protein according to definition h) further comprises F c2 of sequence SEQ ID NO: 392.

[0324] In one embodiment, L 5 of the antibody binding protein according to any of the definitions a) to h) contains 0 amino acids.

[0325] In a further embodiment the antibody binding protein according to any of the definitions a) to g) further comprises the linker L 1 of sequence SEQ ID NO: 307, L 2 of sequence SEQ ID NO: 309, L 3 of the amino acid sequence 'S', L 4 of the amino acid sequence 'RT', C H1 of sequence SEQ ID NO: 329 and F c of sequence SEQ ID NO: 330.

[0326] In a further embodiment, the antibody binding protein according to definition h) further comprises the linker L 1 of sequence SEQ ID NO: 389, L 2 of sequence SEQ ID NO: 389, L 3 and L 4 consisting of 0 amino acid, C H1 of sequence SEQ ID NO: 313 and F c of sequence SEQ ID NO: 394.

[0327] In a further embodiment, the antibody binding protein according to definition h) further comprises a Fc stump of sequence SEQ ID NO: 397 or SEQ ID NO: 398, or a sequence at least 85% identical to SEQ ID NO: 397 or SEQ ID NO: 398.

[0328] In an embodiment, the antibody binding protein that binds specifically to human CD3ε and human CD123 comprises, or essentially consists of: a) one polypeptide of formula [I] consisting of sequence SEQ ID NO: 388 (V D1 of sequence SEQ ID NO: 385, L 1 of sequence SEQ ID NO: 389, V D2 of sequence SEQ ID NO: 141, L 2 of sequence SEQ ID NO: 389 and C L of sequence SEQ ID NO: 310), or a sequence at least 85% identical to SEQ ID NO: 388 in which the 3 CDRs of sequences SEQ ID NO: 378, 'WAS' and SEQ ID NO: 379 of hz7G3 light chain variable domain (V D1 of sequence SEQ ID NO: 385), and the 3 CDRs of sequences. SEQ ID NO:142, 'KVS' and SEQ ID NO:11 of hz20G6 light chain variable domain (V D2 of sequence SEQ ID NO: 141) are unaltered; and b) one polypeptide of formula [II] consisting of sequence SEQ ID NO: 390 (V D3 of sequence SEQ ID NO: 138, L 3 is 0 amino acid, V D4 of sequence SEQ ID NO: 383, L 4 is 0 amino acid and C H1 of sequence SEQ ID NO: 313), or a sequence at least 85% identical to SEQ ID NO: 390 in which the 3 CDRs of sequences SEQ ID NO:381, SEQ ID NO:384, and SEQ ID NO: 382 of the hz7G3 heavy chain variable domain (V D4 of sequence SEQ ID NO: 383), and the 3 CDRs of sequences SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 of hz20G6 heavy chain variable domain (V D3 of sequence SEQ ID NO: 138) are unaltered; and wherein the polypeptide of formula [I] and the polypeptide of formula [II] form a cross-over light chain-heavy chain pair.

[0329] In an embodiment, the antibody binding protein that binds specifically to human CD3ε and human CD123 comprises, or essentially consists of: a) one polypeptide according to formula [IV] of the amino acid sequence SEQ ID NO: 391 (V D1 of sequence SEQ ID NO: 385, L 1 of sequence SEQ ID NO: 389, V D2 of sequence SEQ ID NO: 141, L 2 of sequence SEQ ID NO: 389, C L of sequence SEQ ID NO: 310, L 5 which contains 0 amino acid, and F c2 of sequence SEQ ID NO: 392), or a sequence at least 85% identical to SEQ ID NO: 391 in which the 3 CDRs of sequences SEQ ID NO: 378, 'WAS' and SEQ ID NO: 379 of hz7G3 light chain variable domain (V D1 of sequence SEQ ID NO: 385), and the 3 CDRs of sequences. SEQ ID NO:142, 'KVS' and SEQ ID NO:11 of hz20G6 light chain variable domain (V D2 of sequence SEQ ID NO: 141) are unaltered; and b) one polypeptide according to formula [III] of the amino acid sequence SEQ ID NO: 393 (V D3 of sequence SEQ ID NO: 138, L 3 is 0 amino acid, V D4 of sequence SEQ ID NO: 383, L 4 is 0 amino acid, C H1 of sequence SEQ ID NO: 313, and and F c of sequence SEQ ID NO: 394), or a sequence at least 85% identical to SEQ ID NO : 393 in which the the 3 CDRs of sequences SEQ ID NO:381, SEQ ID NO:384, and SEQ ID NO: 382 of the hz7G3 heavy chain variable domain (V D4 of sequence SEQ ID NO: 383), and the 3 CDRs of sequences SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 of hz20G6 heavy chain variable domain (V D3 of sequence SEQ ID NO: 138) are unaltered; and wherein the polypeptide of formula [IV] and the polypeptide of formula [III] form a cross-over light chain-heavy chain pair.

[0330] In said antibody like binding protein, the polypeptide chains represented by the formulae [III] and [IV] dimerize through their respective F c2 and F c regions.

[0331] In an embodiment, the antibody binding protein that binds specifically to human CD3ε and human CD123 comprises, or essentially consists of: a) one polypeptide according to formula [I] consisting of the amino acid sequence SEQ ID NO: 388 (V D1 of sequence SEQ ID NO: 385, L 1 of sequence SEQ ID NO: 389, V D2 of sequence SEQ ID NO: 141, L 2 of sequence SEQ ID NO: 389 and C L of sequence SEQ ID NO: 310) or a sequence at least 85% identical to SEQ ID NO: 388 in which the 3 CDRs of sequences SEQ ID NO: 378, 'WAS' and SEQ ID NO: 379 of hz7G3 light chain variable domain (VD1 of sequence SEQ ID NO: 385), and the 3 CDRs of sequences. SEQ ID NO:142, 'KVS' and SEQ ID NO:11 of hz20G6 light chain variable domain (VD2 of sequence SEQ ID NO: 141) are unaltered; and b) one polypeptide according to formula [III] of the amino acid sequence SEQ ID NO: 395 (V D3 of sequence SEQ ID NO: 138, L 3 is 0 amino acid, V D4 of sequence SEQ ID NO: 383, L 4 is 0 amino acid, C H1 of sequence SEQ ID NO: 313, and F c of sequence SEQ ID NO: 396), or a sequence at least 85% identical to SEQ ID NO : 395 in which the the 3 CDRs of sequences SEQ ID NO:381, SEQ ID NO:384, and SEQ ID NO: 382 of the hz7G3 heavy chain variable domain (V D4 of sequence SEQ ID NO: 383), and the 3 CDRs of sequences SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 of hz20G6 heavy chain variable domain (V D3 of sequence SEQ ID NO: 138) are unaltered; and c) one Fc stump (polypeptide F c3 ) of the amino acid sequence SEQ ID NO : 397, or a sequence at least 85% identical thereto, wherein said F c3 stump or the sequence at least 85% identical thereto heterodimerises with the F c region of the polypeptide according to formula [III]; and wherein the polypeptide of formula [I] and the polypeptide of formula [III] form a cross-over light chain-heavy chain pair.

[0332] In an embodiment, the antibody binding protein that binds specifically to human CD3ε and human CD123 comprises, or essentially consists of: a) one polypeptide according to formula [I] consisting of the amino acid sequence SEQ ID NO: 388 (V D1 of sequence SEQ ID NO: 385, L 1 of sequence SEQ ID NO: 389, V D2 of sequence SEQ ID NO: 141, L 2 of sequence SEQ ID NO: 389 and C L of sequence SEQ ID NO: 310) or a sequence at least 85% identical to SEQ ID NO: 388 in which the 3 CDRs of sequences SEQ ID NO: 378, 'WAS' and SEQ ID NO: 379 of hz7G3 light chain variable domain (V D1 of sequence SEQ ID NO: 385), and the 3 CDRs of sequences. SEQ ID NO:142, 'KVS' and SEQ ID NO:11 of hz20G6 light chain variable domain (V D2 of sequence SEQ ID NO: 141) are unaltered; b) a polypeptide according to formula III of the amino acid sequence SEQ ID NO: 399 (V D3 of sequence SEQ ID NO: 138, L 3 is 0 amino acid, V D4 of sequence SEQ ID NO: 383, L 4 is 0 amino acid, C H1 of sequence SEQ ID NO: 313, and F c of sequence SEQ ID NO: 400), or a sequence at least 85% identical thereto in which the the 3 CDRs of sequences SEQ ID NO:381, SEQ ID NO:384, and SEQ ID NO: 382 of the hz7G3 heavy chain variable domain (V D4 of sequence SEQ ID NO: 383), and the 3 CDRs of sequences SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 of hz20G6 heavy chain variable domain (V D3 of sequence SEQ ID NO: 138) are unaltered; and c) one Fc stump (polypeptide F c3 ) of the amino acid sequence SEQ ID NO : 398, or a sequence at least 85% identical thereto, wherein said F c3 stump or the sequence at least 85% identical thereto heterodimerises with the Fc region of the polypeptide according to formula [III]; and wherein the polypeptide of formula [I] and the polypeptide of formula [III] form a cross-over light chain-heavy chain pair.Immunoconjugates

[0333] In one embodiment, the anti-CD123 antibody of the invention is conjugated or linked to a growth inhibitory agent, cytotoxic agent, or a prodrug-activating enzyme. In particular, anti-CD123 antibodies of the invention are indeed useful for targeting said growth inhibitory agent, cytotoxic agent, or a prodrug to the cancerous cells expressing or over-expressing CD123 on their surface.Nucleic acids, vectors and recombinant host cells

[0334] A further object of the invention relates to a nucleic acid sequence comprising or consisting of a sequence encoding an anti-CD3 antibody, anti-CD123 antibody or an antibody-like binding protein as defined above.

[0335] Typically, said nucleic acid is a DNA or RNA molecule, which may be included in any suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or a viral vector.

[0336] The terms "vector", "cloning vector" and "expression vector" mean the vehicle by which a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence.

[0337] So, a further object of the invention relates to a vector comprising a nucleic acid of the invention.

[0338] Such vectors may comprise regulatory elements, such as a promoter, enhancer, terminator and the like, to cause or direct expression of said polypeptide upon administration to a subject. Examples of promoters and enhancers used in the expression vector for animal cell include early promoter and enhancer of SV40 (Mizukami T. et al. 1987), LTR promoter and enhancer of Moloney mouse leukemia virus (Kuwana Y et al. 1987), promoter (Mason JO et al. 1985) and enhancer (Gillies SD et al. 1983) of immunoglobulin H chain and the like.

[0339] Any expression vector for animal cell can be used, so long as a gene encoding the human antibody C region can be inserted and expressed. Examples of suitable vectors include pAGE107 (Miyaji H et al. 1990), pAGE103 (Mizukami T et al. 1987), pHSG274 (Brady G et al. 1984), pKCR (O'Hare K et al. 1981), pSG1 beta d2-4-(Miyaji H et al. 1990) and the like. Other examples of plasmids include replicating plasmids comprising an origin of replication, or integrative plasmids, such as for instance pUC, pcDNA, pBR, and the like.

[0340] Other examples of viral vector include adenoviral, retroviral, herpes virus and AAV vectors. Such recombinant viruses may be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc. Detailed protocols for producing such replication-defective recombinant viruses may be found for instance in WO 95 / 14785, WO 96 / 22378, US 5,882,877, US 6,013,516, US 4,861,719, US 5,278,056 and WO 94 / 19478.

[0341] A further object of the present invention relates to a cell which has been transfected, infected or transformed by a nucleic acid and / or a vector according to the invention.

[0342] The term "transformation" means the introduction of a "foreign" (i.e. extrinsic) gene, DNA or RNA sequence to a host cell, so that the host cell will express the introduced gene or sequence to produce a desired substance, typically a protein or enzyme coded by the introduced gene or sequence. A host cell that receives and expresses introduced DNA or RNA bas been "transformed".

[0343] The nucleic acids of the invention may be used to produce a recombinant antibody of the invention in a suitable expression system. The term "expression system" means a host cell and compatible vector under suitable conditions, e.g. for the expression of a protein coded for by foreign DNA carried by the vector and introduced to the host cell.

[0344] Common expression systems include E. coli host cells and plasmid vectors, insect host cells and Baculovirus vectors, and mammalian host cells and vectors. Other examples of host cells include, without limitation, prokaryotic cells (such as bacteria) and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include E. coli, Kluyveromyces or Saccharomyces yeasts, mammalian cell lines (e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.) as well as primary or established mammalian cell cultures (e.g., produced from lymphoblasts, fibroblasts, embryonic cells, epithelial cells, nervous cells, adipocytes, etc.). Examples also include mouse SP2 / 0-Ag14 cell (ATCC CRL1581), mouse P3X63-Ag8.653 cell (ATCC CRL1580), CHO cell in which a dihydrofolate reductase gene (hereinafter referred to as "DHFR gene") is defective (Urlaub G et al; 1980), rat YB2 / 3HL.P2.G11.16Ag.20 cell (ATCC CRL1662, hereinafter referred to as "YB2 / 0 cell"), and the like. The YB2 / 0 cell is preferred, since ADCC activity of chimeric or humanized antibodies is enhanced when expressed in this cell.

[0345] In particular, for expression of humanized antibody or antibody-like binding protein, the expression vector may be either of a type in which a gene encoding an antibody heavy chain and a gene encoding an antibody light chain exists on separate vectors or of a type in which both genes exist on the same vector (tandem type). In respect of easiness of construction of a humanized antibody and antibody-like binding protein expression vector, easiness of introduction into animal cells, and balance between the expression levels of antibody H and L chains in animal cells, humanized antibody expression vector of the tandem type is preferred (Shitara K et al. J Immunol Methods. 1994 Jan. 3;167(1-2):271-8). Examples of tandem type humanized antibody expression vector include pKANTEX93 (WO 97 / 10354), pEE18 and the like.

[0346] The present invention also relates to a method of producing a recombinant host cell expressing an anti-CD3 antibody, anti-CD123 antibody or an antibody-like binding protein according to the invention, said method comprising the steps consisting of : (i) introducing in vitro or ex vivo a recombinant nucleic acid or a vector as described above into a competent host cell, (ii) culturing in vitro or ex vivo the recombinant host cell obtained and (iii), optionally, selecting the cells which express and / or secrete said antibody.

[0347] Such recombinant host cells can be used for the production of anti-CD3 antibody, at least one anti-CD123 antibody or at least one antibody-like binding protein of the invention.Methods of producing antibodies antibody-like binding protein of the invention

[0348] One embodiment of the invention provides a method for making an antibody-like binding protein comprising two polypeptide chains that form two antigen-binding sites, wherein a first polypeptide has a structure represented by the formula [I]:         V D1 -L 1 -V D2 -L 2 -C L      [I] and a second polypeptide has a structure represented by the formula [II]:         V D3 -L 3 -V D4 -L 4 -C H1      [II] wherein: V D1 is a variable domain of heavy or light chain of a first immunoglobulin; V D2 is a variable domain of heavy or light chain of a second immunoglobulin; V D3 is a variable domain of heavy or light chain of said second immunoglobulin; V D4 is a variable domain of heavy or light chain of said first immunoglobulin; C L is a light chain constant domain of an immunoglobulin; C H1 is a C H1 heavy chain constant domain of an immunoglobulin; L 1 , L 2 , L 3 , and L 4 are amino acid linkers; and wherein the first and the second polypeptide form a cross-over light chain-heavy chain pair, and wherein VD1 and VD2 are both either variable domains of light chains, or variable domains of heavy chains, and VD3 and VD4 are both variable domains of heavy chains if VD1 and VD2 are variable domains of light chains, or VD3 and VD4 are both variable domains of light chains if VD1 and VD2 are variable domains of heavy chains.

[0349] In a further embodiment, the invention provides a method for making an antibody-like binding protein comprising four polypeptide chains that form four antigen-binding sites, wherein two polypeptide chains have a structure represented by the formula [I]:         V D1 -L 1 -V D2 -L 2 -C L      [I] and two polypeptide chains have a structure represented by the formula (III):         V D3 -L 3 -V D4 -L 4 -C H1 -F c      [III] wherein: V D1 is a variable domain of heavy or light chain of a first immunoglobulin; V D2 is a variable domain of heavy or light chain of a second immunoglobulin; V D3 is a variable domain of heavy or light chain of said second immunoglobulin; V D4 is a variable domain of heavy or light chain of said first immunoglobulin; C L is a light chain constant domain of an immunoglobulin; C H1 is a C H1 heavy chain constant domain of an immunoglobulin; F c is the immunoglobulin hinge region and CH 2 , CH 3 immunoglobulin heavy chain constant domains of an immunoglobulin; L 1 , L 2 , L 3 , and L 4 are amino acid linkers; and wherein the polypeptides of formula I and the polypeptides of formula III form a cross-over light chain-heavy chain pair, and wherein VD1 and VD2 are both either variable domains of light chains, or variable domains of heavy chains, and VD3 and VD4 are both variable domains of heavy chains if VD1 and VD2 are variable domains of light chains, or VD3 and VD4 are both variable domains of light chains if VD1 and VD2 are variable domains of heavy chains.

[0350] In a further embodiment, the invention provides a method for making an antibody-like binding protein comprising four polypeptide chains that form four antigen-binding sites, wherein two polypeptide chains have a structure represented by the formula [IV]:         V D1 -L 1 -V D2 -L 2 -C L -L 5 -F c2      [IV] and two polypeptide chains have a structure represented by the formula [III]:         V D3 -L 3 -V D4 -L 4 -C H1 -F c      [III] wherein: V D1 is a variable domain of heavy or light chain of a first immunoglobulin; V D2 is a variable domain of heavy or light chain of a second immunoglobulin; V D3 is a variable domain of heavy or light chain of said second immunoglobulin; V D4 is a variable domain of heavy or light chain of said first immunoglobulin; C L is a light chain constant domain of an immunoglobulin; C H1 is a C H1 heavy chain constant domain of an immunoglobulin; F c is the immunoglobulin hinge region and CH 2 , CH 3 immunoglobulin heavy chain constant domains of an immunoglobulin; F c2 is the immunoglobulin hinge region and CH 2 , CH 3 immunoglobulin heavy chain constant domains of an immunoglobulin; L 1 , L 2 , L 3 , L 4 and L 5 are amino acid linkers; and wherein the polypeptides of formula I and the polypeptides of formula III form a cross-over light chain-heavy chain pair, and wherein VD1 and VD2 are both either variable domains of light chains, or variable domains of heavy chains, and VD3 and VD4 are both variable domains of heavy chains if VD1 and VD2 are variable domains of light chains, or VD3 and VD4 are both variable domains of light chains if VD1 and VD2 are variable domains of heavy chains.

[0351] In one embodiment of the invention, the first immunoglobulin or the second immunoglobulin is one anti-CD3 antibody as defined in the section «anti-CD3 antibodies» above.

[0352] In another embodiment of the invention, the first immunoglobulin or the second immunoglobulin is one anti-CD123 antibody as defined in the section «anti-CD123 antibodies» above.

[0353] An Anti-CD3 antibody, anti-CD123 antibody and / or antibody-like binding proteins of the invention may be produced by any technique known in the art, such as, without limitation, any chemical, biological, genetic or enzymatic technique, either alone or in combination.

[0354] Knowing the amino acid sequence of the desired sequence, one skilled in the art can readily produce said antibodies or immunoglobulin chains, by standard techniques for production of polypeptides. For instance, they can be synthesized using well-known solid phase method, in particular using a commercially available peptide synthesis apparatus (such as that made by Applied Biosystems, Foster City, California) and following the manufacturer's instructions. Alternatively, antibodies, immunoglobulin chains and antibody-like binding proteins of the invention can be synthesized by recombinant DNA techniques as is well-known in the art. For example, these fragments can be obtained as DNA expression products after incorporation of DNA sequences encoding the desired (poly)peptide into expression vectors and introduction of such vectors into suitable eukaryotic or prokaryotic hosts that will express the desired polypeptide, from which they can be later isolated using well-known techniques.

[0355] In particular, the invention further relates to a method of producing anti-CD3 antibodies, anti-CD123 antibodies and / or antibody-like binding proteins of the invention, which method comprises the steps consisting of: (i) culturing a transformed host cell according to the invention; (ii) expressing said antibody or polypeptide; and (iii) recovering the expressed antibody or polypeptide.

[0356] Anti-CD3 antibodies, anti-CD123 antibodies and / or antibody-like binding proteins of the invention are suitably separated from the culture medium by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0357] In a particular embodiment, a humanized chimeric anti-CD3 antibodies and / or anti-CD123 of the present invention can be produced by obtaining nucleic sequences encoding humanized VL and VH domains as previously described, constructing a human chimeric antibody expression vector by inserting them into an expression vector for animal cell having genes encoding human antibody CH and human antibody CL, and expressing the coding sequence by introducing the expression vector into an animal cell. In analogy thereto, a humanized antibody-like binding protein can be obtained by using for the variable domain of heavy or light chain of a first humanized immunoglobulin (V D1 ), for the variable domain of heavy or light chain of a second humanized immunoglobulin (V D2 ), for the variable domain of heavy or light chain of said second immunoglobulin (V D3 ), and for the variable domain of heavy or light chain of said first immunoglobulin the variable domains of the heavy and light chains of two humanized antibodies.

[0358] As the CH domain of a human chimeric antibody or the CH domain of antibody-like binding protein of the invention, it may be any region which belongs to human immunoglobulin heavy chains, but those of IgG class are suitable and any one of subclasses belonging to IgG class, such as IgG1, IgG2, IgG3 and IgG4, can also be used. Also, as the CL of a human chimeric antibody or the CL of an antibody-like binding protein of the invention, it may be any region which belongs to human immunoglobulin light chains, and those of kappa class or lambda class can be used.

[0359] Methods for producing humanized or chimeric antibodies involve conventional recombinant DNA and gene transfection techniques are well known in the art (See Morrison SL. et al. (1984) and patent documents US5,202,238; and US5,204, 244).

[0360] Methods for producing humanized antibodies based on conventional recombinant DNA and gene transfection techniques are well known in the art (See, e. g., Riechmann L. et al. 1988; Neuberger MS. et al. 1985). Antibodies can be humanized using a variety of techniques known in the art including, for example, the technique disclosed in the application WO2009 / 032661, CDR-grafting (EP 239,400; PCT publication WO91 / 09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan EA (1991); Studnicka GM et al. (1994); Roguska MA. et al. (1994)), and chain shuffling (U.S. Pat. No.5,565,332). The general recombinant DNA technology for preparation of such antibodies is also known (see European Patent Application EP 125023 and International Patent Application WO 96 / 02576).

[0361] The Fab of the present invention can be obtained by treating an antibody which specifically reacts with CD3 or CD123 with a protease, such as papaine. Also, the Fab can be produced by inserting DNA sequences encoding both chains of the Fab of the antibody into a vector for prokaryotic expression, or for eukaryotic expression, and introducing the vector into procaryotic or eukaryotic cells (as appropriate) to express the Fab.

[0362] The F(ab')2 of the present invention can be obtained treating an antibody which specifically reacts with CD3 or CD123 with a protease, pepsin. Also, the F(ab')2 can be produced by binding Fab' described below via a thioether bond or a disulfide bond.

[0363] The Fab' of the present invention can be obtained treating F(ab')2 which specifically reacts with CD3 or CD123 with a reducing agent, such as dithiothreitol. Also, the Fab' can be produced by inserting DNA sequences encoding Fab' chains of the antibody into a vector for prokaryotic expression, or a vector for eukaryotic expression, and introducing the vector into prokaryotic or eukaryotic cells (as appropriate) to perform its expression.

[0364] The scFv of the present invention can be produced by taking sequences of the CDRs or VH and VL domains as previously described, constructing a DNA encoding an scFv fragment, inserting the DNA into a prokaryotic or eukaryotic expression vector , and then introducing the expression vector into prokaryotic or eukaryotic cells (as appropriate) to express the scFv. To generate a humanized scFv fragment, a well known technology called CDR grafting may be used, which involves selecting the complementary determining regions (CDRs) according to the invention, and grafting them onto a human scFv fragment framework of known three dimensional structure (see, e. g., WO98 / 45322; WO 87 / 02671; US5,859,205; US5,585,089; US4,816,567; EP0173494).Modification of the antibodies of the invention

[0365] Amino acid sequence modification(s) of the antibodies or antibody-like binding proteins as described herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody nor or antibody-like binding protein. For instance, it is known that when a humanized antibody is produced by simply grafting only CDRs in VH and VL of an antibody derived from a non-human animal in FRs of the VH and VL of a human antibody, the antigen binding activity may be reduced in comparison with that of the original antibody derived from a non-human animal. It is considered that several amino acid residues of the VH and VL of the non-human antibody, not only in CDRs but also in FRs, may be directly or indirectly associated with the antigen binding activity. Hence, substitution of these amino acid residues with different amino acid residues derived from FRs of the VH and VL of the human antibody would reduce the binding activity. In order to solve the problem, in human antibodies grafted with non-human CDRs, attempts have to be made to identify, among amino acid sequences of the FR of the VH and VL of human antibodies, an amino acid residue which is directly associated with binding of the antibody, or which interacts with an amino acid residue of a CDR, or which maintains the three-dimensional structure of the antibody and which is directly associated with binding to the antigen. The reduced antigen binding activity could be increased by replacing the identified amino acids with amino acid residues of the original antibody derived from a non-human animal. An antibody-like binding protein of the invention may comprise the variable regions of a humanized antibody and therefore herein mentioned considerations apply equally to antibody-like binding proteins of the invention.

[0366] Modifications and changes may be made in the structure of the antibodies of the present invention, and in the DNA sequences encoding them, and still result in a functional antibody, antibody-like binding protein or polypeptide with desirable characteristics.

[0367] In making the changes in the amino sequences of polypeptide, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art. It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8) ; phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophane (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate -3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0368] A further object of the present invention also encompasses function-conservative variants of the polypeptides of the anti-CD3 antibodies, anti-CD123 antibodies and antibody-like binding proteins of the present invention.

[0369] For example, certain amino acids may be substituted by other amino acids in a protein structure without appreciable loss of activity. Since the interactive capacity and nature of a protein define its biological functional activity, certain amino acid substitutions can be made in a protein sequence, and of course in its DNA encoding sequence, while nevertheless obtaining a protein with like properties. It is thus contemplated that various changes may be made in the antibodies sequences of the invention, or corresponding DNA sequences which encode said polypeptides, without appreciable loss of their biological activity.

[0370] It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e. still obtain a biological functionally equivalent protein. It is also possible to use well-established technologies, such as alanine-scanning approaches, to identify, in an antibody or polypeptide of the invention, all the amino acids that can be substituted without significant loss of binding to the antigen. Such residues can be qualified as neutral, since they are not involved in antigen binding or in maintaining the structure of the antibody. One or more of these neutral positions can be substituted by alanine or by another amino acid can without changing the main characteristics of the antibody or polypeptide of the invention.

[0371] As outlined above, amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions which take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

[0372] It may be also desirable to modify the anti-CD3 antibody, anti-CD123 antibody and antibody-like binding protein of the present invention with respect to effector function, e.g. so as to enhance or reduce antigen-dependent cell-mediated cytotoxicity (ADCC) and / or complement dependent cytotoxicity (CDC) of the antibody. This may be achieved by introducing one or more amino acid substitutions in an Fc region of the antibody, herein also called Fc-variants in context with the antibody-like binding proteins of the present invention. Alternatively or additionally, cysteine residue(s) may be introduced in the Fc region, thereby allowing inter-chain disulfide bond formation in this region. The homodimeric antibody thus generated may have improved or reduced internalization capability and / or increased complement-mediated cell killing and / or antibody-dependent cellular cytotoxicity (ADCC) (Caron PC. et al. 1992; and Shopes B. 1992).

[0373] Another type of amino acid modification of the anti-CD3 antibody, anti-CD123 antibody and antibody-like binding protein of the invention may be useful for altering the original glycosylation pattern of the anti-CD3 antibody, anti-CD123 antibody and antibody-like binding protein, i.e. by deleting one or more carbohydrate moieties found in the anti-CD3 antibody, anti-CD123 antibody and antibody-like binding protein, and / or adding one or more glycosylation sites that are not present in the anti-CD3 antibody, anti-CD123 antibody and antibody-like binding protein. The presence of either of the tripeptide sequences asparagine-X-serine, and asparagine-X-threonine, where X is any amino acid except proline, creates a potential glycosylation site. Addition or deletion of glycosylation sites to the anti-CD3 antibody, anti-CD123 antibody and antibody-like binding protein is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites).

[0374] Another type of modification involves the removal of sequences identified, either in silico or experimentally, as potentially resulting in degradation products or heterogeneity of anti-CD3 antibody, anti-CD123 antibody and antibody-like binding protein preparations. As examples, deamidation of asparagine and glutamine residues can occur depending on factors such as pH and surface exposure. Asparagine residues are particularly susceptible to deamidation, primarily when present in the sequence Asn-Gly, and to a lesser extent in other dipeptide sequences such as Asn-Ala. When such a deamidation site, in particular Asn-Gly, is present in an anti-CD3 antibody, anti-CD123 antibody and antibody-like binding protein or polypeptide of the invention, it may therefore be desirable to remove the site, typically by conservative substitution to remove one of the implicated residues. Such substitutions in a sequence to remove one or more of the implicated residues are also intended to be encompassed by the present invention.

[0375] Another type of covalent modification involves chemically or enzymatically coupling glycosides to the anti-CD3 antibody, anti-CD123 antibody and antibody-like binding protein. These procedures are advantageous in that they do not require production of anti-CD3 antibody, anti-CD123 antibody or antibody-like binding protein in a host cell that has glycosylation capabilities for N-or O-linked glycosylation. Depending on the coupling mode used, the sugar(s) may 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, orhydroxyproline, (e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan, or (f) the amide group of glutamine. For example, such methods are described in WO87 / 05330.

[0376] Removal of any carbohydrate moieties present on the anti-CD3 antibody, anti-CD123 antibody or antibody-like binding protein may be accomplished chemically or enzymatically. Chemical deglycosylation requires exposure of the anti-CD3 antibody, anti-CD123 antibody or antibody-like binding 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 antibody intact. Chemical deglycosylation is described by Sojahr H. et al. (1987) and by Edge, AS. et al. (1981). Enzymatic cleavage of carbohydrate moieties on antibodies can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura, NR. et al. (1987).

[0377] Another type of covalent modification of the anti-CD3 antibody, anti-CD123 antibody or antibody-like binding protein comprises linking the antibody to one of a variety of non proteinaceous polymers, eg. , polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in the manner set forth in US Patent Nos. 4,640, 835; 4,496, 689; 4,301, 144; 4,670, 417; 4,791, 192 or 4,179,337.Pharmaceutical compositions

[0378] The anti-CD3 antibody, anti-CD123 antibody and / or antibody-like binding protein of the invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.

[0379] Thus, another object of the invention relates to a pharmaceutical composition comprising an anti-CD3 antibody, an anti-CD123 antibody or antibody-like binding protein of the invention and a pharmaceutically acceptable carrier.

[0380] The invention also relates to an anti-CD3 antibody, an anti-CD123 antibody or antibody-like binding protein according to the invention, for use as a medicament. The invention also relates to a pharmaceutical composition of the invention for use as a medicament.

[0381] The terms "pharmaceutical composition" or "therapeutic composition" as used herein refer to a compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient.

[0382] Such therapeutic or pharmaceutical compositions may comprise a therapeutically effective amount of an anti-CD3 antibody, an anti-CD123 antibody or antibody-like binding protein or drug conjugates thereof, in admixture with a pharmaceutically or physiologically acceptable formulation agent selected for suitability with the mode of administration.

[0383] "Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

[0384] As used herein, "pharmaceutically-acceptable carriers" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, and the like that are physiologically compatible. Examples of suitable carriers, diluents and / or excipients include one or more of water, amino acids, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, as well as combination thereof. In many cases, it will be preferable to include isotonic agents, such as sugars, polyalcohols, or sodium chloride in the composition and formulation may also contain an antioxidant such as tryptamine and a stabilizing agent such as Tween 20.

[0385] The form of the pharmaceutical compositions, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and gender of the patient, etc.

[0386] The pharmaceutical compositions of the invention can be formulated for a topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous or intraocular administration and the like.

[0387] In particular, the pharmaceutical compositions contain vehicles, which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.

[0388] The doses used for the administration can be adapted as a function of various parameters, and in particular as a function of the mode of administration used, of the relevant pathology, or alternatively of the desired duration of treatment.

[0389] To prepare pharmaceutical compositions, an effective amount of the antibody or immunoconjugate of the invention may be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

[0390] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

[0391] Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0392] A anti-CD3 antibody, anti-CD123 antibody or antibody-like binding of the invention can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, glycine, histidine, procaine and the like.

[0393] The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.

[0394] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0395] The preparation of more, or highly concentrated solutions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small tumor area.

[0396] Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.

[0397] For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

[0398] In one embodiment, an anti-CD3 antibody, anti-CD123 antibody or antibody-like binding protein of the invention is formulated within a therapeutic mixture to comprise about 0.01 to 100 milligrams, per dose or so.

[0399] In addition to the anti-CD3 antibody, anti-CD123 antibody or antibody-like binding protein formulated for parenteral administration, such as intravenous or intramuscular injection, other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; time-release capsules; and any other form currently used.

[0400] In certain embodiments, the use of liposomes and / or nanoparticles is contemplated for the introduction of polypeptides such as anti-CD3 antibody, anti-CD123 antibody or antibody-like binding protein into host cells. The formation and use of liposomes and / or nanoparticles are known to those of skill in the art.

[0401] Nanocapsules can generally entrap compounds in a stable and reproducible way. To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 µm) are generally designed using polymers able to be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present invention, and such particles may be are easily made.

[0402] Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs)). MLVs generally have diameters of from 25 nm to 4 µm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 Å, containing an aqueous solution in the core. The physical characteristics of liposomes depend on pH, ionic strength and the presence of divalent cations.

[0403] Once the pharmaceutical composition has been formulated, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. Such formulations can be stored either in a ready-to-use form or in a form (e.g., lyophilized) requiring reconstitution prior to administration.Therapeutic methods and uses

[0404] The inventors have shown for several bi-specific compounds of the invention, such as "hz20G6x7G3", "7G3xhz4B4","hz4B4x3E3" and "hz20G6x7G3-TL4" T-cell mediated cytotoxicity on a CD123 positive tumor cell line model. Furthermore, the inventors demonstrated the capacity of for several bi-specific compounds of the invention to activate T-cells in presence of target cells leading to cytotoxicity of the tumor cells. The inventors further demonstrated the low activation of T-cells in the absence of T-cell activation in absence of target cells.

[0405] It is well known that therapeutic anti-CD123 monoclonal antibodies can lead to the depletion of cells bearing the antigen specifically recognized by the antibody. This depletion can be mediated through at least three mechanisms: antibody mediated cellular cytotoxicity (ADCC), complement dependent cytotoxicity (CDC) and direct antitumour inhibition of tumour growth through signals given via the antigen targeted by the antibody. In one embodiment, the anti-CD123 antibodies of the invention induce cytotoxicity in a CD123 expressing cell by antibody mediated cellular cytotoxicity (ADCC), complement dependent cytotoxicity (CDC).

[0406] "Complement dependent cytotoxicity" or "CDC" refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system to antibodies which are bound to their cognate antigen. To assess complement activation, a CDC assay, e.g. as described in Gazzano-Santoro et al. (1997) may be performed.

[0407] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted antibodies bound onto Fc receptors (FcRs) present on certain cytotoxic cells (e.g. Natural Killer (NK) cells, neutrophils, and macrophages) enable these cytotoxic effector cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell. To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in US Patent No. 5,500,362 or 5,821,337 may be performed.

[0408] As described above in the section "anti-CD3 antibodies" the anti-CD3 antibodies of the invention have a low T-cell activation they thus have a therapeutic potential in a subject for use as an immunosuppressive agent.

[0409] Furthermore, an antibody-like binding protein as defined above aims at enhancing the patient's immune response to tumors by targeting T-cells to tumor cells. In one embodiment, the antibody-like binding protein as defined above targets CD3ε subunit of the T-cell Rece...

Claims

1. An antibody-like binding protein comprising two polypeptide chains that form two antigen-binding sites, wherein a first polypeptide has a structure represented by the formula [I]:         VD1-L1-VD2-L2-CL     [I] and a second polypeptide has a structure represented by the formula [II]:         VD3-L3-VD4-L4-CH1     [II] wherein: VD1 is a variable domain of heavy or light chain of a first immunoglobulin; VD2 is a variable domain of heavy or light chain of a second immunoglobulin; VD3 is a variable domain of heavy or light chain of said second immunoglobulin; VD4 is a variable domain of heavy or light chain of said first immunoglobulin; CL is a light chain constant domain of an immunoglobulin; CH1 is a CH1 heavy chain constant domain of an immunoglobulin; L1, L2, L3, and L4 are amino acid linkers; and wherein the first and the second polypeptide form a cross-over light chain-heavy chain pair; wherein VD1 and VD2 are both either variable domains of light chains, or variable domains of heavy chains, and VD3 and VD4 are both variable domains of heavy chains if VD1 and VD2 are variable domains of light chains, or VD3 and VD4 are both variable domains of light chains if VD1 and VD2 are variable domains of heavy chains; and wherein the polypeptide of formula [II] further comprises a Fc domain, or the polypeptide of formula [II] further comprises a Fc domain and the polypeptide of formula [I] further comprises a Fc domain (Fc2).

2. The antibody-like binding protein according to claim 1, wherein the polypeptide of formula [II] further comprises a Fc domain and wherein the antibody-like binding protein comprises a third polypeptide chain comprising a Fc domain (Fc3).

3. The antibody-like binding protein according to either claim 1 or 2, wherein the antibody-like binding protein binds specifically to human CD3ε.

4. The antibody-like binding protein according to claim 3, wherein the VD1 and VD4, or VD2 and VD3 comprise a heavy chain variable domain and a light chain variable domain of an antibody comprising: a) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 6, CDR2-H of sequence SEQ ID NO: 7, and CDR3-H of sequence SEQ ID NO: 8; and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 142, CDR2-L of sequence 'KVS', and CDR3-L of sequence SEQ ID NO: 11; or b) a heavy chain variable domain comprising the sequence SEQ ID NO: 138 or a sequence at least 85% identical to SEQ ID NO: 138 in which the 3 CDRs of sequences SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 are unaltered; and a light chain variable domain comprising the sequence SEQ ID NO: 141 or a sequence at least 85% identical to SEQ ID NO: 143 in which the 3 CDRs of sequences SEQ ID NO: 142, 'KVS', and SEQ ID NO: 11 are unaltered.

5. The antibody-like binding protein according to claim 3, wherein the antibody-like binding protein binds to at least one further target antigen.

6. The antibody-like binding protein according to either claim 1 or 2, wherein the antibody-like binding protein binds specifically to CD123.

7. The antibody-like binding protein according to either claim 3 or 6 wherein the antibody-like binding protein comprises a light chain variable domain of a first immunoglobulin (VD1) consisting of the amino acid sequence SEQ ID NO: 385 or a sequence at least 85% identical thereto, a light chain variable domain of a second immunoglobulin (VD2) consisting of the amino acid sequence SEQ ID NO : 141 or a sequence at least 85% identical thereto, a heavy chain variable domain of the second immunoglobulin (VD3) consisting of the amino acid sequence SEQ ID NO : 138 or a sequence at least 85% identical thereto, and a heavy chain variable domain of the first immunoglobulin (VD4) consisting of the amino acid sequence SEQ ID NO : 383 or a sequence at least 85% identical thereto.

8. The antibody-like binding protein according to claim 6, wherein the VD1 and VD4, or VD2 and VD3 comprise a heavy chain variable domain and a light chain variable domain of an antibody comprising a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 381, CDR2-H of sequence SEQ ID NO: 384, and CDR3-H of sequence SEQ ID NO: 382; and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 378, CDR2-L of sequence 'WAS', and CDR3-L of sequence SEQ ID NO: 379.

9. An antibody-like binding protein according to claim 1 or 2 that binds specifically to human CD123 and at least one further target antigen, wherein the antibody-like binding protein comprises a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 381, CDR2-H of sequence SEQ ID NO: 384, and CDR3-H of sequence SEQ ID NO: 382; and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 378, CDR2-L of sequence 'WAS', and CDR3-L of sequence SEQ ID NO: 379.

10. An antibody-like binding protein according to claim 1 or 2 that binds specifically to human CD3ε and at least one further target antigen, wherein the antibody-like binding protein comprises: a) a heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 6, CDR2-H of sequence SEQ ID NO: 7, and CDR3-H of sequence SEQ ID NO: 8; and a light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 142, CDR2-L of sequence 'KVS', and CDR3-L of sequence SEQ ID NO: 11; or b) a heavy chain variable domain comprising the sequence SEQ ID NO: 138 or a sequence at least 85% identical to SEQ ID NO: 138 in which the 3 CDRs of sequences SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 are unaltered; and a light chain variable domain comprising the sequence SEQ ID NO: 141 or a sequence at least 85% identical to SEQ ID NO: 143 in which the 3 CDRs of sequences SEQ ID NO: 142, 'KVS', and SEQ ID NO: 11 are unaltered.

11. A pharmaceutical composition comprising an antibody-like binding protein according to any one of claims 1 to 10 and a pharmaceutically acceptable carrier.

12. An antibody-like binding protein according to any one of claims 1 to 10 or a pharmaceutical composition according to claim 11 for use as a medicament.

13. An antibody-like binding protein according to any one of claims 1 to 10 or a pharmaceutical composition according to claim 11 for use in a method of treating or preventing a disease or disorder.

14. An isolated nucleic acid comprising a sequence encoding an antibody-like binding protein according to any one of claims 1 to 10.

15. A host cell which has been transformed by a nucleic acid according to claim 14.

16. A kit comprising at least one antibody-like binding protein according to any one of claims 1 to 10.

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