Anti-CD3 antibody, anti-CD123 antibody, and bispecific antibody that specifically binds to CD3 and / or CD123

Abstract

To provide antibody-like binding proteins that efficiently target and kill cancer cells.SOLUTION: Disclosed is an antibody-like binding protein comprising two polypeptide chains that form two antigen binding sites, where the first polypeptide chain has a structure represented by VD1-L1-VD2-L2-CL-L5-Fc2, and a second polypeptide chain has a structure represented by VD3-L3-VD4-L4-CH1-Fc, where VD1 and VD4 are heavy chain variable region (VH) or light chain variable region (VL) of a first immunoglobulin, VD2 and VD3 are heavy chain variable region (VH) or light chain variable region (VL) of a second immunoglobulin; CL, CH1 are light chain or CH1 heavy chain constant domain; Fc, Fc2 are a hinge region and CH2, CH3 heavy chain constant domain; L1-5 is an amino acid linker; and when VD1 and VD2 are VL, both VD3 and VD4 are VH, and when VD1 and VD2 are VH, VD3 and VD4 are both VL.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to an antibody-like binding protein that specifically binds to CD3 and specifically binds to at least one additional antigen, such as CD123. The present invention further relates to anti-CD3 antibodies and anti-CD123 antibodies. The present invention also relates to an antibody-like binding protein that specifically binds to CD123 and specifically binds to at least one additional antigen. The present invention also relates to pharmaceutical compositions comprising the antibody-like binding proteins, anti-CD3 antibodies or anti-CD123 antibodies of the present invention, and their use for treating cancer. The present invention further relates to isolated nucleic acids, vectors and host cells comprising sequences encoding the above antibody-like binding proteins, anti-CD3 or anti-CD123 antibodies, and the use of the above anti-CD123 antibodies as diagnostic tools.

Background Art

[0002] The first generation of bispecific antibodies was developed over 20 years ago. Since then, numerous 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 vicinity of targeted cancer cells to achieve anti-cancer activity.

[0003] Since bispecific antibody technology was developed, a different group of fusion proteins called bispecific T-cell engagers (BiTEs), in which one scFv binds to a target cell and the other binds to CD3 on the surface of T cells were generated by connecting two antibody single-chain variable regions (scFvs) with only a flexible linker (Fc amino acid segments were not included). Blinatumomab, one BiTE with CD19xCD3 bispecific binding activity, showed promising results in a Phase II clinical trial for patients with minimal residual disease in B-lineage acute lymph blastosis.

[0004] CD123 (interleukin-3 receptor alpha chain, IL-3Rα) is a tumor antigen that is overexpressed in various hematologic neoplasms. Most AML blasts express surface CD123, and this expression does not vary by AML subtype. Higher expression of CD123 on AML at diagnosis has been reported to be associated with a worse prognosis. CD123 has been reported to be expressed on leukemia stem cells (LSCs). Evidence is increasing that AML arises from these leukemia stem cells (LSCs), which have been shown to be quiescent and relatively resistant to DNA-damaging chemotherapy. Therefore, increased expression of CD123 in LSCs compared to hematopoietic stem cells (HSCs) presents an opportunity for the targeted therapy of AML-LSCs. The monoclonal antibody (MAb) 7G3 produced against CD123 has previously been shown to inhibit IL-3-mediated proliferation and activation of both leukemia cell lines and primary cells (Patent Document 1). However, it remains unclear whether AML-LSCs can be functionally impaired by targeting CD123. The use of a CD123xCD3 antibody-like binding protein results in cell death, as shown herein by the inventors.

[0005] The idea of manufacturing a bispecific antibody-like binding protein with CD123xCD3 bispecific binding activity has already been proposed and described in Patent Document 2.

[0006] Furthermore, a CD123xCD3 dual-affinity retargeting (DART) bispecific antibody-based molecule from MacroGenics entered a Phase I clinical trial in 2014.

[0007]

[0008]

[0009]

[0010] ​​​​​However, as shown to the inventors, for example, a CD123xCD3 bispecific dual affinity retargeting (DART) bispecific antibody-based molecule from MacroGenics 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 can result in severe side effects such as cytokine release syndrome. Cytokine release syndrome results in a systemic inflammatory response similar to that seen in severe infections and is characterized by hypotension, fever, and chills, and refers to the release of cytokines by activated T cells. Deaths due to cytokine release syndrome have been reported, for example, for OKT3.

Prior Art Documents

Patent Documents

[0011]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0012] Therefore, despite these advances in bispecific antibody technology, there remains a need for further cancer therapeutics, particularly those that efficiently target and kill cancer cells, either directly or indirectly.

[0013] The inventors have succeeded in generating, screening, and selecting specific rat anti-CD3 antibodies that show high affinity for both human and cynomolgus macaque (Macaca fascicularis) CD3 proteins. Succeeded.

Means for Solving the Problems

[0014] The inventors have determined the biological and An antibody-like binding protein having immunological specificity was developed. In one example, to demonstrate the use of these anti-CD3 antibodies in the production of bispecific antibody-like binding proteins, the inventors generated anti-CD3 / anti-CD123 antibody-like binding proteins and demonstrated their therapeutic use. These bispecific anti-CD3 / anti-CD123 antibody-like binding proteins have low T cell activation, as observed for anti-CD3 antibodies alone. However, if target cells expressing CD123, such as THP-1 cells, are present, the bispecific anti-CD3 / anti-CD123 antibody-like binding proteins show high activation of T cells. Thus, the anti-CD3 antibodies of the present invention as defined above are particularly useful for the production of the antibody-like binding proteins of the present invention. binding proteins of the present invention.

[0015] Definitions Throughout this application, the term " and / or " is to be construed as a grammatical conjunction that includes the possibility that one or more of those it connects may be present. For example, the expression "such a natural sequence protein can be produced using standard recombinant and / or synthetic methods" indicates that the natural sequence protein can be produced using standard recombinant methods or that the natural sequence protein can be produced using standard synthetic methods.

[0016] Furthermore, throughout this application, the term " comprising " is to be construed as including any additional unspecified ones as well as all specifically recited features. The use of the term " comprise " as used herein also discloses embodiments in which no features other than the specifically recited features are present (i.e., " consisting of "). Furthermore, the indefinite article "a" " or "an" does not exclude the possibility of a plurality. The mere fact that certain means are recited in different claims does not indicate that a combination of these means cannot be used advantageously.

[0017] The term " gene " includes all or part of one or more proteins or enzymes and may or may not include a control sequence such as a promoter sequence that determines, for example, the conditions under which a gene is expressed, and encodes a specific amino acid sequence or the corresponding DNA sequence means. Some genes that are not structural genes are transcribed from DNA to RNA but not translated into an amino acid sequence. Other genes can function as regulators of structural genes or as regulators of DNA transcription control factors. In particular, the term gene can be intended to mean a genomic sequence encoding a protein, i.e., a sequence comprising genomic sequence regulators, promoters, introns, and exon sequences.

[0018] " at least 85% identical to the reference sequence " sequence is the full length of the reference sequence and, over its full length, has 85% or more, particularly 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the reference sequence. is a sequence having.

[0019] In the context of the present application, " percentage identity " is calculated using global pairwise alignment (i.e., the two sequences are compared over their full lengths ). Methods for comparing the identity of two or more sequences are well known in the art . For example, using the Needleman-Wunsch global alignment algorithm (Needleman and Wunsch, 1970 J. Mol. Biol. 48:443-453), the optimal alignment (including gaps) of two sequences can be found when considering their full lengths< sequences. < <needle>>The program can be used. The needle program is available, for example, on the ebi.ac.uk World Wide Web site The percentage identity between two polypeptides according to the present 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 the Blosum62 matrix. Proteins consisting of amino acid sequences that are "at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical" to the reference sequence may contain mutations such as deletions, insertions and / or substitutions compared to the reference sequence. In the case of substitutions, proteins consisting of amino acid sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the reference sequence may correspond to homologous sequences derived from a different species than the reference sequence.

[0020] The "substitutions" may be conservative or non-conservative. Preferably, the substitutions are conservative substitutions, where one amino acid is replaced by another amino acid having similar structural and / or chemical properties. The substitutions preferably correspond to conservative substitutions as shown in the following table.

[0021] " amino acid substitution " may also be called "

[0022]

Table 1

[0023] " immunoglobulin " and also called " antibody " can 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 chains, lambda (l) and kappa (k). There are five main heavy chain classes (or isotypes) that determine the functional activity of the antibody molecule: IgM, IgD, IgG, IgA, and IgE. Each chain contains distinct sequence domains. The light chain contains two domains or regions, a variable domain (VL) and a constant domain (CL). The heavy chain contains four domains, a variable domain (VH) and three constant domains (CH1, CH2, and CH3, collectively called CH). Both the variable regions of the light chain (VL) and the heavy chain (VH) determine antigen binding recognition and specificity. The constant region domains of the light chain (CL) and the heavy chain (CH) confer important biological properties such as antibody chain association, secretion, transplacental mobility, complement binding, and binding to Fc receptors (FcR). The Fv fragment is the N-terminal portion of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain. The specificity of an antibody lies in the enzymatic complementarity between the antibody binding site and the antigen determinant. The antibody binding site is mainly composed of residues from the hypervariable or complementarity determining regions (CDRs). Residues from the non-hypervariable or framework regions (FRs) can affect the overall domain structure and thus may affect the binding site. The complementarity determining region or CDR refers to the amino acid sequence that together defines the binding affinity and specificity of the native Fv region of the antibody 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. Thus, a conventional antibody binding site contains six CDRs, including CDRs from each of the heavy and light chain V regions. In the context of the present invention, an antibody or immunoglobulin is IgM, IgD, IgG, IgA, and IgE.

[0024] ​​​​

[0025] " framework region " (FR) is between the CDRs, i.e., between different immune nucleotides in a single species. The term "light chain variable domain" refers to the amino acid sequence inserted into the portion of the immunoglobulin light and heavy chain variable domain that is relatively conserved among immunoglobulins. The light and heavy chains of an immunoglobulin each have four FRs, designated FR1-L, FR2-L, FR3-L, and FR4-L, and FR1-H, FR2-H, FR3-H, and FR4-H, respectively. Thus, the light chain variable domain The heavy chain variable domain is designated as (FR1-L)-(CDR1-L)-(FR2-L)-(CDR2-L)-(FR3-L)-(CDR3-L)-(FR4-L) and the heavy chain variable domain is designated as (FR1-H)-(CDR1-H)-(FR2-H)-(CDR2-H)-(FR3-H)-(CDR3-H)-(FR4-H).

[0026] Knowing the amino acid sequences of the CDRs, one skilled in the art can readily determine the framework regions FR1-L, FR2-L, FR3-L, FR4-L and / or FR1-H, FR2-H, FR3-H, FR4-H.

[0027] As used herein, " human framework region " are substantially identical (about 85% or more, particularly 90%, 95%, 97%, 99% or more) to the framework regions of a naturally occurring human antibody. or 100%) framework regions.

[0028] In the context of the present invention, the CDR / FR definitions in an immunoglobulin light or heavy chain are based on the IMGT definitions (Lefranc et al. Dev. Comp. Immunol., 2003, 27(1):55-77; www.imgt.org) This should be decided.

[0029] As used herein, the term " antibody " refers to conventional antibodies and their fragments, as well as single-domain antibodies and their fragments, in particular the variable heavy chains of single-domain antibodies, and chimeric, humanized, bispecific or multispecific antibodies.

[0030] As used herein, an antibody or immunoglobulin also refers to an antibody that has been more recently described and whose complementarity-determining regions are part of a single-domain polypeptide, " single domain intrabody ". Examples of single-domain antibodies include heavy-chain antibodies, antibodies that are naturally lacking in light chains, single-domain antibodies derived from conventional four-chain antibodies, and engineered single-domain antibodies. Single-domain antibodies can be derived from any species including, but not limited to, mouse, human, camel, llama, goat, rabbit, and cow. A single-domain antibody may be a naturally occurring single-domain antibody known as a heavy-chain antibody lacking a light chain. In particular, camelid species such as camel, dromedary, llama, alpaca, and guanaco produce heavy-chain antibodies that are naturally lacking in light chains. Camelid heavy-chain antibodies also lack the CH1 domain. missing.

[0031] The variable heavy chains of these single-domain antibodies lacking a light chain 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 approximately 10 times smaller than IgG molecules, and as a result, properly folded functional nanobodies can be produced by in vitro expression while achieving high yields. Furthermore, nanobodies are very stable and resistant to the action of proteases. The properties and production of nanobodies are reviewed by Harmsen and De Haard HJ (Appl. Microbiol. Biotechnol. 2007 Nov;77(1):13-22).

[0032] monoclonal antibody The term " monoclonal antibody 」 or 「 mAb 」 refers to an antibody molecule of single amino acid resuscitation specific to a particular antibody and should not be construed as requiring the production of the antibody by any particular method. Monoclonal antibodies can be produced by a single clone of B cells or a hybridoma, but may also be produced recombinantly, i.e., by protein engineering.

[0033] The term 「 chimeric antibody 」, in its broad sense, refers to an engineered antibody containing one or more regions from one antibody and one or more regions from one or more other antibodies. In particular, a chimeric antibody includes the VH and VL domains of an antibody from a non-human animal in association with the CH and CL domains of another antibody, particularly a human antibody. Any animal such as a mouse, rat, hamster, rabbit or the like can be used as the non-human animal. A chimeric antibody can also exhibit multispecific antibodies having specificities for at least two different antigens.

[0034] The term 「 humanized antibody 」 refers to an antibody that is wholly or partially of non-human origin and has been modified to replace certain amino acids, particularly the framework regions of the heavy and light chains, so as to avoid or minimize the immune response in humans. The constant domains of a humanized antibody are, in the majority of cases, human CH and CL domains.

[0035] Numerous methods for humanization of antibody sequences are known in the art; see, for example, the review by Almagro and Fransson (2008) Front Biosci. 13:1619-1633. One commonly used method is CDR grafting, or antibody reshaping which involves the grafting of CDR sequences from a donor antibody, typically a mouse antibody, onto the framework backbone of a human antibody of a different specificity. CDR grafting involves CDR grafting The binding specificity and affinity of the resulting non-human antibody can be reduced and thus its biological activity can be decreased. Therefore, in order to maintain the binding specificity and affinity of the parental antibody, back mutations can be introduced at selected positions of the CDR-grafted antibody. Identification of possible positions for back mutations can be done using information available in the literature and antibody databases. Amino acid residues that are candidates for back mutations are typically those located on the surface of the antibody molecule, although buried or low surface-exposed residues are usually not altered. Alternative humanization techniques for CDR grafting and back mutations are resurfacing, where residues not exposed on the surface of non-human origin are retained, while surface residues are changed to human residues. Another alternative technique is known as "guided selection" (Jespers et al. (1994) Biotechnology 12, 899) and can be used, for example, to derive fully human antibodies from mice or rats that preserve the epitope and binding characteristics of the parental antibody. A further method of humanization is so-called 4D humanization. The 4D humanization protocol is described in patent application US20110027266 A1 (WO2009032661A1) and is exemplified below by applying 4D humanization to humanize rat antibody variable light (VL) and heavy (VH) domains. In one example, a rat antibody homology model is typically performed using the PDB structure (Berman et al., Nucleic Acids Research, 2000, 28:235-242) as a template using MOE software (v.2011.10 - Chemical Computing Group, Quebec, Canada), and then using standard procedures implemented in MOE to perform the humanization. After that, the standard procedures implemented in MOE are used It was minimized using energy. Next, molecular dynamics (MD) simulations were performed on the minimized 3D homology model of the rat antibody (performed using MOE software), and compared with 49 human models derived from seven representative light chains (vk1, vk2, vk3, vk4, vlambda1, vlambda2, vlambda3) and seven representative heavy chains (vh1a, vh1b, vh2, vh3, vh4, vh5, vh6) designated, for example, as LGCR / SDI and available within MOE. For example, one model of a chain pair (Vkx-Vhx) with the best hydrophobicity, both electrostatic components, and sequence identity outside the CDR was selected for "humanization". For the pairing between the rat variable domain and the selected model, the sequences were typically aligned based on the optimal 3D superposition of the alpha carbons of the corresponding homology models. Then, it was mutated considering undesirable motifs. Finally, the resulting humanized sequence was blasted for sequence similarity against, for example, the IEDB database (http: / / www.immuneepitope.org; accessible in the 2012 / 01 / 30 region) to ensure that none of the sequences contain the listed known B-cell or T-cell epitopes. For humanization of chimeric antibodies, it typically involves modification of the framework regions of the variable region sequences. Amino acid residues that are part of the CDR are typically not changed with respect to humanization, but specific ones

[0036]

[0037] In some cases, for example, it may be desirable to modify the CDR amino acid residues here in order to remove glycosylation sites, deamidation sites or unwanted cysteine residues. N-linked glycosylation occurs by the attachment of an oligosaccharide chain to an asparagine residue in the tripeptide sequence Asn-X-Ser or Asn-X-Thr, where X is any amino acid other than Pro. Removal of N-glycosylation sites can be achieved by mutating either the Asn or Ser / Thr residue to a different residue, particularly using conservative substitutions. Deamidation of asparagine and glutamine residues can occur depending on factors such as pH and surface exposure. Asparagine residues are particularly sensitive to deamidation, mainly when the sequence Asn-Gly is present and to a lesser extent in other dipeptide sequences such as Asn-Ala. Thus, when such deamidation sites, particularly Asn-Gly, are present in the CDR sequence, it may be desirable to remove that site, typically by removing one of the related residues by conservative substitution. Substitutions in the CDR sequence to remove one of the related residues are also intended to be encompassed by the present invention.

[0038] (Conventional) antibody " fragment " includes a part of an intact antibody, particularly the antigen-binding region or variable region of an intact antibody. Examples of antibody fragments include Fv, Fab, F(ab ’)2, Fab’, dsFv, (dsFv)2, scFv, sc(Fv)2, bispecific antibodies (diabodies), bispecific and multispecific antibodies formed from antibody fragments. Fragments of conventional antibodies can also be single domain antibodies such as heavy chain antibodies or VHHs.

[0039] The term " Fab " refers to an antibody fragment having a molecular weight of about 50,000 and antigen-binding activity, where between the fragments obtained by treating IgG with the protease papain about the first half of the N-terminal sides of the entire H and L chains are joined together through disulfide bonds are.

[0040] The term " F(ab’)2 " refers to an antibody fragment having a molecular weight of about 100,000 and antigen-binding activity and which is a fragment obtained by treating IgG with the protease pepsin and which is slightly larger than Fab which is bound via the disulfide bond in the hinge region among the fragments .

[0041] The term " Fab’ " refers to an antibody fragment having a molecular weight of about 50,000 and antigen-binding activity and which is obtained by cleaving the disulfide bond in the hinge region of F(ab’)2.

[0042] The single-chain Fv (" scFv ") polypeptide is covalently linked to a VH::VL heterodimer which is expressed from a gene fusion comprising VH and VL which are usually linked by a peptide coding linker. The human scFv fragments of the present invention contain CDRs which are retained in an appropriate conformation, particularly by using recombinant DNA technology. Bivalent and multivalent antibody fragments can be formed spontaneously by the binding of monovalent scFv or can be generated by coupling monovalent scFv to a peptide linker as in bivalent sc(Fv) 2 . "dsFv" is a VH::VL heterodimer stabilized by a disulfide bond. "(dsFv)2" refers to two dsFvs coupled to a peptide linker. peptide linker.

[0043] The term " bispecific antibody " or " BsAb " typically refers to an antibody which combines the antigen-binding sites of two antibodies within a single molecule. Thus, a BsAb can bind to two different antigens simultaneously. Genetic manipulation can be used to combine the binding specific Antibodies have been used with increasing frequency to design, engineer and generate antibodies or antibody derivatives that have desired sets of properties and effector functions.

[0044] term" multispecific antibody " refers to an antibody that combines the antigen-binding sites of two or more antibodies in a single molecule.

[0045] term" diabodies " is a small antibody fragment with two antigen-binding sites. These fragments consist of the same polypeptide chain (VH-VL) and the same light chain variable domain. Each antibody comprises a heavy-chain variable domain (VH) connected to a complementary heavy-chain variable domain (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.

[0046] term" hybridoma " is a B antigen produced by immunizing a non-human mammal with an antigen. The cells obtained by subjecting the cells to cell fusion with myeloma cells from a mouse or the like that produce the desired monoclonal antibody with antigen specificity are shown.

[0047] " purified " and " isolated " when referring to a polypeptide (i.e., an antibody of the invention) or a nucleotide sequence, means that the indicated molecule is present in the substantial absence of other biological macromolecules of the same type. purified " means that there are at least 75%, 85%, 95%, or 98% by weight of the same type of biological macromolecule. isolated " A nucleic acid molecule refers to a nucleic acid molecule that is substantially free of other nucleic acid molecules that do not encode a polypeptide of interest; however, the molecule may include some additional bases or moieties that do not deleteriously affect the basic characteristics of the composition.

[0048] As used herein, the term " antigen " or " target antigen " refers to a molecule or a part of a molecule that can be bound by an antibody or an antibody-like binding protein. The term further refers to a molecule or a part of a molecule that can be used in an animal to produce an antibody that can bind to an epitope of the antigen. The target antigen has one or more epitopes obtainable. For each target antigen recognized by an antibody or an antibody-like binding protein, the antibody-like binding protein can compete with an intact antibody that recognizes the target antigen.

[0049] " affinity " is theoretically defined by the equilibrium binding between the whole antibody and the antigen. Affinity can be represented, for example, by the half-maximal effective concentration (EC 50 ), or the equilibrium dissociation constant (KD).

[0050] " EC 50 ", also referred to as " half-maximal effective concentration ", refers to the concentration of a drug, antibody, or poison that induces a response intermediate between the baseline and the maximum value after a specific exposure time. EC 50 and affinity are inversely correlated, and the lower the EC 50 value, the higher the affinity of the antibody.

[0051] " K D " is the equilibrium dissociation constant, the ratio of k off / k on between the antibody and its antigen. K D and affinity are inversely correlated. The K D value is related to the concentration of the antibody, and the lower the K D value, the higher the affinity of the antibody. Affinity can be determined experimentally by various known methods, for example, measuring the association and dissociation rates by surface plasmon resonance, or measuring EC 50 by immunoassay (ELISA, FACS). can be evaluated. Enzyme-linked immunosorbent assay (ELISA) is a biochemical assay that uses a solid-phase enzyme immunoassay to detect the presence of a substance, usually an antigen, in a liquid sample or a wet sample. The antigen derived from the sample is bound to the surface. Then, additional specific antibodies are applied onto the surface so that they can bind to the antigen. This antibody is linked to an enzyme, and in the final step, a substance containing the substrate of the enzyme is added. The subsequent reaction produces a detectable signal, a color change in the most common substrate. Fluorescence-activated cell sorting (FACS) provides a method for sorting a heterogeneous mixture of biological cells, one cell at a time, into two or more containers based on the specific light-scattering and fluorescence characteristics of each cell. In these assays, EC 50 is the concentration of the antibody that mediates the intermediate response between the baseline and the maximum value after a certain specified exposure time at a defined concentration of cells expressing the antigen by ELISA (enzyme-linked immunosorbent assay) or by FACS (fluorescence-activated cell sorting). Surface plasmon resonance is a label-free method in which the binding of a molecule (the "analyte") in the soluble phase to a "ligand" molecule immobilized on the sensor surface is measured directly. In a sensor device, the binding of the ligand is monitored by an optical phenomenon called surface plasmon. In particular, when the "analyte" molecule dissociates from the "ligand" molecule, a phenomenon of the SPR signal (expressed in resonance units RU) is observed. The association rate ("on-rate", k a ) and the dissociation rate ("off-rate", k d ) are obtained from the signals obtained during association and dissociation, and the equilibrium dissociation constant ("binding constant" , K D ) can be calculated from them. The signal expressed in resonance units (RU) depends on the size of the ligand present in the analyte , but when the experimental conditions are the same, that is, when the ligand is the same molecule under the same conditions, the RU obtained can indicate the affinity, where the higher the signal obtained in RU, the higher the binding.

[0052] A monoclonal antibody that binds to antigen 1 (Ag1) is "cross-reactive" with antigen 2 (Ag2) when the EC 50 is in a similar range for both antigens. In the present application, an antibody that binds to Ag1 is cross-reactive with Ag2 when the ratio of the affinity of Ag2 to the affinity of Ag1 is equal to or less than 10 (in particular, 5, 2, 1, or 0.5) in terms of the affinity measured in the same way for both antigens. cross-reactivity In the present application, an antibody that binds to Ag1 is "cross-reactive" with Ag2 when the ratio of the affinity of Ag2 to the affinity of Ag1 is equal to or less than 10 (in particular, 5, 2, 1, or 0.5) in terms of the affinity measured in the same way for both antigens. In the present application, an antibody that binds to Ag1 is cross-reactive with Ag2 when the ratio of the affinity of Ag2 to the affinity of Ag1 is equal to or less than 10 (in particular, 5, 2, 1, or 0.5) in terms of the affinity measured in the same way for both antigens. In the present application, an antibody that binds to Ag1 is cross-reactive with Ag2 when the ratio of the affinity of Ag2 to the affinity of Ag1 is equal to or less than 10 (in particular, 5, 2, 1, or 0.5) in terms of the affinity measured in the same way for both antigens.

[0053] An antibody that binds to Ag1 is "not cross-reactive" with Ag2 when the affinities are very different for the two antigens. The affinity for Ag2 may not be measurable if the binding response is too low. In the present application, a monoclonal antibody that binds to Ag1 is not significantly cross-reactive with Ag2 if 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. not significantly cross-reactive An antibody that binds to Ag1 is "not cross-reactive" with Ag2 when the affinities are very different for the two antigens. The affinity for Ag2 may not be measurable if the binding response is too low. In the present application, a monoclonal antibody that binds to Ag1 is not significantly cross-reactive with Ag2 if 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. An antibody that binds to Ag1 is "not cross-reactive" with Ag2 when the affinities are very different for the two antigens. The affinity for Ag2 may not be measurable if the binding response is too low. In the present application, a monoclonal antibody that binds to Ag1 is not significantly cross-reactive with Ag2 if 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. An antibody that binds to Ag1 is "not cross-reactive" with Ag2 when the affinities are very different for the two antigens. The affinity for Ag2 may not be measurable if the binding response is too low. In the present application, a monoclonal antibody that binds to Ag1 is not significantly cross-reactive with Ag2 if 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 may be the concentration required to reach the saturation plateau obtained with EC 50 or Ag1. In practice, the antibody concentration used may be the concentration required to reach the saturation plateau obtained with EC

[0054] As used herein, "specificity" indicates the ability of an antibody to distinguish the target peptide sequence ("epitope") to which it binds from closely related highly homologous peptide sequences. specificity As used herein, "specificity" indicates the ability of an antibody to distinguish the target peptide sequence ("epitope") to which it binds from closely related highly homologous peptide sequences.

[0055] A monoclonal antibody is "specific" for Ag1 if it is not significantly cross-reactive with Ag2. specifically binds A monoclonal antibody is "specific" for Ag1 if it is not significantly cross-reactive with Ag2.

[0056] "Specificity" domain is generally defined based on sequence homology and can often be any region of a protein related to a particular structure or functional entity.

[0057] "Specificity" recombinant The "molecule" is produced, expressed, generated, or isolated by recombinant means.

[0058] As used herein, the term " subject " refers to mammals such as rodents, felines, canines, and primates. In particular, the subject according to the present invention is a human.

[0059] Anti-CD3 antibody " CD3 " is expressed on T cells as part of the multi-molecular T cell receptor complex and refers to an antigen consisting of at least two different chains, CD3ε, CD3δ, and CD3γ. CD3δ and CD3γ have low sequence identity and / or similarity to CD3ε (less than 20% similarity and identity) . CD3ε and CDR3δ can form a complex together (referred to herein as the "CD3ε / δ complex"). CD3ε can also form a complex with CDR3γ, the so-called "CD3ε / γ complex" clustering of CD3 on T cells, for example, by immobilizing an anti-CD3 antibody, resulting in T cell activation similar to the binding of the T cell receptor, but with a specificity typical of that clone being independent. " " contains three domains, an intracellular domain, a transmembrane domain, and CD3ε an extracellular domain.

[0060] Most prior art anti-CD3 antibodies recognize the CD3ε chain. One such prior art anti-CD3 antibody is OKT3. The prior art has exemplified T cell activation events using antibody molecules, for example, the antibody molecule OKT3. Anti-CD3 antibodies and their variants are 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 is an effective in clinical transplantation for treating allograft rejection.It has also been used as an immunosuppressant (Thistlethwaite 1984, Transplantation 38, 695-701; Woodle 1991, Transplantation 51, 1207-1212; Choi 2001, Eur. J. Immunol. 31(1), 94-106).

[0061] The main drawbacks of this treatment are T cell activation and human anti-mouse antibody (HAMA) responses, manifested by cytokine release resulting from cross-linking between T cells and FcγR-bearing cells. Several publications have described modifications to reduce these side effects, such as humanization of OKT3: US 5,929,212; US 5,885,573 and others. On the other hand, OKT3 or other anti-CD3 antibodies can be used as immunostimulants that 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). 。An anti-CD3 antibody has also been described as an agent used in combination with an anti-CD28 antibody to induce T cell proliferation (US 6,352,694). OKT3 has further been used alone 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). 。The previous approaches of using antibodies as agents to mobilize T cells have been hampered by several

[0062] findings. First, natural or engineered antibodies with high binding affinity for T cells often do not activate the T cells to which they bind. Second, those directed against T cells are often unable to activate the T cells to which they bind. Second, those directed against Natural or engineered antibodies with low binding affinity are also ineffective with respect to their ability to initiate T cell-mediated cell lysis.

[0063] The reference sequence of the full-length human CD3ε protein containing the signal peptide is available from the Uniprot database under accession number P07766 and is incorporated herein as SEQ ID NO: 1 (available on December 12, 2014).

[0064] The reference sequence of the full-length cynomolgus (Macaca fascicularis) CD3ε protein containing the signal peptide is available from the Uniprot database under accession number Q95LI5 and is incorporated herein as SEQ ID NO: 2 (available on December 12, 2014).

[0065] The sequence of the mature human CD3ε His-tagged Fc fusion protein cloned from genomic DNA by the inventors is disclosed in SEQ ID NO: 3. The above mature human CD3ε His-tagged Fc fusion protein contains amino acids 23-126 of the full-length human CD3ε protein and thus contains the extracellular domain of human CD3ε.

[0066] The sequence of the mature cynomolgus CD3ε Fc fusion protein cloned from genomic DNA by the inventors is disclosed in SEQ ID NO: 4. The above mature cynomolgus CD3ε Fc fusion protein contains amino acids 23-117 of the full-length cynomolgus CD3ε protein and thus contains the extracellular domain of human or cynomolgus CD3ε containing an exchange of one alanine to valine at amino acid position 35 compared to amino acid position 57 of the wild-type sequence.

[0067] The domain organization of human and cynomolgus CD3ε is as follows (based on the Uniprot P07766 sequence (human) and the Uniprot Q95LI5 sequence (cynomolgus)): [Table 2]​​​​

[0068] Thus, the extracellular domain of human CD3ε consists of the amino acids at positions 23 to 126 of SEQ ID NO: 1, and the extracellular domain of cynomolgus monkey CD3ε consists of the amino acids at positions 22 to 117 of SEQ ID NO: 2. The inventors have succeeded in generating, screening, and selecting specific mouse and rat anti-CD3 antibodies. These anti-CD3 antibodies exhibit high affinity for both human and cynomolgus monkey CD3 proteins, but have low T cell activation in the absence of target cells.

[0069] The inventors have determined the sequences of the variable heavy and light chains of such monoclonal antibodies, 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".

[0070]

[0071] The so-called "20G6-F3" anti-CD3 antibody: - a heavy chain variable domain consisting of CDR1-H of the sequence of SEQ ID NO: 6, CDR2-H of the sequence of SEQ ID NO: 7, and CDR3-H of the sequence of SEQ ID NO: 8, and

Chemical Formula

[0072] The so-called "4B4-D7" anti-CD3 antibody is: - A heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 13, CDR2-H of the sequence of SEQ ID NO: 14, and CDR3-H of the sequence of SEQ ID NO: 15, the sequence [Chemical formula] (SEQ ID NO: 12, CDRs are shown in bold and underlined), and - a light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 17, CDR2-L consisting of the sequence "KVS", and CDR3-L of the sequence of SEQ ID NO: 11, the sequence [Chemical formula] (SEQ ID NO: 16, CDRs are shown in bold and underlined) comprising.

[0073] The so-called "4E7-C9" anti-CD3 antibody is: - A heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 13, CDR2-H of the sequence of SEQ ID NO: 19, and CDR3-H of the sequence of SEQ ID NO: 20, the sequence [Chemical formula] (SEQ ID NO: 18, CDRs are shown in bold and underlined), and - a light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 22, CDR2-L consisting of the sequence "KVS", and CDR3-L of the sequence of SEQ ID NO: 11, the C DR3-L, the sequence [Chemical formula] The light chain variable domain consisting of (sequence number 21, CDRs are shown in bold and underlined) is included.

[0074] The so-called "18F5-H10" anti-CD3 antibody is: - A sequence including CDR1-H of the sequence of sequence number 24, CDR2-H of the sequence of sequence number 19, and CDR3-H of the sequence of sequence number 25 [Chemical formula] The heavy chain variable domain consisting of (sequence number 23, CDRs are shown in bold and underlined), and - A sequence including CDR1-L of the sequence of sequence number 27, CDR2-L of the sequence "KVS", and CDR3-L of the sequence of sequence number 28 is included. [Chemical formula] The light chain variable domain consisting of (sequence number 26, CDRs are shown in bold and underlined) is included.

[0075] The so-called "12D2-E5" anti-CD3 antibody is: - A sequence including CDR1-H of the sequence of sequence number 30, CDR2-H of the sequence of sequence number 31, and CDR3-H of the sequence of sequence number 32 [Chemical formula] The heavy chain variable domain consisting of (sequence number 29, CDRs are shown in bold and underlined), and - A sequence including CDR1-L of the sequence of sequence number 34, CDR2-L of the sequence "RDD", and CDR3-L of the sequence of sequence number 35 [Chemical formula] The light chain variable domain consisting of (sequence number 33, CDRs are shown in bold and underlined) is included.

[0076] The so-called "11D7-C3" anti-CD3 antibody is: - a heavy chain variable domain consisting of CDR1-H of the sequence of SEQ ID NO: 13, CDR2-H of the sequence of SEQ ID NO: 37, and CDR3-H of the sequence of SEQ ID NO: 38, and

Chemical Structure

Chemical Structure

[0077] The so-called "11H3-E5" anti-CD3 antibody is: - a heavy chain variable domain consisting of CDR1-H of the sequence of SEQ ID NO: 13, CDR2-H of the sequence of SEQ ID NO: 37, and CDR3-H of the sequence of SEQ ID NO: 41, and

Chemical Structure

Chemical Structure

[0078] The so-called "13H2-C2" anti-CD3 antibody is: - a heavy chain variable domain consisting of CDR1-H of the sequence of SEQ ID NO: 13, CDR2-H of the sequence of SEQ ID NO: 37, and CDR3-H of the sequence of SEQ ID NO: 44, and [Chemical formula] A heavy chain variable domain consisting of (Accession No. 43, CDRs are shown in bold and underlined), and - A sequence containing CDR1-L of the sequence of Accession No. 10, CDR2-L consisting of the sequence "KVS", and CDR3-L of the sequence of Accession No. 11 [Chemical formula] A light chain variable domain consisting of (Accession No. 45, CDRs are shown in bold and underlined) including.

[0079] The so-called "13C1-F6" anti-CD3 antibody is: - A sequence containing CDR1-H of the sequence of Accession No. 13, CDR2-H of the sequence of Accession No. 37, and CDR3-H of the sequence of Accession No. 47 [Chemical formula] A heavy chain variable domain consisting of (Accession No. 46, CDRs are shown in bold and underlined), and - A sequence containing CDR1-L of the sequence of Accession No. 10, CDR2-L consisting of the sequence "KVS", and CDR3-L of the sequence of Accession No. 11 [Chemical formula] A light chain variable domain consisting of (Accession No. 48, CDRs are shown in bold and underlined) including.

[0080] The so-called "18H11-F10" anti-CD3 antibody is: - A sequence containing CDR1-H of the sequence of Accession No. 50, CDR2-H of the sequence of Accession No. 51, and CDR3-H of the sequence of Accession No. 52 [Chemical formula] A heavy chain variable domain consisting of (Accession No. 49, CDRs are shown in bold and underlined), and - a CDR1-L of the sequence of Accession No. 54, a CDR2-L consisting of the sequence "NAN", and a CDR3-L of the sequence of Accession No. 55, the sequence [Chemical formula] A light chain variable domain consisting of (Accession No. 53, CDRs are shown in bold and underlined) including

[0081] The so-called "1E6-C9" anti-CD3 antibody is: - A CDR1-H of the sequence of Accession No. 57, a CDR2-H of the sequence of Accession No. 37, and a CDR3-H of the sequence of Accession No. 58, the sequence [Chemical formula] A heavy chain variable domain consisting of (Accession No. 56, CDRs are shown in bold and underlined), and - a CDR1-L of the sequence of Accession No. 10, a CDR2-L consisting of the sequence "KVS", and a CDR3-L of the sequence of Accession No. 28, the sequence [Chemical formula] A light chain variable domain consisting of (Accession No. 59, CDRs are shown in bold and underlined) including

[0082] The so-called "10F4-C10" anti-CD3 antibody is: - A CDR1-H of the sequence of Accession No. 13, a CDR2-H of the sequence of Accession No. 37, and a CDR3-H of the sequence of Accession No. 61, the sequence [Chemical formula] A heavy chain variable domain consisting of (Accession No. 60, CDRs are shown in bold and underlined), and - A CDR1-L of the sequence of Accession No. 10, a CDR2-L consisting of the sequence "KVS", and a CDR3-L of the sequence of Accession No. 11, the sequence [Chemistry] A light chain variable domain consisting of (Accession No. 62, CDRs are shown in bold and underlined) and includes.

[0083] The so-called "10E6-G6" anti-CD3 antibody is: - A sequence including CDR1-H of the sequence of Accession No. 64, CDR2-H of the sequence of Accession No. 65, and CDR3-L of the sequence of Accession No. 47 [Chemistry] A heavy chain variable domain consisting of (Accession No. 63, CDRs are shown in bold and underlined), and - A sequence including CDR1-L of the sequence of Accession No. 67, CDR2-L consisting of the sequence "KVS", and CDR3-L of the sequence of Accession No. 28 [Chemistry] A light chain variable domain consisting of (Accession No. 66, CDRs are shown in bold and underlined) and includes.

[0084] The so-called "18G9-H11" anti-CD3 antibody is: - A sequence including CDR1-H of the sequence of Accession No. 13, CDR2-H of the sequence of Accession No. 37, and CDR3-H of the sequence of Accession No. 69 [Chemistry] A heavy chain variable domain consisting of (Accession No. 68, CDRs are shown in bold and underlined), and - A sequence including CDR1-L of the sequence of Accession No. 10, CDR2-L consisting of the sequence "KVS", and CDR3-L of the sequence of Accession No. 71 [Chemistry] A light chain variable domain consisting of (Accession No. 70, CDRs are shown in bold and underlined) and includes.

[0085] The so-called "11F3-B9" anti-CD3 antibody is: - A heavy chain variable domain consisting of CDR1-H of the sequence of SEQ ID NO: 13, CDR2-H of the sequence of SEQ ID NO: 37, and CDR3-H of the sequence of SEQ ID NO: 84, and [Chemical formula] (SEQ ID NO: 72, with the CDRs shown in bold and underlined), and - CDR1-L of the sequence of SEQ ID NO: 17, CDR2-L of the sequence "KVS", and CDR3-L of the sequence of SEQ ID NO: 11 contained in the sequence [Chemical formula] (SEQ ID NO: 73, with the CDRs shown in bold and underlined) consisting of a light chain variable domain including it.

[0086] The so-called "12G3-E8" anti-CD3 antibody is: - A heavy chain variable domain consisting of CDR1-H of the sequence of SEQ ID NO: 75, CDR2-H of the sequence of SEQ ID NO: 76, and CDR3-H of the sequence of SEQ ID NO: 77, and [Chemical formula] (SEQ ID NO: 74, with the CDRs shown in bold and underlined), and - CDR1-L of the sequence of SEQ ID NO: 10, CDR2-L consisting of the sequence "KVS", and CDR3-L of the sequence of SEQ ID NO: 11 contained in the sequence [Chemical formula] (SEQ ID NO: 78, with the CDRs shown in bold and underlined) consisting of a light chain variable domain including it.

[0087] The so-called "5B1-G2" anti-CD3 antibody is: - A heavy chain variable domain consisting of CDR1-H of the sequence of SEQ ID NO: 80, CDR2-H of the sequence of SEQ ID NO: 76, and CDR3-H of the sequence of SEQ ID NO: 81, and [Chemical formula] A heavy chain variable domain consisting of (SEQ ID NO: 79, CDRs are shown in bold and underlined), and - a CDR1-L of the sequence of SEQ ID NO: 10, a CDR2-L consisting of the sequence "KVS", and a CDR3-L of the sequence of SEQ ID NO: 11, the sequence [Chemical formula] A light chain variable domain consisting of (SEQ ID NO: 82, CDRs are shown in bold and underlined) including.

[0088] The so-called "16F8-A7" anti-CD3 antibody is: - A CDR1-H of the sequence of SEQ ID NO: 13, a CDR2-H of the sequence of SEQ ID NO: 37, and a CDR3-H of the sequence of SEQ ID NO: 84, the sequence [Chemical formula] A heavy chain variable domain consisting of (SEQ ID NO: 83, CDRs are shown in bold and underlined), and - a CDR1-L of the sequence of SEQ ID NO: 10, a CDR2-L consisting of the sequence "KVS", and a CDR3-L of the sequence of SEQ ID NO: 11, the sequence [Chemical formula] A light chain variable domain consisting of (SEQ ID NO: 85, CDRs are shown in bold and underlined) including.

[0089] The so-called "11F9-F8" anti-CD3 antibody is: - A CDR1-H of the sequence of SEQ ID NO: 13, a CDR2-H of the sequence of SEQ ID NO: 37, and a CDR3-H of the sequence of SEQ ID NO: 47, the sequence [Chemical formula] A heavy chain variable domain consisting of (SEQ ID NO: 46, CDRs are shown in bold and underlined), and - a CDR1-L of the sequence of SEQ ID NO: 10, a CDR2-L consisting of the sequence "KVS", and a CDR3-L of the sequence of SEQ ID NO: 88, the sequence [Chem.] A light chain variable domain consisting of (Accession No. 87, CDRs are shown in bold and underlined) and containing.

[0090] The so-called "3G5-E10" anti-CD3 antibody is: - A sequence containing CDR1-H of the sequence of Accession No. 90, CDR2-H of the sequence of Accession No. 91, and CDR3-H of the sequence of Accession No. 32 [Chem.] A heavy chain variable domain consisting of (Accession No. 89, CDRs are shown in bold and underlined), and - a sequence containing CDR1-L of the sequence of Accession No. 93, CDR2-L consisting of the sequence "GAS", and CDR3-L of the sequence of Accession No. 94 [Chem.] A light chain variable domain consisting of (Accession No. 92, CDRs are shown in bold and underlined) and containing.

[0091] The so-called "9D7-F3" anti-CD3 antibody is: - A sequence containing CDR1-H of the sequence of Accession No. 96, CDR2-H of the sequence of Accession No. 97, and CDR3-H of the sequence of Accession No. 98 [Chem.] A heavy chain variable domain consisting of (Accession No. 95, CDRs are shown in bold and underlined), and - a sequence containing CDR1-L of the sequence of Accession No. 100, CDR2-L consisting of the sequence "NTN", and CDR3-L of the sequence of Accession No. 101 and containing. [Chem.] A light chain variable domain consisting of (Accession No. 99, CDRs are shown in bold and underlined) and containing.

[0092] The so-called "8C2-F7" anti-CD3 antibody is: - A heavy chain variable domain consisting of CDR1-H of the sequence of SEQ ID NO: 103, CDR2-H of the sequence of SEQ ID NO: 104, and CDR3-H of the sequence of SEQ ID NO: 105, and the sequence containing

Chemical formula

Chemical formula

[0093] The so-called "20E5-F10" anti-CD3 antibody is: - A heavy chain variable domain consisting of CDR1-H of the sequence of SEQ ID NO: 80, CDR2-H of the sequence of SEQ ID NO: 19, and CDR3-H of the sequence of SEQ ID NO: 108, and the sequence

Chemical formula

Chemical formula

[0094] The so-called "20B5-F10" anti-CD3 antibody is: - An array comprising CDR1-H of the array of SEQ ID NO: 13, CDR2-H of the array of SEQ ID NO: 37, and CDR3-H of the array of SEQ ID NO: 111

Chemical formula

Chemical formula

[0095] The so-called "6C9-C9" anti-CD3 antibody is: - An array comprising CDR1-H of the array of SEQ ID NO: 116, CDR2-H of the array of SEQ ID NO: 117, and CDR3-H of the array of SEQ ID NO: 118 of the array

Chemical formula

Chemical formula

[0096] The so-called "3E8-G1" anti-CD3 antibody is: - The heavy chain variable domain consisting of CDR1-H of the sequence of SEQ ID NO: 122, CDR2-H of the sequence of SEQ ID NO: 123, and the sequence of CDR3-H of SEQ ID NO: 124 of the sequence

[0097]

Chemical formula

Chemical formula

[0098] The so-called "3H6-D2" anti-CD3 antibody is: - The heavy chain variable domain consisting of CDR1-H of the sequence of SEQ ID NO: 13, CDR2-H of the sequence of SEQ ID NO: 19, and the sequence of CDR3-H of SEQ ID NO: 129

Chemical formula

Chemical formula

[0099] The so-called "8H2" anti-CD3 antibody is: - The CDR1-H of the sequence of SEQ ID NO: 103, the CDR2-H of the sequence of SEQ ID NO: 104, and the sequence of SEQ ID NO: 105 comprising the CDR3-H of

Chemical formula

Chemical formula

[0100] In one embodiment, the anti-CD3 antibody of the present invention binds to human CD3. In another embodiment, the anti-CD3 antibody of the present invention further binds to cynomolgus CD3. In particular, the anti-CD3 antibody of the present invention binds to the extracellular domain of human CD3 or both human and cynomolgus CD3. More specifically, the antibody binds to CD3ε. More specifically, the anti-CD3 antibody binds to the human and cynomolgus extracellular domains of CD3ε. The anti-CD3 antibody binds to CD3ε whether it is expressed in isolated form or present in complex form such as a CD3ε / δ complex, without distinguishing whether it is present in the soluble extracellular domain or in the form of full-length membrane-anchored CD3ε as present in, for example, T cells, or whether it is present as a single protein.

[0101] The anti-CD3 antibody according to the present invention is specific for the surface human CD3 protein or for the human and cynomolgus CD3 proteins, particularly for CD3ε.

[0102] In one embodiment, the anti-CD3 antibody according to the invention has a ratio of the affinity for cynomolgus CD3 to the affinity for human CD3 (KD(cynomolgus) / KD(human)) of ≤10, in particular ≤6, ≤5, ≤4, ≤3, for example ≤2, ≤1 or ≤0.5. Such polypeptides according to the invention can be used in toxicity studies in monkeys, and the associated toxicity profiles observed in monkeys predict possible adverse effects in humans. In particular, the anti-CD3 antibody of the invention does not bind to or does not significantly cross-react with CD3γ and / or CD3δ proteins.

[0103] In particular, the antibody does not bind to or does not significantly cross-react with the extracellular domains of the above-mentioned human and cynomolgus CD3γ and / or CD3δ proteins. The sequence of the full-length human CD3δ protein is available in the Uniprot database under accession number P04234 (SEQ ID NO: 86, as available on December 14, 2014). The extracellular domain of human CD3δ

[0104] consists of the amino acids at positions 22-105 of SEQ ID NO: 86.

[0105] The sequence of the full-length human CD3γ protein is available in the Uniprot database under accession number P09693 (SEQ ID NO: 185, as available on December 14, 2014). The extracellular domain of human CD3γ consists of the amino acids at positions 23-116 of SEQ ID NO: 185. Furthermore, the anti-CD3 antibody according to the invention has a KD of ≤90 nM, ≤50 nM, or ≤30 nM, for example ≤20 nM, ≤10 nM, ≤8 nM, ≤6 nM, ≤4 nM or

[0106] ≤2 nM for human CD3 or cynomolgus CD3, or both.

[0107] For human CD3 or cynomolgus CD3, or both. ≤2 nM for human CD3 or cynomolgus CD3, or both. It has an affinity (KD) of ≤2 nM, for example, an affinity of 0.1 nM to 10 nM, particularly 0.1 nM to 8 nM, or 0.1 nM to 4 nM.

[0108] The affinity for human CD3 or cynomolgus CD3 is measured as the KD value using surface plasmon resonance with a soluble recombinant CD3ε / δ complex derived from human and cynomolgus as the capture antigen.

[0109] In one example, the binding affinity of the anti-CD3 antibody is measured by surface plasmon resonance (SPR) using, for example, a Biacore 3000 instrument (GE Healthcare). The assay buffer is, for example, HBS-EP (BR-1001-88, GE Healthcare). For example, a human CD3ε and human CD3δ subunit extracellular domain construct containing a signal peptide in the form of the Fc fusion protein described in the examples can be used as the antigen. Alternatively, a cynomolgus CD3ε and cynomolgus CD3δ subunit extracellular domain construct containing a signal peptide in the form of the Fc fusion protein described in the examples can be utilized as the antigen. The cynomolgus CD3ε / δ-Fc fusion protein is achieved, for example, using a human antibody capture kit (GE Healthcare). For example, the capture antibody can be coupled to a CM5 chip (BR-1001-88, GE Healthcare) up to, for example, about 12,000 RU using, for example, an amine coupling kit (BR-100-50, GE Healthcare). The CD3ε / δ-Fc fusion protein is supplemented at 10 μl / min up to about 70 RU, resulting in an Rmax value of 30 RU. The binding kinetics for the anti-CD3 antibody can be measured for 240 seconds and 600 seconds at 30 μl / min for the binding and dissociation regions, respectively. For example, a two-fold dilution of the anti-CD3 antibody from 3 to 400 nM in the assay buffer can be used. The regeneration of the capture surface is, for example, 3M MgCl 2. 2 ​​​It can be carried out using, for example, an injection of 30 μl / min of the solution for 1 minute. For data analysis, for example, BIAevaluation software v.4.1 (GE Healthcare) can be used. The data can be globally fitted using a 1:1 Langmuir model with mass transfer.

[0110] In one embodiment, the anti-CD3 antibody of the invention also has an apparent EC50, determined by FACS analysis in, for example, human T cells, of ≤60 nM, such as ≤50 nM, ≤40 nM, ≤30 nM, ≤20 nM or ≤15 nM. Typically, the apparent EC50 is in the range of 1-60 nM, particularly 1-30 nM, such as 1-20 nM.

[0111] In one embodiment, the anti-CD3 antibody of the invention has T cell activation that is less than 10%, less than 8%, less than 6%, less than 4%, less than 2%, less than 1%, such as less than 0.5% in the absence of target cells. to have.

[0112] The term " T cell activation " herein refers to inducing CD3 signaling including cytotoxic granule fusion, transient cytokine release, and proliferation. The antibody-like binding proteins and anti-CD3 antibodies of the invention target CD3ε and activate T cells in the presence of target cells; this activation is also referred to as " ". The T cell T cell engaging effect inducing effect induces cytotoxicity in target cells.

[0113] As is known to those skilled in the art, activation of T cells induces the expression of surface markers such as CD69 and CD25. Thus, activation of T cells can be measured by detecting and measuring the expression of CD4+ / CD25+, CD4+ / CD69+, CD8+ / CD25+, or CD8+ / CD69+ T cells. Methods for measuring T cell activation are known to those skilled in the art.

[0114] Methods for measuring T cell activation are further disclosed in the Examples section (Example 3.3). Thus, in the context of the present invention, T cell activation is measured as the percentage (%) of cells expressing CD69 out of the total number of cells, or as the percentage (%) of cells expressing CD4 and CD69 out of the total number of cells, or as any of the percentage (%) of cells expressing CD8 and CD69 out of the total number of cells. In the context of the anti-CD3 antibodies of the present invention, "

[0115] " refers to T cell activation of 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. low T cell activation The VH and VL region sequences of the "20G6-F3", "4B4-D7", "4E7-C9", "18F5-H10", "12D2-E5", "11D7-C3",

[0116] "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 aligned. Comparison of the CDR-H and CDR-L sequences structurally shows that "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. are related next, and the above antibodies tend to show that they probably bind to the same epitope. The comparison of the CDR-H and CDR-L sequences of the above related antibodies is shown in FIGS. 1 and 2 respectively. The CDR positions are strictly conserved among the antibodies and were thus presumed to be important for specificity, but , on the other hand, other positions were identified as being able to support substitutions.

[0117] Therefore, the antibodies according to the present invention are: - the sequence GFX 1 X 2 X 3 X 4 AW (SEQ ID NO: 331) [where 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] consisting of CDR1-H; and the sequence IKX 1 X 2 X 3 NX 4 YX 5 T (SEQ ID NO: 332) [where 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] consisting of CDR2-H; and the 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) [where X 1 is Y, G or A, X 2 is V, T or L and X 3 is H, N, Y or Q, and X 4 is G, R or A, and X 5 is F or V, or is not an amino acid, and X 6 is R or is not an amino acid, and X 7 is F, S or I or is not an amino acid, and X 8 is F, L, N, M, Y, S, A or G, and X 9 is Y, A, K, S, N, T, F or L, and X 10 is A, P, G or T, and X 11 is M, L, F or S, and X 12 is A, V or Y, or a combination of any of them] consisting of a CDR3-H containing a heavy chain variable domain, and - sequence QX 1 LX 2 HX 3 NGX 4 TY (SEQ ID NO: 334) [where X 1 is R or S, and X 2 is V or E and X 3 is N, D or T, and X 4 is N or Y, or a combination of any of them] consisting of a CDR1-L; and a CDR2-L consisting of the sequence "KVS"; and a CDR3-L consisting of the sequence GQGX X 1 X 2 YPFT (SEQ ID NO: 335) [where X 1 is T, A or S, and X 2 is H, E or Q, or a combination of any of them] consisting of a light chain variable domain containing a CDR3-L in including.

[0118] According to one embodiment, the anti-CD3 antibody according to the present invention is one of the 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", and contains the CDR sequences of one of the heavy and / or light chains of the anti-CD3 antibody.

[0119] Therefore, the present invention provides: a) a heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 6 or a sequence differing from SEQ ID NO: 6 by one amino acid substitution; CDR2-H of the sequence of SEQ ID NO: 7 or a sequence differing from SEQ ID NO: 7 by one or more amino acid substitutions; CDR3-H of the sequence of 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 the sequence of 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; CDR2-L of the sequence "KVS" or a sequence differing from the sequence "KVS" by one amino acid substitution; and CDR3-L of the sequence of 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 the sequence of SEQ ID NO: 13 or a sequence differing from SEQ ID NO: 13 by one amino acid substitution; CDR2-H of the sequence of SEQ ID NO: 14 or a sequence differing from SEQ ID NO: 14 by one or more amino acid substitutions; CDR3-H of the sequence of 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 the sequence of SEQ ID NO: 16 or SEQ ID NO: 143 or a sequence differing from SEQ ID NO: 16 or SEQ ID NO: 143 by one amino acid substitution; CDR2-L of the sequence "KVS" or a sequence differing from the sequence "KVS" by one amino acid substitution; and CDR3-L of the sequence of SEQ ID NO: 17 or a sequence differing from SEQ ID NO: 17 by one amino acid substitution. The light chain variable domain comprising CDR1-L of the sequence of 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 the sequence "KVS" or a sequence differing from the sequence "KVS" by one amino acid substitution; and CDR3-L of the sequence of SEQ ID NO: 11 or a sequence differing from SEQ ID NO: 11 by one amino acid substitution; or c) The heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 13 or a sequence differing from SEQ ID NO: 13 by one amino acid substitution; CDR2-H of the sequence of SEQ ID NO: 19 or a sequence differing from SEQ ID NO: 19 by one or more amino acid substitutions; CDR3-H of the sequence of SEQ ID NO: 20 or a sequence differing from SEQ ID NO: 20 by one amino acid substitution; and The light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 22 or a sequence differing from SEQ ID NO: 22 by one amino acid substitution; CDR2-L of the sequence "KVS" or a sequence differing from the sequence "KVS" by one amino acid substitution; and CDR3-L of the sequence of SEQ ID NO: 11 or a sequence differing from SEQ ID NO: 11 by one amino acid substitution; or d) The heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 24 or a sequence differing from SEQ ID NO: 24 by one amino acid substitution; CDR2-H of the sequence of SEQ ID NO: 19 or a sequence differing from SEQ ID NO: 19 by one or more amino acid substitutions; CDR3-H of the sequence of SEQ ID NO: 25 or a sequence differing from SEQ ID NO: 25 by one amino acid substitution; and The light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 27 or a sequence differing from SEQ ID NO: 27 by one amino acid substitution; CDR2-L of the sequence "KVS" or a sequence differing from the sequence "KVS" by one amino acid substitution; and CDR3-L of the sequence of SEQ ID NO: 28 or a sequence differing from SEQ ID NO: 28 by one amino acid substitution; or e) The heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 30 or a sequence differing from SEQ ID NO: 30 by one amino acid substitution; CDR2-H of the sequence of SEQ ID NO: 31 or a sequence differing from SEQ ID NO: 31 by one or more amino acid substitutions; CDR3-H of the sequence of SEQ ID NO: 32 or a sequence differing from SEQ ID NO: 32 by one amino acid substitution; and The light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 34 or a sequence differing from the sequence of SEQ ID NO: 34 by one amino acid substitution; CDR2-L of the sequence "RDD" or a sequence differing from the sequence "RDD" by one amino acid substitution; and CDR3-L of the sequence of SEQ ID NO: 35 or a sequence differing from the sequence of SEQ ID NO: 35 by one amino acid substitution; or

[0120] f) CDR1-H of the sequence of SEQ ID NO: 13 or a sequence differing from the sequence of SEQ ID NO: 13 by one amino acid substitution; CDR2-H of the sequence of SEQ ID NO: 37 or a sequence differing from the sequence of SEQ ID NO: 37 by one or more amino acid substitutions; CDR3-H of the sequence of SEQ ID NO: 38 or a sequence differing from the sequence of SEQ ID NO: 38 by one amino acid substitution comprising heavy chain variable domain; and The light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 10 or a sequence differing from the sequence of SEQ ID NO: 10 by one amino acid substitution; CDR2-L of the sequence "KVS" or a sequence differing from the sequence "KVS" by one amino acid substitution; and CDR3-L of the sequence of SEQ ID NO: 28 or a sequence differing from the sequence of SEQ ID NO: 28 by one amino acid substitution; or g) CDR1-H of the sequence of SEQ ID NO: 13 or a sequence differing from the sequence of SEQ ID NO: 13 by one amino acid substitution; CDR2-H of the sequence of SEQ ID NO: 37 or a sequence differing from the sequence of SEQ ID NO: 37 by one or more amino acid substitutions thereby differing sequence; CDR3-H of the sequence of SEQ ID NO: 41 or a sequence differing from the sequence of SEQ ID NO: 41 by one amino acid substitution comprising heavy chain variable domain; and The light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 17 or a sequence differing from the sequence of SEQ ID NO: 17 by one amino acid substitution; CDR2-L of the sequence "KVS" or a sequence differing from the sequence "KVS" by one amino acid substitution; and CDR3-L of the sequence of SEQ ID NO: 11 or a sequence differing from the sequence of SEQ ID NO: 11 by one amino acid substitution; or h) A heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 13 or a sequence differing from the sequence of SEQ ID NO: 13 by one amino acid substitution; CDR2-H of the sequence of SEQ ID NO: 37 or a sequence differing from the sequence of SEQ ID NO: 37 by one or more amino acid substitutions; CDR3-H of the sequence of SEQ ID NO: 44 or a sequence differing from the sequence of SEQ ID NO: 44 by one amino acid substitution; and a light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 10 or a sequence differing from the sequence of SEQ ID NO: 10 by one amino acid substitution; CDR2-L of the sequence "KVS" or a sequence differing from the sequence "KVS" by one amino acid substitution; and CDR3-L of the sequence of SEQ ID NO: 11 or a sequence differing from the sequence of SEQ ID NO: 11 by one amino acid substitution; or i) A heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 13 or a sequence differing from the sequence of SEQ ID NO: 13 by one amino acid substitution; CDR2-H of the sequence of SEQ ID NO: 37 or a sequence differing from the sequence of SEQ ID NO: 37 by one or more amino acid substitutions; CDR3-H of the sequence of SEQ ID NO: 47 or a sequence differing from the sequence of SEQ ID NO: 47 by one amino acid substitution; and a light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 10 or a sequence differing from the sequence of SEQ ID NO: 10 by one amino acid substitution; CDR2-L of the sequence "KVS" or a sequence differing from the sequence "KVS" by one amino acid substitution; and CDR3-L of the sequence of SEQ ID NO: 11 or a sequence differing from the sequence of SEQ ID NO: 11 by one amino acid substitution; or j) A heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 50 or a sequence differing from the sequence of SEQ ID NO: 50 by one amino acid substitution; CDR2-H of the sequence of SEQ ID NO: 51 or a sequence differing from the sequence of SEQ ID NO: 51 by one or more amino acid substitutions; CDR3-H of the sequence of SEQ ID NO: 52 or a sequence differing from the sequence of SEQ ID NO: 52 by one amino acid substitution; and a light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 54 or a sequence differing from the sequence of SEQ ID NO: 54 by one amino acid substitution; CDR2-L of the sequence "NAN" or a sequence differing from the sequence "NAN" by one amino acid substitution; and CDR3-L of the sequence of SEQ ID NO: 55 or a sequence differing from the sequence of SEQ ID NO: 55 by one amino acid substitution; or

[0121] k) A heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 57 or a sequence differing from the sequence of SEQ ID NO: 57 by one amino acid substitution; CDR2-H of the sequence of SEQ ID NO: 37 or a sequence differing from the sequence of SEQ ID NO: 37 by one or more amino acid substitutions; and CDR3-H of the sequence of SEQ ID NO: 58 or a sequence differing from the sequence of SEQ ID NO: 58 by one amino acid substitution; and A light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 10 or a sequence differing from the sequence of SEQ ID NO: 10 by one amino acid substitution; CDR2-L of the sequence "KVS" or a sequence differing from the sequence "KVS" by one amino acid substitution; and CDR3-L of the sequence of SEQ ID NO: 28 or a sequence differing from the sequence of SEQ ID NO: 28 by one amino acid substitution; or l) A heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 13 or a sequence differing from the sequence of SEQ ID NO: 13 by one amino acid substitution; CDR2-H of the sequence of SEQ ID NO: 37 or a sequence differing from the sequence of SEQ ID NO: 37 by one or more amino acid substitutions; and CDR3-H of the sequence of SEQ ID NO: 61 or a sequence differing from the sequence of SEQ ID NO: 61 by one amino acid substitution; and A light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 10 or a sequence differing from the sequence of SEQ ID NO: 10 by one amino acid substitution; CDR2-L of the sequence "KVS" or a sequence differing from the sequence "KVS" by one amino acid substitution; and CDR3-L of the sequence of SEQ ID NO: 11 or a sequence differing from the sequence of SEQ ID NO: 11 by one amino acid substitution; or m) A heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 64 or a sequence differing from the sequence of SEQ ID NO: 64 by one amino acid substitution; CDR2-H of the sequence of SEQ ID NO: 65 or a sequence differing from the sequence of SEQ ID NO: 65 by one or more amino acid substitutions; and CDR3-H of the sequence of SEQ ID NO: 47 or a sequence differing from the sequence of SEQ ID NO: 47 by one amino acid substitution; and A light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 67 or a sequence differing from the sequence of SEQ ID NO: 67 by one amino acid substitution; CDR2-L of the sequence "KVS" or a sequence differing from the sequence "KVS" by one amino acid substitution; and CDR3-L of the sequence of SEQ ID NO: 28 or a sequence differing from the sequence of SEQ ID NO: 28 by one amino acid substitution; or n) A heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 13 or a sequence differing from the sequence of SEQ ID NO: 13 by one amino acid substitution; CDR2-H of the sequence of SEQ ID NO: 37 or a sequence differing from the sequence of SEQ ID NO: 37 by one or more amino acid substitutions; CDR3-H of the sequence of SEQ ID NO: 69 or a sequence differing from the sequence of SEQ ID NO: 69 by one amino acid substitution; and A light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 10 or a sequence differing from the sequence of SEQ ID NO: 10 by one amino acid substitution; CDR2-L of the sequence "KVS" or a sequence differing from the sequence "KVS" by one amino acid substitution; and CDR3-L of the sequence of SEQ ID NO: 71 or a sequence differing from the sequence of SEQ ID NO: 71 by one amino acid substitution; or o) A heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 13 or a sequence differing from the sequence of SEQ ID NO: 13 by one amino acid substitution; CDR2-H of the sequence of SEQ ID NO: 37 or a sequence differing from the sequence of SEQ ID NO: 37 by one or more amino acid substitutions; CDR3-H of the sequence of SEQ ID NO: 84 or a sequence differing from the sequence of SEQ ID NO: 84 by one amino acid substitution; and A light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 17 or a sequence differing from the sequence of SEQ ID NO: 17 by one amino acid substitution; CDR2-L of the sequence "KVS" or a sequence differing from the sequence "KVS" by one amino acid substitution; and CDR3-L of the sequence of SEQ ID NO: 11 or a sequence differing from the sequence of SEQ ID NO: 11 by one amino acid substitution; or

[0122] p) A heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 75 or a sequence differing from the sequence of SEQ ID NO: 75 by one amino acid substitution; CDR2-H of the sequence of SEQ ID NO: 76 or a sequence differing from the sequence of SEQ ID NO: 76 by one or more amino acid substitutions; CDR3-H of the sequence of SEQ ID NO: 77 or a sequence differing from the sequence of SEQ ID NO: 77 by one amino acid substitution; and A light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 10 or a sequence differing from the sequence of SEQ ID NO: 10 by one amino acid substitution; CDR2-L of the sequence "KVS" or a sequence differing from the sequence "KVS" by one amino acid substitution; and CDR3-L of the sequence of SEQ ID NO: 11 or a sequence differing from the sequence of SEQ ID NO: 11 by one amino acid substitution; or q) A heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 80 or a sequence differing from the sequence of SEQ ID NO: 80 by one amino acid substitution; CDR2-H of the sequence of SEQ ID NO: 76 or a sequence differing from the sequence of SEQ ID NO: 76 by one or more amino acid substitutions; and CDR3-H of the sequence of SEQ ID NO: 81 or a sequence differing from the sequence of SEQ ID NO: 81 by one amino acid substitution; and A light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 10 or a sequence differing from the sequence of SEQ ID NO: 10 by one amino acid substitution; CDR2-L of the sequence "KVS" or a sequence differing from the sequence "KVS" by one amino acid substitution; and CDR3-L of the sequence of SEQ ID NO: 11 or a sequence differing from the sequence of SEQ ID NO: 11 by one amino acid substitution; or r) A heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 13 or a sequence differing from the sequence of SEQ ID NO: 13 by one amino acid substitution; CDR2-H of the sequence of SEQ ID NO: 37 or a sequence differing from the sequence of SEQ ID NO: 37 by one or more amino acid substitutions; and CDR3-H of the sequence of SEQ ID NO: 84 or a sequence differing from the sequence of SEQ ID NO: 84 by one amino acid substitution; and A light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 10 or a sequence differing from the sequence of SEQ ID NO: 10 by one amino acid substitution; CDR2-L of the sequence "KVS" or a sequence differing from the sequence "KVS" by one amino acid substitution; and CDR3-L of the sequence of SEQ ID NO: 11 or a sequence differing from the sequence of SEQ ID NO: 11 by one amino acid substitution; or s) A heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 13 or a sequence differing from the sequence of SEQ ID NO: 13 by one amino acid substitution; CDR2-H of the sequence of SEQ ID NO: 37 or a sequence differing from the sequence of SEQ ID NO: 37 by one or more amino acid substitutions; and CDR3-H of the sequence of SEQ ID NO: 47 or a sequence differing from the sequence of SEQ ID NO: 47 by one amino acid substitution; and A light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 10 or a sequence differing from the sequence of SEQ ID NO: 10 by one amino acid substitution; the CDR2-L of the sequence "KVS" or a sequence differing from the sequence "KVS" by one amino acid substitution; and CDR3-L of the sequence of SEQ ID NO: 88 or a sequence differing from the sequence of SEQ ID NO: 88 by one amino acid substitution; or t) A heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 90 or a sequence differing from the sequence of SEQ ID NO: 90 by one amino acid substitution; CDR2-H of the sequence of SEQ ID NO: 91 or a sequence differing from the sequence of SEQ ID NO: 91 by one or more amino acid substitutions; CDR3-H of the sequence of SEQ ID NO: 32 or a sequence differing from the sequence of SEQ ID NO: 32 by one amino acid substitution; and CDR1-L of the sequence of SEQ ID NO: 93 or a sequence differing from the sequence of SEQ ID NO: 93 by one amino acid substitution; CDR2-L of the sequence "GAS" or a sequence differing from the sequence "GAS" by one amino acid substitution; and CDR3-L of the sequence of SEQ ID NO: 94 or a sequence differing from the sequence of SEQ ID NO: 94 by one amino acid substitution; or

[0123] u) A heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 96 or a sequence differing from the sequence of SEQ ID NO: 96 by one amino acid substitution; CDR2-H of the sequence of SEQ ID NO: 97 or a sequence differing from the sequence of SEQ ID NO: 97 by one or more amino acid substitutions; CDR3-H of the sequence of SEQ ID NO: 98 or a sequence differing from the sequence of SEQ ID NO: 98 by one amino acid substitution; and CDR1-L of the sequence of SEQ ID NO: 100 or a sequence differing from the sequence of SEQ ID NO: 100 by one amino acid substitution; CDR2-L of the sequence "NTN" or a sequence differing from the sequence "NTN" by one amino acid substitution; and CDR3-L of the sequence of SEQ ID NO: 101 or a sequence differing from the sequence of SEQ ID NO: 101 by one amino acid substitution; or v) A heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 103 or a sequence differing from the sequence of SEQ ID NO: 103 by one amino acid substitution; CDR2-H of the sequence of SEQ ID NO: 104 or a sequence differing from the sequence of SEQ ID NO: 104 by one or more amino acid substitutions; CDR3-H of the sequence of SEQ ID NO: 105 or a sequence differing from the sequence of SEQ ID NO: 105 by one amino acid substitution; and CDR1-L of the sequence of SEQ ID NO: 10 or a sequence differing from the sequence of SEQ ID NO: 10 by one amino acid substitution; CDR2-L of the sequence "KVS" or a sequence differing from the sequence "KVS" by one amino acid substitution; and CDR3-L of the sequence of SEQ ID NO: 11 or a sequence differing from the sequence of SEQ ID NO: 11 by one amino acid substitution; or w) a heavy chain variable domain comprising a CDR1-H of the sequence of SEQ ID NO: 80 or a sequence differing from the sequence of SEQ ID NO: 80 by one amino acid substitution; a CDR2-H of the sequence of SEQ ID NO: 19 or a sequence differing from the sequence of SEQ ID NO: 19 by one or more amino acid substitutions; a CDR3-H of the sequence of SEQ ID NO: 108 or a sequence differing from the sequence of SEQ ID NO: 108 by one amino acid substitution; and a light chain variable domain comprising a CDR1-L of the sequence of SEQ ID NO: 10 or a sequence differing from the sequence of SEQ ID NO: 10 by one amino acid substitution; a CDR2-L of the sequence "KVS" or a sequence differing from the sequence "KVS" by one amino acid substitution; and a CDR3-L of the sequence of SEQ ID NO: 11 or a sequence differing from the sequence of SEQ ID NO: 11 by one amino acid substitution; or x) a heavy chain variable domain comprising a CDR1-H of the sequence of SEQ ID NO: 13 or a sequence differing from the sequence of SEQ ID NO: 13 by one amino acid substitution; a CDR2-H of the sequence of SEQ ID NO: 37 or a sequence differing from the sequence of SEQ ID NO: 37 by one or more amino acid substitutions; a CDR3-H of the sequence of SEQ ID NO: 111 or a sequence differing from the sequence of SEQ ID NO: 111 by one amino acid substitution; and a light chain variable domain comprising a CDR1-L of the sequence of SEQ ID NO: 113 or a sequence differing from the sequence of SEQ ID NO: 113 by one amino acid substitution; a CDR2-L of the sequence "KVS" or a sequence differing from the sequence "KVS" by one amino acid substitution; and a CDR3-L of the sequence of SEQ ID NO: 114 or a sequence differing from the sequence of SEQ ID NO: 114 by one amino acid substitution; or y) a heavy chain variable domain comprising a CDR1-H of the sequence of SEQ ID NO: 116 or a sequence differing from the sequence of SEQ ID NO: 116 by one amino acid substitution; a CDR2-H of the sequence of SEQ ID NO: 117 or a sequence differing from the sequence of SEQ ID NO: 117 by one or more amino acid substitutions; a CDR3-H of the sequence of SEQ ID NO: 118 or a sequence differing from the sequence of SEQ ID NO: 118 by one amino acid substitution; and a light chain variable domain comprising a CDR1-L of the sequence of SEQ ID NO: 100 or a sequence differing from the sequence of SEQ ID NO: 100 by one amino acid substitution; a CDR2-L of the sequence "VTN" or a sequence differing from the sequence "VTN" by one amino acid substitution; and ​​A light chain variable domain comprising a CDR3-L of the sequence of 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 a CDR1-H of the sequence of SEQ ID NO: 122 or a sequence differing from SEQ ID NO: 122 by one amino acid substitution; a CDR2-H of the sequence of SEQ ID NO: 123 or a sequence differing from SEQ ID NO: 123 by one or more amino acid substitutions; a CDR3-H of the sequence of 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 a CDR1-L of the sequence of SEQ ID NO: 126 or a sequence differing from SEQ ID NO: 126 by one amino acid substitution; a CDR2-L of the sequence "RDD" or a sequence differing from the sequence "RDD" by one amino acid substitution; and a CDR3-L of the sequence of SEQ ID NO: 127 or a sequence differing from SEQ ID NO: 127 by one amino acid substitution; or

[0124] aa) A heavy chain variable domain comprising a CDR1-H of the sequence of SEQ ID NO: 13 or a sequence differing from SEQ ID NO: 13 by one amino acid substitution; a CDR2-H of the sequence of SEQ ID NO: 19 or a sequence differing from SEQ ID NO: 19 by one or more amino acid substitutions; a CDR3-H of the sequence of 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 a CDR1-L of the sequence of SEQ ID NO: 10 or a sequence differing from SEQ ID NO: 10 by one amino acid substitution; a CDR2-L of the sequence "KVS" or a sequence differing from the sequence "KVS" by one amino acid substitution; and a CDR3-L of the sequence of 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 a CDR1-H of the sequence of SEQ ID NO: 103 or a CDR1-H differing from SEQ ID NO: 103 by one amino acid substitution; a CDR2-H of the sequence of SEQ ID NO: 104 or a sequence differing from SEQ ID NO: 104 by one or more amino acid substitutions; a CDR3-H of the sequence of SEQ ID NO: 105 or a sequence differing from SEQ ID NO: 105 by one amino acid substitution; and The light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 133 or a sequence differing from the sequence of SEQ ID NO: 133 by one amino acid substitution; CDR2-L of the sequence "LVS" or a sequence differing from the sequence "LVS" by one amino acid substitution; and CDR3-L of the sequence of SEQ ID NO: 134 or a sequence differing from the sequence of SEQ ID NO: 134 by one amino acid substitution relates to an anti-CD3 antibody comprising the same.

[0125] One or more individual amino acids may be substituted in one or more of the above CDR sequences by substitution, particularly conservative substitution. Such modifications may be intended, for example, to remove glycosylation sites or deamidation sites, in connection with humanization of the antibody.

[0126] Based on the alignment of the sequences of the VH and VL regions of "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", various amino acid substitutions have been identified. Thus, in one embodiment, the amino acids are: - in one or more of positions 3 to 6 in CDR1-H, for example in position 3, 5 or 6 of CDR1-H of the sequence GFTFSNAW (SEQ ID NO: 13), or for example in positions 4 and 5 of CDR1-H of the sequence GFTFSNAW (SEQ ID NO: 13); and / or - in one or more of positions 3 to 5, 7 and 9 in CDR2-H, for example in position 3, 4, 5, 7 or 9 of CDR2-H of the sequence IKAKSNNYAT (SEQ ID NO: 37), or for example in positions 3 and 5 or 3 and 7 of CDR2-H of the sequence IKAKSNNYAT (SEQ ID NO: 37); and / or - In CDR3-H, at one or more of positions 2-4, 7-10, and 12, for example, 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 the CDR3-H of the sequence RGVYYALSPFDY (SEQ ID NO: 8), or at positions 7 and 8 of the CDR3-H of the sequence RGLYYGLSPSDY (SEQ ID NO: 38), or at positions 2, 3, 4, 6, 7, 8, 9, and 10 of the CDR3-H of the 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 the CDR1-L of the sequences QSLVHDNGNTY (SEQ ID NO: 17) or QSLVHTNGNTY (SEQ ID NO: 27), or at position 2 of the CDR1-L of the sequences QSLVHNNGNTY (SEQ ID NO: 10) or QRLVHNNGNTY (SEQ ID NO: 113), or at positions 4, 6, and 9 of the CDR1-L of the sequence QSLVHNNGNTY (SEQ ID NO: 10); and or / and - In CDR3-L, at one or more of positions 4 and 5, for example, at position 4 of the CDR3-L of the sequences GQGSQYPFT (SEQ ID NO: 71) or GQGTQYPFT (SEQ ID NO: 11), or at positions 4 and 5 of the CDR3-L of the sequence GQGAHYPFT (SEQ ID NO: 88), or at position 5 of the CDR3-L of the sequences GQGTHYPFT (SEQ ID NO: 28) or GQGTEYPFT (SEQ ID NO: 114) is substituted. The anti-CD3 antibody according to the present invention is a conventional antibody, in particular a conventional monoclonal antibody,

[0127] or an antibody fragment, bispecific or multispecific antibody. The anti-CD3 antibody according to the present invention particularly comprises or consists of IgG, or a fragment thereof.

[0128]

[0129] ​ According to a further embodiment, the present invention is: a) a heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 6, CDR2-H of the sequence of SEQ ID NO: 7, and CDR3-H of the sequence of SEQ ID NO: 8, and CDR1-L of the sequence of SEQ ID NO: 10 or SEQ ID NO: 142, CDR2-L of the sequence "KVS" ", and a light chain variable domain comprising CDR3-L of the sequence of SEQ ID NO: 11; or b) a heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 13, CDR2-H of the sequence of SEQ ID NO: 14, and CDR3-H of the sequence of SEQ ID NO: 15, and a light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 17 or SEQ ID NO: 184, CDR2-L of the sequence "KVS", and CDR3-L of the sequence of SEQ ID NO: 11; or c) a heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 13, CDR2-H of the sequence of SEQ ID NO: 19, and CDR3-H of the sequence of SEQ ID NO: 20, and a light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 22, CDR2-L of the sequence "KVS", and CDR3-L of the sequence of SEQ ID NO: 11 ; or d) a heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 24, CDR2-H of the sequence of SEQ ID NO: 19, and CDR3-H of the sequence of SEQ ID NO: 25, and a light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 27, CDR2-L of the sequence "KVS", and CDR3-L of the sequence of SEQ ID NO: 28 ; or e) a heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 30, CDR2-H of the sequence of SEQ ID NO: 31, and CDR3-H of the sequence of SEQ ID NO: 32, and a light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 34, CDR2-L of the sequence "RDD", and CDR3-L of the sequence of SEQ ID NO: 35 ; or f) a heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 13, CDR2-H of the sequence of SEQ ID NO: 37, and CDR3-H of the sequence of SEQ ID NO: 38, and comprising CDR1-L of the sequence of SEQ ID NO: 10, CDR2-L of the sequence "KVS", and CDR3-L of the sequence of SEQ ID NO: 28 a light chain variable domain; or g) a heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 13, CDR2-H of the sequence of SEQ ID NO: 37, and CDR3-H of the sequence of SEQ ID NO: 41, and comprising CDR1-L of the sequence of SEQ ID NO: 17, CDR2-L of the sequence "KVS", and CDR3-L of the sequence of SEQ ID NO: 11 a light chain variable domain; or h) a heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 13, CDR2-H of the sequence of SEQ ID NO: 37, and CDR3-H of the sequence of SEQ ID NO: 44, and comprising CDR1-L of the sequence of SEQ ID NO: 10, CDR2-L of the sequence "KVS", and CDR3-L of the sequence of SEQ ID NO: 11 a light chain variable domain; or

[0130] i) a heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 13, CDR2-H of the sequence of SEQ ID NO: 37, and CD R3-H of the sequence of SEQ ID NO: 47, and a light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 10, CDR2-L of the sequence "KVS", and CDR3-L of the sequence of SEQ ID NO: 11; or j) a heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 50, CDR2-H of the sequence of SEQ ID NO: 51, and CDR3-H of the sequence of SEQ ID NO: 52, and a light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 54, CDR2-L of the sequence "NAN", and CDR3-L of the sequence of SEQ ID NO: 55; or k) a heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 57, CDR2-H of the sequence of SEQ ID NO: 37, and CDR3-H of the sequence of SEQ ID NO: 58, and a light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 10, CDR2-L of the sequence "KVS", and l) A heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 13, CDR2-H of the sequence of SEQ ID NO: 37, and CDR3-H of the sequence of SEQ ID NO: 61, and a light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 10, CDR2-L of the sequence "KVS", and CDR3-L of the sequence of SEQ ID NO: 11; or m) A heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 64, CDR2-H of the sequence of SEQ ID NO: 65, and CDR3-H of the sequence of SEQ ID NO: 47, and a light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 67, CDR2-L of the sequence "KVS", and CDR3-L of the sequence of SEQ ID NO: 28; or n) A heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 13, CDR2-H of the sequence of SEQ ID NO: 37, and CDR3-H of the sequence of SEQ ID NO: 69, and a light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 10, CDR2-L of the sequence "KVS", and CDR3-L of the sequence of SEQ ID NO: 71; or o) A heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 13, CDR2-H of the sequence of SEQ ID NO: 37, and CDR3-H of the sequence of SEQ ID NO: 84, and a light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 17, CDR2-L of the sequence "KVS", and CDR3-L of the sequence of SEQ ID NO: 11; or p) A heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 75, CDR2-H of the sequence of SEQ ID NO: 76, and CDR3-H of the sequence of SEQ ID NO: 77, and a light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 10, CDR2-L of the sequence "KVS", and CDR3-L of the sequence of SEQ ID NO: 11; or q) A heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 80, CDR2-H of the sequence of SEQ ID NO: 76, and CDR3-H of the sequence of SEQ ID NO: 81, and a light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 10, CDR2-L of the sequence "KVS", and CDR3-L of the sequence of SEQ ID NO: 11; or r) A heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 13, CDR2-H of the sequence of SEQ ID NO: 37, and CDR3-H of the sequence of SEQ ID NO: 84, and a light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 10, CDR2-L of the sequence "KVS", and CDR3-L of the sequence of SEQ ID NO: 11; or s) A heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 13, CDR2-H of the sequence of SEQ ID NO: 37, and CDR3-H of the sequence of SEQ ID NO: 47, and a light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 10, CDR2-L of the sequence "KVS", and CDR3-L of the sequence of SEQ ID NO: 88; or

[0131] t) A heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 90, CDR2-H of the sequence of SEQ ID NO: 91, and CDR3-H of the sequence of SEQ ID NO: 32, and a light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 93, CDR2-L of the sequence "GAS", and CDR3-L of the sequence of SEQ ID NO: 94; or u) A heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 96, CDR2-H of the sequence of SEQ ID NO: 97, and CDR3-H of the sequence of SEQ ID NO: 98, and a light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 100, CDR2-L of the sequence "NTN", and CDR3-L of the sequence of SEQ ID NO: 101; or v) A heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 103, CDR2-H of the sequence of SEQ ID NO: 104, and CDR3-H of the sequence of SEQ ID NO: 105 and a light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 10, CDR2-L of the sequence "KVS", and CDR3-L of the sequence of SEQ ID NO: 11; or w) A heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 80, CDR2-H of the sequence of SEQ ID NO: 19, and CDR3-H of the sequence of SEQ ID NO: 108, and a light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 10, CDR2-L of the sequence "KVS", and CDR3-L of the sequence of SEQ ID NO: 11; or x) A heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 13, CDR2-H of the sequence of SEQ ID NO: 37, and CDR3-H of the sequence of SEQ ID NO: 111, and CDR1-L of the sequence of SEQ ID NO: 113, CDR2-L of the sequence "KVS" and a light chain variable domain comprising CDR3-L of the sequence of SEQ ID NO: 114; or y) A heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 116, CDR2-H of the sequence of SEQ ID NO: 117, and CDR3-H of the sequence of SEQ ID NO: 118, and CDR1-L of the sequence of SEQ ID NO: 100, CDR2 -L of the sequence "VTN" and a light chain variable domain comprising CDR3-L of the sequence of SEQ ID NO: 120; or -L and a light chain variable domain comprising CDR3-L of the sequence of SEQ ID NO: 120; or z) A heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 122, CDR2-H of the sequence of SEQ ID NO: 123, and CDR3-H of the sequence of SEQ ID NO: 124, and CDR1-L of the sequence of SEQ ID NO: 126, CDR2-L of the sequence "RDD" and a light chain variable domain comprising CDR3-L of the sequence of SEQ ID NO: 127; or

[0132] aa) A heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 13, CDR2-H of the sequence of SEQ ID NO: 19, and CDR3-H of the sequence of SEQ ID NO: 129, and CDR1-L of the sequence of SEQ ID NO: 10, CDR2-L of the sequence "KVS" and a light chain variable domain comprising CDR3-L of the sequence of SEQ ID NO: 11; or bb) A heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 103, CDR2-H of the sequence of SEQ ID NO: 104, and CDR3-H of the sequence of SEQ ID NO: 105; and a light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 133, CDR2-L of the sequence "LVS" and CDR3-L of the sequence of SEQ ID NO: 134

[0133] relates to an anti-CD3 antibody comprising

[0133] The present invention also relates to at least the heavy chain variable Provided is an anti-CD3 antibody comprising a domain and / or a light chain variable domain.

[0134] Accordingly, the present invention particularly provides: a) a heavy chain variable domain of the sequence of SEQ ID NO: 5 or a sequence that is at least 85% identical thereto, and / or a light chain variable domain of the sequence of SEQ ID NO: 9, or a sequence that is at least 85% identical thereto; or b) a heavy chain variable domain of the sequence of SEQ ID NO: 12 or a sequence that is at least 85% identical thereto, and / or a light chain variable domain of the sequence of SEQ ID NO: 16, or a sequence that is at least 85% identical thereto; or c) a heavy chain variable domain of the sequence of SEQ ID NO: 18 or a sequence that is at least 85% identical thereto, and / or a light chain variable domain of the sequence of SEQ ID NO: 21, or a sequence that is at least 85% identical thereto; or d) a heavy chain variable domain of the sequence of SEQ ID NO: 23 or a sequence that is at least 85% identical thereto, and / or a light chain variable domain of the sequence of SEQ ID NO: 26, or a sequence that is at least 85% identical thereto; or e) a heavy chain variable domain of the sequence of SEQ ID NO: 29 or a sequence that is at least 85% identical thereto, and / or a light chain variable domain of the sequence of SEQ ID NO: 33, or a sequence that is at least 85% identical thereto; or f) a heavy chain variable domain of the sequence of SEQ ID NO: 36 or a sequence that is at least 85% identical thereto, and / or a light chain variable domain of the sequence of SEQ ID NO: 39, or a sequence that is at least 85% identical thereto; or g) a heavy chain variable domain of the sequence of SEQ ID NO: 40 or a sequence that is at least 85% identical thereto, and / or a light chain variable domain of the sequence of SEQ ID NO: 42, or a sequence that is at least 85% identical thereto; or h) a heavy chain variable domain of the sequence of SEQ ID NO: 43 or a sequence that is at least 85% identical thereto, and / or a light chain variable domain of the sequence of SEQ ID NO: 45, or a sequence that is at least 85% identical thereto; or i) a heavy chain variable domain of the sequence of SEQ ID NO: 46 or a sequence that is at least 85% identical thereto, and / or a light chain variable domain of the sequence of SEQ ID NO: 48, or a sequence that is at least 85% identical thereto; or i) The heavy chain variable domain of the sequence of SEQ ID NO: 46 or a sequence that is at least 85% identical thereto, and / or the light chain variable domain of the sequence of SEQ ID NO: 48, or a sequence that is at least 85% identical thereto; or

[0135] j) The heavy chain variable domain of the sequence of SEQ ID NO: 49 or a sequence that is at least 85% identical thereto, and / or the light chain variable domain of the sequence of SEQ ID NO: 53, or a sequence that is at least 85% identical thereto; or k) The heavy chain variable domain of the sequence of SEQ ID NO: 56 or a sequence that is at least 85% identical thereto , and / or the light chain variable domain of the sequence of SEQ ID NO: 59, or a sequence that is at least 85% identical thereto; l) The heavy chain variable domain of the sequence of SEQ ID NO: 60 or a sequence that is at least 85% identical thereto, and / or the light chain variable domain of the sequence of SEQ ID NO: 62, or a sequence that is at least 85% identical thereto; or m) The heavy chain variable domain of the sequence of SEQ ID NO: 63 or a sequence that is at least 85% identical thereto, and / or the light chain variable domain of the sequence of SEQ ID NO: 66, or a sequence that is at least 85% identical thereto; or n) The heavy chain variable domain of the sequence of SEQ ID NO: 68 or a sequence that is at least 85% identical thereto, and / or the light chain variable domain of the sequence of SEQ ID NO: 70, or a sequence that is at least 85% identical thereto; or o) The heavy chain variable domain of the sequence of SEQ ID NO: 72 or a sequence that is at least 85% identical thereto, and / or the light chain variable domain of the sequence of SEQ ID NO: 73, or a sequence that is at least 85% identical thereto; or p) The heavy chain variable domain of the sequence of SEQ ID NO: 74 or a sequence that is at least 85% identical thereto, and / or the light chain variable domain of the sequence of SEQ ID NO: 78, or a sequence that is at least 85% identical thereto; or q) The heavy chain variable domain of the sequence of SEQ ID NO: 79 or a sequence that is at least 85% identical thereto, and / or the light chain variable domain of the sequence of SEQ ID NO: 82, or a sequence that is at least 85% identical thereto; or r) The heavy chain variable domain of the sequence of SEQ ID NO: 83 or a sequence that is at least 85% identical thereto, and / or the light chain variable domain of the sequence of SEQ ID NO: 85, or a sequence that is at least 85% identical thereto; or s) The heavy chain variable domain of the sequence of SEQ ID NO: 46 or a sequence that is at least 85% identical thereto, and / or the light chain variable domain of the sequence of SEQ ID NO: 87, or a sequence that is at least 85% identical thereto; or

[0136] t) The heavy chain variable domain of the sequence of SEQ ID NO: 89 or a sequence that is at least 85% identical thereto, and / or the light chain variable domain of the sequence of SEQ ID NO: 92, or a sequence that is at least 85% identical thereto; or u) The heavy chain variable domain of the sequence of SEQ ID NO: 95 or a sequence that is at least 85% identical thereto, and / or the light chain variable domain of the sequence of SEQ ID NO: 99, or a sequence that is at least 85% identical thereto; or v) The heavy chain variable domain of the sequence of SEQ ID NO: 102 or a sequence that is at least 85% identical thereto and / or the light chain variable domain of the sequence of SEQ ID NO: 106, or a sequence that is at least 85% identical thereto ; or w) The heavy chain variable domain of the sequence of SEQ ID NO: 107 or a sequence that is at least 85% identical thereto and / or the light chain variable domain of the sequence of SEQ ID NO: 109, or a sequence that is at least 85% identical thereto ; or x) The heavy chain variable domain of the sequence of SEQ ID NO: 110 or a sequence that is at least 85% identical thereto and / or the light chain variable domain of the sequence of SEQ ID NO: 112, or a sequence that is at least 85% identical thereto ; or y) The heavy chain variable domain of the sequence of SEQ ID NO: 115 or a sequence that is at least 85% identical thereto and / or the light chain variable domain of the sequence of SEQ ID NO: 119, or a sequence that is at least 85% identical thereto ; or z) The heavy chain variable domain of the sequence of SEQ ID NO: 121 or a sequence that is at least 85% identical thereto and / or a light chain of the sequence of SEQ ID NO: 125 or a sequence at least 85% identical thereto. Variable domain; or

[0137] aa) a heavy chain variable domain of sequence SEQ ID NO: 128 or a sequence at least 85% identical thereto and / or the light chain of the sequence of SEQ ID NO: 130 or a sequence at least 85% identical thereto. A variable domain; or bb) a heavy chain variable domain having the sequence SEQ ID NO: 131 or a sequence at least 85% identical thereto; and / or the light chain of the sequence of SEQ ID NO: 132 or a sequence at least 85% identical thereto. Variable domain The present invention relates to an anti-CD3 antibody, comprising:

[0138] For example, the sequence of the heavy or light chain variable domain may be, as appropriate, the sequence of 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 one or more amino acid substitutions, in particular by one or more conservative amino acid substitutions and / or substitutions at canonical residues. In particular, the sequence of the heavy or light chain variable domain 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 only by conservative amino acid substitutions.

[0139] Array changes compared to the sequences of SEQ ID NOs: 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 are present, in particular, 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.

[0140] However, amino acid substitutions in one or more CDRs are also possible. In particular, the sequence of the light chain variable domain differs from the sequence of SEQ ID NO: 9, by at least a substitution of S to R at position 28 (in CDR1-L) of SEQ ID NO: 9, and / or by at least a substitution of V to E at position 30 (in CDR1-L) of SEQ ID NO: 9, and / or by at least a substitution of N to D or T at position 33 (in CDR1-L) of SEQ ID NO: 9, and / or by at least a substitution of N to Y at position 35 (in CDR1-L) of SEQ ID NO: 9, and / or the sequence of the light chain variable domain differs from the sequence of SEQ ID NO: 9, by at least a substitution of T to S or A at position 97 (in CDR3-L) of SEQ ID NO: 9, and / or by at least a substitution of Q to H or E at position 98 (in CDR3-L) of SEQ ID NO: 9, and / or the heavy chain variable domain differs from the sequence of SEQ ID NO: 5, by at least a substitution of T to N or S at position 28 (in CDR1-H) of SEQ ID NO: 5, and / or by at least a substitution of F to V at position 29 (in CDR1-H) of SEQ ID NO: 5 and / or by at least a substitution of Q to H or E at position 98 (in CDR3-L) of SEQ ID NO: 9, and / or the heavy chain variable domain differs from the sequence of SEQ ID NO: 5, by at least a substitution of T to N or S at position 28 (in CDR1-H) of SEQ ID NO: 5, and / or by at least a substitution of F to V at position 29 (in CDR1-H) of SEQ ID NO: 5. and / or by at least a substitution of T to N or S at position 28 (in CDR1-H) of SEQ ID NO: 5, and / or by at least a substitution of F to V at position 29 (in CDR1-H) of SEQ ID NO: 5 、or by substitution of T to N or Y at position 30 (in CDR1-H) of SEQ ID NO: 5, or by substitution of K to L or Y at least at position 31 (in CDR1-H) of SEQ ID NO: 5, and / or the heavy chain variable domain may differ by, and / or the heavy chain variable domain is the sequence of SEQ ID NO: 5 and by substitution of D to A at least at position 53 (in CDR2-H) of SEQ ID NO: 5, or by substitution of K to R at least at position 54 (in CDR2-H) of SEQ ID NO: 5 by, or by substitution of S to A at least at position 55 (in CDR2-H) of SEQ ID NO: 5 by, or by substitution of S to N at least at position 57 (in CDR2-H) of SEQ ID NO: 5 by substitution, or by substitution of A to E at least at position 59 (in CDR2-H) of SEQ ID NO: 5 and may differ by, and / or the heavy chain variable domain is the sequence of SEQ ID NO: 5 and by substitution of G to A at least at position 100 (in CDR3-H) of SEQ ID NO: 5, and / or by substitution of T to V at least at position 101 (in CDR3-H) of SEQ ID NO: 5, and may differ by substitution of Q to Y at least at position 102 (in CDR3-H) of SEQ ID NO: 5.

[0141] In one embodiment, the anti-CD3 antibodies and fragments thereof of the present invention are, respectively, rat antibodies and fragments of rat antibodies.

[0142] In one aspect of the present invention, the anti-CD3 antibody of the present invention is a chimeric antibody, and in particular a rat / human antibody, for example an antibody comprising rat variable domains of the heavy and light chains and CH and CL domains derived from a human antibody.

[0143] In a further aspect of the present invention, the anti-CD3 antibody may also be, for example, a CDR-grafted or 4D a humanized antibody or a fragment of a humanized antibody obtained by the method (US20110027266), is also acceptable.

[0144] Therefore, in one embodiment, the anti-CD3 antibody of the present invention is a) a heavy chain variable domain comprising 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 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 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 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 and is a humanized antibody.

[0145] According to a further embodiment, the anti-CD3 antibody of the present invention is: a) a heavy chain variable domain of the sequence of SEQ ID NO: 138 and / or a light chain variable domain of the sequence of SEQ ID NO: 143; or b) a heavy chain variable domain of the sequence of SEQ ID NO: 171 and / or a light chain variable domain of the sequence of SEQ ID NO: 158; or c) A humanized antibody comprising the heavy chain variable domain of the sequence of SEQ ID NO: 176 and / or the light chain variable domain of the sequence of SEQ ID NO: 164 which is a humanized antibody.

[0146] In one embodiment, the anti-CD3 antibody according to the invention comprises one of the so-called anti-CD3 antibodies listed above, three CDR sequences or variable domains of the heavy chain, or six CDR sequences or variable domains of the heavy and light chains.

[0147] The invention further refers to fragments of the humanized anti-CD3 antibody defined above. In one embodiment, the above-mentioned humanized anti-CD3 antibody is a chimeric antibody.

[0148] The anti-CD3 antibody according to the invention may also be a single domain antibody or a fragment thereof which may in particular consist of a variable heavy chain (VHH) comprising one CDR1-H, CDR2-H and CDR3-H of the above antibodies. The anti-CD3 antibody may also be a heavy chain antibody, i.e. an antibody lacking a light chain, which may or may not contain a CH1 domain.

[0149] The single domain antibody or fragment thereof may also contain a framework region of a camelid single domain antibody and optionally a constant domain of a camelid single domain antibody

[0150] The anti-CD3 antibody according to the invention may also be an antibody fragment selected from the group consisting of Fv, Fab, F(ab’)2, Fab’, dsFv, (dsFv)2, scFv, sc(Fv)2, and bispecific antibodies, in particular a humanized anti- body fragment.

[0151] Therefore, the anti-CD3 antibody of the invention is ​​a) A heavy chain amino acid sequence SEQ ID NO: 186 and / or a light chain amino acid sequence SEQ ID NO: 187; or b) A heavy chain amino acid sequence SEQ ID NO: 188 and / or a light chain amino acid sequence SEQ ID NO: 189; or c) A heavy chain amino acid sequence SEQ ID NO: 190 and / or a light chain amino acid sequence SEQ ID NO: 191; or d) A heavy chain amino acid sequence SEQ ID NO: 192 and / or a light chain amino acid sequence SEQ ID NO: 193 and is a Fab comprising or consisting of these.

[0152] In one embodiment, the CD3 antibody is a bispecific or multispecific antibody formed from at least one fragment or at least one variable domain of the anti-CD3 antibodies of the present invention. The multispecific antibody is a multi valent protein complex as described, for example, in EP 2 050 764 A1 or US 2005 / 0003403 A1.

[0153] The bispecific or multispecific CD3 antibody according to the present invention can be specific for (a) the extracellular domains of human and cynomolgus CD3 targeted by one of the above anti-CD3 antibodies, and (b) at least one other antigen.

[0154] In one embodiment, the other antigen is CD123, and thus the resulting bispecific antibody is a CD3 / CD123 bispecific antibody. Conventional bispecific antibodies can be produced by techniques known to those skilled in the art.

[0155] The antibodies and fragments of the anti-CD3 antibodies according to the present invention can be isolated (e.g., purified) from or contained in vectors such as membranes or lipid vesicles (e.g., liposomes) and used therewith.

[0156] In a further embodiment, the anti-CD3 antibody of the present invention is an "antibody-like binding protein" For use in the manufacture of the antibody-like binding proteins of the invention as further defined in the section above. Any combination of the above embodiments forms part of the invention.

[0157] Anti-CD123 antibody “ CD123 ” (surface antigen classification 123) is also known as “ interleukin-3 receptor, alpha (IL3RA) ” or “ IL3R ”, “ IL3RX ”, “ IL3RY ”, “ IL3RAY ”, “ hIL-3Ra ” and represents the interleukin-3 specific subunit of the hematopoietin dimer cytokine receptor. The functional interleukin-3 receptor is a heterodimer comprising a specific alpha chain (IL-3A; CD123), and the IL-3 receptor beta chain (β ; CD131) common to the receptors for granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin-5 (IL-5). CD123 is a type I integral membrane protein with an estimated molecular weight of about 43 kDa 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 whose sequence shows similarity to the equivalent regions of the GM-CSF and IL-5 receptor alpha chains; and a region near the transmembrane domain containing four conserved cysteine residues and the WSXWS motif common to this cytokine receptor family. 0 The IL-3 binding domain contains a cytokine receptor motif (CRM) of about 200 amino acid residues composed of two Ig-like folded domains. The extracellular domain of CD123 is highly glycosylated including N-glycosylation necessary for both ligand binding and receptor signaling. The protein family consists of three members: IL3RA (CD123A), CSF2RA and IL5RA. The overall structure is well conserved among the three members, but the sequence homology is variable. The property is very low. One 300 - amino - acid - long isoform of CD123 has been discovered so far, but only at the RNA level, which is accessible in the Getentry database under accession number ACM24116.1. It is accessible.

[0158] U.S. Patent No. 6,177,078 discloses the anti - IL - 3 receptor alpha chain (IL - 3Rα, CD123) monoclonal antibody 7G3 and the ability of 7G3 to bind to the N - terminal domain of IL - 3Rα, particularly amino acid residues 19 - 49. U.S. Patent No. 6,733,743 discloses a method of disabling hematopoietic cancer progenitor cells that express CD123 but only slightly express CD131 by contacting the cells with a composition of an antibody and a cytotoxic agent (selected from a chemotherapeutic agent, a toxin, or an alpha - emitting radioactive isotope) in an amount effective to selectively bind to CD123 thereby causing cell death. However, it remains unclear whether targeting CD123 can functionally impair AML - LSC. The reference sequence of the full - length human CD123 protein including the signal peptide is available from the NCBI database under accession number NP_002174.1 and Uniprot accession number P26951 and is disclosed herein as SEQ ID NO: 194 (as available on December 14, 2014). The reference sequence of the full - length cynomolgus monkey CD123 protein including the signal peptide is available from the GenBank database under accession number EHH61867.1 and Uniprot accession number G8F3K3 and is disclosed herein as SEQ ID NO: 195 (as available on December 14, 2014).

[0159] The mature human CD123 Strep - II - tagged Fc fusion protein cloned from genomic DNA by the inventors is available from the NCBI database under accession number NP_002174.1 and Uniprot accession number P26951 and is disclosed herein as SEQ ID NO: 194 (as available on December 14, 2014). The reference sequence of the full - length cynomolgus monkey CD123 protein including the signal peptide is available from the GenBank database under accession number EHH61867.1 and Uniprot accession number G8F3K3 and is disclosed herein as SEQ ID NO: 195 (as available on December 14, 2014).

[0160] The mature human CD123 Strep - II - tagged Fc fusion protein cloned from genomic DNA by the inventors The sequence of the fusion protein is disclosed in SEQ ID NO: 196. The above-mentioned mature human CD123 Fc fusion protein contains amino acids 19 to 305 of the full-length human CD123 protein, and thus contains the extracellular domain of human CD123.

[0161] The sequence of the mature cynomolgus CD123 Strep-II tagged Fc fusion protein cloned from cDNA by the inventors of the present invention is disclosed in SEQ ID NO: 197. The above-mentioned mature cynomolgus CD123 Fc fusion protein contains amino acids 19 to 305 of the full-length cynomolgus CD123 protein, and thus contains the extracellular domain of cynomolgus CD123.

[0162] The domain structures of human and cynomolgus CD123 are as follows (based on the human CD123 sequence accessible with accession NP_002174.1 (SEQ ID NO: 194) in the NCBI database, and based on the cynomolgus CD123 sequence accessible with accession number G8F3K3, SEQ ID NO: 195 in the Uniprot database):

Table 3

[0163] Therefore, the extracellular domain of human CD123 consists of the amino acids at positions 19 to 305 of SEQ ID NO: 194.

[0164] CD123 (interleukin-3 receptor alpha chain IL-3Rα) is a tumor antigen overexpressed in various hematological neoplasms. Most of the 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 a worse prognosis. CD123 expression is associated with myelodysplastic syndrome, systemic obesity ​​It has been reported in other hematological malignancies including erythrocytosis, blastic plasmacytoid dendritic cell neoplasm (BPDCN), ALL, and hairy cell leukemia.

[0165] CD123 is expressed in AML leukemia stem cells, and there is increasing evidence suggesting that AML arises from these LSCs, which are quiescent and relatively resistant to DNA-damaging chemotherapy. It has been shown to be so. The persistence of LSCs is hypothesized to support relapse after initial remission, and thus eradication of LSCs is a requirement for cure and can be considered an important therapeutic goal. It can be considered an important therapeutic goal.

[0166] “ leukemia stem cell (LSC) ” are cancer cells with characteristics associated with normal stem cells, namely the property of self-renewal and the ability to generate multiple differentiation lineages. Such cells are proposed to persist as a distinct population in hematological cancers such as AML. The LCS present in AML patients are the so-called “AML-LCS”.

[0167] “ acute myeloid leukemia (AML) ” is a clonal abnormality clinically presented as increased proliferation of heterogeneous and undifferentiated myeloblasts. The leukemia hierarchy is maintained by a small population of LSCs (AML-LCS), which have a unique ability of self-renewal and can differentiate into leukemic precursors. These precursor cells generate a large number of leukemic blasts that are easily detectable in patients at diagnosis and relapse and ultimately lead to death. AML-LSCs are generally reported as quiescent cells, in contrast to rapidly dividing clonogenic precursor cells. This property of AML-LSCs weakens the effectiveness of conventional chemotherapy targeting proliferating cells, and although a high proportion of AML patients enter complete remission, almost all of them relapse, and less than 30% of adults survive longer than 4 years. This property of AML-LSCs weakens the effectiveness of conventional chemotherapy targeting proliferating cells, and a high proportion of AML patients enter complete remission, but almost all of them relapse, and less than 30% of adults survive longer than 4 years. It may explain the current experience. Furthermore, minimal residual disease occurrence and poor survival were due to the high LSC frequency at diagnosis in AML patients. As a result, the development of new treatments to specifically eliminate LSCs is essential for the long-term management of AML (and similarly other blood cancer states described above).

[0168] Overexpression of CD123 has been reported in AML blasts and CD34 + / CD38 AML-LSCs compared to normal hematopoietic cells.

[0169] Therefore, CD123 provides an important therapeutic target for cancer treatment, especially in patients with a poor prognosis.

[0170] The inventors have generated, screened, and selected specific mouse and rat anti-CD123 antibodies that show high affinity for both human and cynomolgus CD123 proteins, which do not significantly cross-react with human CSF2RA and IL5RA proteins, as well as cynomolgus CD3 protein.

[0171] The inventors have determined the sequences of the variable heavy and light chains of such monoclonal antibodies, 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.

[0172] The so-called "3E3-D3" anti-CD123 antibody has: - CDR1-H of the sequence of SEQ ID NO: 227, CDR2-H of the sequence of SEQ ID NO: 228, and CDR3-H of the sequence of SEQ ID NO: 229, and the sequence

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0173] The so-called "1E1-G5" anti-CD123 antibody is: - CDR1-H of the sequence of sequence number 199, CDR2-H of the sequence of sequence number 200, and CDR3-H of the sequence of sequence number 201 Containing

Chemical formula

Chemical formula

[0174] The so-called "2B8-F3" anti-CD123 antibody is: - The heavy chain variable domain consisting of CDR1-H of the sequence of Accession No. 206, CDR2-H of the sequence of Accession No. 207, and CDR3-H of the sequence of Accession No. 208, and sequence

Chemical formula

Chemical formula

[0175] The so-called "2F8-D6" anti-CD123 antibody is: - The heavy chain variable domain consisting of CDR1-H of the sequence of Accession No. 213, CDR2-H of the sequence of Accession No. 214, and CDR3-H of the sequence of Accession No. 215, and sequence

Chemical formula

Chemical formula

[0176] The so-called "3B10-E6" anti-CD123 antibody: - A heavy chain variable domain consisting of CDR1-H of the sequence of SEQ ID NO: 220, CDR2-H of the sequence of SEQ ID NO: 221, and CDR3-H of the sequence of SEQ ID NO: 222, and sequence

Chem.

Chem.

[0177] The so-called "5A5-B4" anti-CD123 antibody: - A heavy chain variable domain consisting of CDR1-H of the sequence of SEQ ID NO: 234, CDR2-H of the sequence of SEQ ID NO: 235, and CDR3-H of the sequence of SEQ ID NO: 236, and sequence

Chem.

Chem.

[0178] The so-called "6B10-E4" anti-CD123 antibody: CDR1-H of sequence SEQ ID NO: 241, CDR2-H of sequence SEQ ID NO: 242 and CDR2-H of sequence SEQ ID NO: 243 Column CDR3-H, including sequence [ka] (SEQ ID NO: 240, CDRs shown in bold), and - a CDR1-L of the sequence SEQ ID NO: 245, a CDR2-L consisting of the sequence "YAS" and a CDR2-L of the sequence SEQ ID NO: 246 The sequence [ka] (SEQ ID NO:244, CDRs are shown in bold) Includes.

[0179] The so-called "6C10-C4" anti-CD123 antibody: - CDR1-H of sequence SEQ ID NO: 248, CDR2-H of sequence SEQ ID NO: 249 and CDR2-H of sequence SEQ ID NO: 250 Column CDR3-H, including sequence [ka] (SEQ ID NO: 247, CDRs are shown in bold), and - CDR1-L of sequence SEQ ID NO: 252, CDR2-L consisting of the sequence "WAS" and sequence SEQ ID NO: 253 The sequence [ka] A light chain variable domain consisting of (SEQ ID NO:251, CDRs shown in bold) Includes.

[0180] The so-called "6D6-B8" anti-CD123 antibody: - The CDR1-H of the sequence of SEQ ID NO: 255, the CDR2-H of the sequence of SEQ ID NO: 200, and the CDR3-H of the sequence of SEQ ID NO: 201, the sequence including a heavy chain variable domain consisting of

Chemical formula

Chemical formula

[0181] The so-called "8B11-B7" anti-CD123 antibody is: - The CDR1-H of the sequence of SEQ ID NO: 199, the CDR2-H of the sequence of SEQ ID NO: 260, and the CDR3-H of the sequence of SEQ ID NO: 201, the sequence including a heavy chain variable domain consisting of

Chemical formula

Chemical formula

[0182] The so-called "9B8-G6" anti-CD123 antibody is: - The CDR1-H of the sequence of SEQ ID NO: 265, the CDR2-H of the sequence of SEQ ID NO: 266, and the CDR3-H of the sequence of SEQ ID NO: 267, the sequence including a heavy chain variable domain consisting of [Chemical formula] A heavy chain variable domain consisting of (Accession No. 264, CDRs are shown in bold), and - CDR1-L of the sequence of Accession No. 252, CDR2-L consisting of the sequence "WAS", and the sequence of Accession No. 253 including CDR3-L of [Chemical formula] A light chain variable domain consisting of (Accession No. 251, CDRs are shown in bold) is included.

[0183] The so-called "9D7-C8" anti-CD123 antibody is: - CDR1-H of the sequence of Accession No. 265, CDR2-H of the sequence of Accession No. 266, and CDR3-H of the sequence of No. 269, including the sequence [Chemical formula] A heavy chain variable domain sequence consisting of (Accession No. 268, CDRs are shown in bold), and - CDR1-L of the sequence of Accession No. 271, CDR2-L consisting of the sequence "WAS", and the sequence of Accession No. 253 including CDR3-L of [Chemical formula] A light chain variable domain consisting of (Accession No. 270, CDRs are shown in bold) is included.

[0184] The so-called "9F6-G3" anti-CD123 antibody is: - CDR1-H of the sequence of Accession No. 273, CDR2-H of the sequence of Accession No. 274, and CDR3-H of the sequence of Accession No. 201, including the sequence [Chemical formula] ​A heavy chain variable domain consisting of (Accession No. 272, with CDRs shown in bold), and - CDR1-L of the sequence of Accession No. 276, CDR2-L consisting of the sequence "DAS", and the sequence of Accession No. 258 including CDR3-L of the sequence

Chemical formula

[0185] In one aspect of the present invention, the anti-CD123 antibody binds to human CD123. In another embodiment, the anti-CD123 antibody further binds to cynomolgus CD123. In particular, the anti-CD123 antibody of the present invention binds to the extracellular domain of human CD123 or both human and cynomolgus CD123. More specifically, the anti-CD123 antibody binds to the distal portion of CD123, for example, to the amino acids at positions 19-49 of human CD123 of the sequence Accession No. 194 . The anti-CD123 antibody binds to CD123, whether expressed in isolated form or present in a soluble extracellular domain or full-length membrane-anchored CD123 as present on cells expressing CD123 such as AML cells or CD123-transfected cells. The anti-CD123 antibody according to the present invention is specific for cells expressing human and cynomolgus CD123 proteins on their surfaces, such as cancer cells expressing CD123. The anti-CD123 antibody according to the present invention has a ratio of affinity for cynomolgus CD123 to affinity for human CD123 (KD (

[0186] cynomolgus) / KD (human)) that is ≤ 10, particularly ≤ 6, ≤ 5, ≤ 4, ≤ 3, ≤ 2, ≤ 1 or ≤ 0.5. Accordingly, the polypeptide according to the present invention has, in monkeys ​It can be used in toxicological studies conducted, and the toxicity profile observed in monkeys is relevant to predicting possible adverse effects in humans.

[0187] In particular, the anti-CD123 antibody of the present invention does not bind to or significantly cross-react with CSF2RA and IL5RA proteins.

[0188] In particular, the antibody does not bind to or significantly cross-react with the extracellular domains of the aforementioned human and cynomolgus monkey CSF2RA and IL5RA proteins. It does not bind to or significantly cross-react with these.

[0189] Furthermore, the CD123 antibody according to the present invention has an affinity (KD) of ≤50 nM, ≤40 nM, ≤30 nM, for example ≤20 nM, ≤15 nM, ≤10 nM, ≤8 nM, ≤6 nM, ≤4 nM, ≤2 nM, ≤1 nM or ≤0.5 nM for human CD123 or cynomolgus monkey CD123, or both, for example an affinity (KD) of 0.1 nM to 20 nM, for example 0.1 nM to 10 nM, particularly 0.1 nM to 2 nM, or 0.1 nM to 1 nM. In one example, the affinity for human CD3 or cynomolgus monkey CD3 is determined as the KD value using surface plasmon resonance with recombinant CD123 proteins derived from humans and cynomolgus monkeys as the captured antigen.

[0190] For example, using human and cynomolgus monkey CD123-Fc fusion proteins. It is determined as the KD value.

[0191] As an antigen, for example, a human or cynomolgus CD123 extracellular domain containing a 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 (cynomolgus monkey)) can be used. The amino acid sequences obtained for the human or cynomolgus CD123 mature protein are listed as SEQ ID NO: 196 and SEQ ID NO: 197, respectively. Biacore measurements are known to those skilled in the art. In the current example, the Biacore measurements can be performed as described in the section on "anti-CD3 antibody" above.

[0192] The anti-CD123 antibody of the present invention can also have an apparent affinity constant (apparent KD) of ≤20 nM, for example, ≤15 nM, ≤10 nM, ≤6 nM, ≤5 nM, ≤4 nM, ≤3 nM, ≤2 nM, or ≤1 nM, as determined by FACS using an unpurified anti-CD123 antibody in the supernatant of a hybridoma in cells expressing CD123, such as CD123-transfected HEK293 cells. Typically, the apparent KD is in the range of 0.1 - 20 nM, particularly 0.1 - 10 nM, for example, 0.1 - 5 nM.

[0193] 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", Alignment of the sequences of the VH and VL regions of the "9F6-G3" anti-CD123 antibody. Ratio of CDR-H and CDR-L sequences The comparison shows that structurally, on the one hand, the anti-CD123 antibodies "1E1-G5", "6D6-B8", "8B11-B7" and "9F6-G3", and on the other hand, the anti-CD123 antibodies "6C10-C4", "9B8-G6", "9D7-C8" are closely related, and the above antibodies tend to indicate that they probably bind to the same epitope. The alignments 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 is strictly conserved between the two groups of antibodies, and thus further identifies CDR positions that are considered important with respect to specificity, while other positions could support substitutions and were such that could support substitutions were such that could support substitutions

[0194] Accordingly, the antibodies according to the invention are: a) CDR1-H consisting of sequence X 1 YTFTDX 2 I (SEQ ID NO: 336) [where X 1 is G or A, and X 2 is H, Y or N, or any combination thereof]; and sequence INPYSX 1 GX 2 (SEQ ID NO: 337) [where X 1 is G or D, and X 2 is T or A, or any combination thereof]; and a heavy chain variable domain comprising CDR3-H consisting of the sequence ALNYGSYYAMDA (SEQ ID NO: 201), and sequence X 1 DIX 2 X 3 N (SEQ ID NO: 338) [where X 1 is E or K, X 2 is F, H or Y, and X 3 CDR1-L consisting of N or S, or any combination thereof; and CDR2-L consisting of the sequence "DAN" or "DAS"; and sequence X 1 QYNX 2 YPYT (SEQ ID NO: 339) [where X 1 is H or Q, and X 2 is I, K or is N, or any combination thereof] comprising a light chain variable domain of CDR3-L ; or b) the sequence GFSLTSYX 1 (SEQ ID NO: 340) [where X 1 is H or S] comprising CDR1-H ; and sequence MWX 1 DGDT (SEQ ID NO: 341) [where X 1 is S or N] comprising CDR2-H; and 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) [where 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 an amino acid is not, X 6 is L, I or not an amino acid, X 7 is Y or not an amino acid 、X 8 is L or not an amino acid, X 9 is W or not an amino acid, X 10 is A or D, or any combination thereof] comprising a heavy chain variable do Main, as well as the array QSFLSSGDX 1 X 2 NY (SEQ ID NO: 343) [where X 1 is E or G, and X 2 is R or is K, or a combination of any of them] consisting of CDR1-L; and CDR2-L consisting of the sequence "WAS"; and the sequence QQYYDTPLT (SEQ ID NO: 253) consisting of CDR3-L including a light chain variable domain including.

[0195] According to one embodiment, the anti-CD123 antibody according to the present invention is one of the 13 so-called "3E3-D3", "1E1-G5", "2B8-F3", "2F8-D6", "3B10-E6", "5A5-B4", "6B10-E4" listed above , "6C10-C4", "6D6-B8", "8B11-B7", "9B8-G6", "9D7-C8", and "9F6-G3" including the CDR sequences of one of the heavy and / or light chains of the anti-CD123 antibody.

[0196] Therefore, the present invention provides: a) CDR1-H of the sequence of SEQ ID NO: 227 or a sequence different from the sequence of SEQ ID NO: 227 by one amino acid substitution; CDR2-H of the sequence of SEQ ID NO: 228 or SEQ ID NO: 353 or SEQ ID NO: 279 or a sequence different from the sequence of SEQ ID NO: 228 or SEQ ID NO: 353 or SEQ ID NO: 279 by one or more amino acid substitutions; a heavy chain variable domain including CDR3-H of the sequence of SEQ ID NO: 229 or a sequence different from the sequence of SEQ ID NO: 229 by one amino acid substitution; and CDR1-L of the sequence of SEQ ID NO: 231 or a sequence different from the sequence of SEQ ID NO: 231 by one amino acid substitution; CDR2-L of the sequence "RDD" or a sequence different from the sequence "RDD" by one amino acid substitution and CDR3-L of the sequence of SEQ ID NO: 232 or a sequence different from the sequence of SEQ ID NO: 232 by one amino acid substitution including a light chain variable domain; or b) The CDR1-H of the sequence of SEQ ID NO: 199 or a sequence differing from SEQ ID NO: 199 by one amino acid substitution; the CDR2-H of the sequence of SEQ ID NO: 200 or a sequence differing from SEQ ID NO: 200 by one or more amino acid substitutions; the heavy chain variable domain comprising the CDR3-H of the sequence of SEQ ID NO: 201 or a sequence differing from SEQ ID NO: 201 by one amino acid substitution; and the light chain variable domain comprising the CDR1-L of the sequence of SEQ ID NO: 203 or a sequence differing from SEQ ID NO: 203 by one amino acid substitution; the CDR2-L of the sequence "DAN" or a sequence differing from "DAN" by one amino acid substitution; and the CDR3-L of the sequence of SEQ ID NO: 204 or a sequence differing from SEQ ID NO: 204 by one amino acid substitution; or c) The CDR1-H of the sequence of SEQ ID NO: 206 or a sequence differing from SEQ ID NO: 206 by one amino acid substitution; the CDR2-H of the sequence of SEQ ID NO: 207 or a sequence differing from SEQ ID NO: 207 by one or more amino acid substitutions; the heavy chain variable domain comprising the CDR3-H of the sequence of SEQ ID NO: 208 or a sequence differing from SEQ ID NO: 208 by one amino acid substitution; and the CDR1-L of the sequence of SEQ ID NO: 210 or a sequence differing from SEQ ID NO: 210 by one amino acid substitution ; the CDR2-L of the sequence "ETS" or a sequence differing from "ETS" by one amino acid substitution; and the light chain variable domain comprising the CDR3-L of the sequence of SEQ ID NO: 211 or a sequence differing from SEQ ID NO: 211 by one amino acid substitution; or d) The CDR1-H of the sequence of SEQ ID NO: 213 or a sequence differing from SEQ ID NO: 213 by one amino acid substitution; the CDR2-H of the sequence of SEQ ID NO: 214 or a sequence differing from SEQ ID NO: 214 by one or more amino acid substitutions; the heavy chain variable domain comprising the CDR3-H of the sequence of SEQ ID NO: 215 or a sequence differing from SEQ ID NO: 215 by one amino acid substitution; and the CDR1-L of the sequence of SEQ ID NO: 217 or a sequence differing from SEQ ID NO: 217 by one amino acid substitution; the CDR2-L of the sequence "NTN" or a sequence differing from "NTN" by one amino acid substitution; and the CDR3-L of the sequence of SEQ ID NO: 218 ​A light chain variable containing CDR3-L of the sequence or array number 218 and a sequence different by one amino acid substitution domain; or e) A heavy chain variable domain containing CDR1-H of the sequence of SEQ ID NO: 220 or a sequence different by one amino acid substitution from SEQ ID NO: 220; CDR2-H of the sequence of SEQ ID NO: 221 or a sequence different by one or more amino acid substitutions from SEQ ID NO: 221; CDR3-H of the sequence of SEQ ID NO: 222 or a sequence different by one amino acid substitution from SEQ ID NO: 222; and A light chain variable domain containing CDR1-L of the sequence of SEQ ID NO: 224 or a sequence different by one amino acid substitution from SEQ ID NO: 224; CDR2-L of the sequence "RVS" or a sequence different by one amino acid substitution from "RVS"; and CDR3-L of the sequence of SEQ ID NO: 225 or a sequence different by one amino acid substitution from SEQ ID NO: 225; or

[0197] f) A heavy chain variable domain containing CDR1-H of the sequence of SEQ ID NO: 234 or a sequence different by one amino acid substitution from SEQ ID NO: 234; CDR2-H of the sequence of SEQ ID NO: 235 or a sequence different by one or more amino acid substitutions from SEQ ID NO: 235; CDR3-H of the sequence of SEQ ID NO: 236 or a sequence different by one amino acid substitution from SEQ ID NO: 236; and CDR1-L of the sequence of SEQ ID NO: 238 or a sequence different by one amino acid substitution from SEQ ID NO: 238; CDR2-L of the sequence "GAS" or a sequence different by one amino acid substitution from "GAS"; and A light chain variable containing the sequence of SEQ ID NO: 239 or a sequence different by one amino acid substitution from SEQ ID NO: 239 domain; or g) A heavy chain variable domain containing CDR1-H of the sequence of SEQ ID NO: 241 or a sequence different by one amino acid substitution from SEQ ID NO: 241; CDR2-H of the sequence of SEQ ID NO: 242 or a sequence different by one or more amino acid substitutions from SEQ ID NO: 242; CDR3-H of the sequence of SEQ ID NO: 243 or a sequence different by one amino acid substitution from SEQ ID NO: 243; and The light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 245 or a sequence differing from the sequence of SEQ ID NO: 245 by one amino acid substitution; CDR2-L of a sequence differing from the sequence "YAS" or "YAS" by one amino acid substitution; and CDR3-L of a sequence differing from the sequence of SEQ ID NO: 246 or a sequence differing from the sequence of SEQ ID NO: 246 by one amino acid substitution; or a light chain variable domain comprising CDR3-L of a sequence differing from the sequence of SEQ ID NO: 246 or a sequence differing from the sequence of SEQ ID NO: 246 by one amino acid substitution; or or h) A heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 248 or a sequence differing from the sequence of SEQ ID NO: 248 by one amino acid substitution; CDR2-H of the sequence of SEQ ID NO: 249 or a sequence differing from the sequence of SEQ ID NO: 249 by one or more amino acid substitutions; and CDR3-H of the sequence of SEQ ID NO: 250 or a sequence differing from the sequence of SEQ ID NO: 250 by one amino acid substitution; and a light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 252 or a sequence differing from the sequence of SEQ ID NO: 252 by one amino acid substitution; CDR2-L of a sequence differing from the sequence "WAS" or "WAS" by one amino acid substitution; and CDR3-L of a sequence differing from the sequence of SEQ ID NO: 253 or a sequence differing from the sequence of SEQ ID NO: 253 by one amino acid substitution; or i) A heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 255 or a sequence differing from the sequence of SEQ ID NO: 255 by one amino acid substitution; CDR2-H of the sequence of SEQ ID NO: 200 or a sequence differing from the sequence of SEQ ID NO: 200 by one or more amino acid substitutions; and CDR3-H of the sequence of SEQ ID NO: 201 or a sequence differing from the sequence of SEQ ID NO: 201 by one amino acid substitution; and a light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 257 or a sequence differing from the sequence of SEQ ID NO: 257 by one amino acid substitution; CDR2-L of a sequence differing from the sequence "DAN" or "DAN" by one amino acid substitution; and CDR3-L of a sequence differing from the sequence of SEQ ID NO: 258 or a sequence differing from the sequence of SEQ ID NO: 258 by one amino acid substitution; or

[0198] j) A heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 199 or a sequence differing from the sequence of SEQ ID NO: 199 by one amino acid substitution; CDR2-H of the sequence of SEQ ID NO: 260 or a sequence differing from the sequence of SEQ ID NO: 260 by one or more amino acid substitutions; CDR3-H of the sequence of SEQ ID NO: 201 or a sequence differing from the sequence of SEQ ID NO: 201 by one amino acid substitution; and A light chain comprising CDR1-L of the sequence of SEQ ID NO: 262 or a sequence differing from the sequence of SEQ ID NO: 262 by one amino acid substitution; CDR2-L of the sequence "DAS" or a sequence differing from the sequence "DAS" by one amino acid substitution; and CDR3-L of the sequence of SEQ ID NO: 263 or a sequence differing from the sequence of SEQ ID NO: 263 by one amino acid substitution variable domain; or k) A heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 265 or a sequence differing from the sequence of SEQ ID NO: 265 by one amino acid substitution; CDR2-H of the sequence of SEQ ID NO: 266 or a sequence differing from the sequence of SEQ ID NO: 266 by one or more amino acid substitutions; CDR3-H of the sequence of SEQ ID NO: 267 or a sequence differing from the sequence of SEQ ID NO: 267 by one amino acid substitution; and A light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 252 or a sequence differing from the sequence of SEQ ID NO: 252 by one amino acid substitution; CDR2-L of the sequence "WAS" or a sequence differing from the sequence "WAS" by one amino acid substitution; and CDR3-L of the sequence of SEQ ID NO: 253 or a sequence differing from the sequence of SEQ ID NO: 253 by one amino acid substitution; or l) A heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 265 or a sequence differing from the sequence of SEQ ID NO: 265 by one amino acid substitution; CDR2-H of the sequence of SEQ ID NO: 266 or a sequence differing from the sequence of SEQ ID NO: 266 by one or more amino acid substitutions; CDR3-H of the sequence of SEQ ID NO: 269 or a sequence differing from the sequence of SEQ ID NO: 269 by one amino acid substitution; and The light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 271 or a sequence differing from the sequence of SEQ ID NO: 271 by one amino acid substitution; CDR2-L of the sequence "WAS" or a sequence differing from "WAS" by one amino acid substitution; and CDR3-L of the sequence of SEQ ID NO: 253 or a sequence differing from the sequence of SEQ ID NO: 253 by one amino acid substitution; or m) The heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 273 or a sequence differing from the sequence of SEQ ID NO: 273 by one amino acid substitution; CDR2-H of the sequence of SEQ ID NO: 274 or a sequence differing from the sequence of SEQ ID NO: 274 by one or more amino acid substitutions; and CDR3-H of the sequence of SEQ ID NO: 201 or a sequence differing from the sequence of SEQ ID NO: 201 by one amino acid substitution; and The light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 276 or a sequence differing from the sequence of SEQ ID NO: 276 by one amino acid substitution; CDR2-L of the sequence "DAS" or a sequence differing from "DAS" by one amino acid substitution; and CDR3-L of the sequence of SEQ ID NO: 258 or a sequence differing from the sequence of SEQ ID NO: 258 by one amino acid substitution relating to an anti-CD123 antibody comprising

[0199] One or more individual amino acids may be altered by substitution, particularly conservative substitution, in one or more of the above CDR sequences. Such alterations may be intended, for example, in connection with humanization of the antibody, to remove glycosylation sites or deamidation sites.

[0200] Based on the alignment of the sequences of the VH and VL regions of the "1E1-G5", "6D6-B8", "8B11-B7", "9F6-G3" anti-CD123 antibodies, various amino acid substitutions were identified. Thus, in one embodiment, the amino acids are: - at one or more of positions 1 and 7 in CDR1-H, for example at position 7 of CDR1-H of the sequence GYTFTDHI (SEQ ID NO: 199) or GYTFTDYI (SEQ ID NO: 273), and / or at positions 1 and 5 of CDR1-H of the sequence AYTFTDNI (SEQ ID NO: 255); and / or - In CDR2-H, at one or more of positions 6 and 8, for example, at positions 6 and / or 8 of CDR2-H of the sequences 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 at positions 1, 4, and 5 of CDR1-L of the sequences KDIYSN (SEQ ID NO: 262) or EDIFNN (SEQ ID NO: 257); and / or - In CDR2-L, at position 3 of the sequences "DAN" or "DAS"; and / or - In CDR3-L, at one or more of positions 1 and 5, for example, at positions 1 and / or 5 of CDR3-L of the sequences HQYNIYPYT (SEQ ID NO: 258) or QQYNKYPYT (SEQ ID NO: 204) or HQYNNYPYT (SEQ ID NO: 263) may be substituted.

[0201] In a further embodiment, the amino acids are: - At position 8 in CDR1-H, for example, at position 8 of CDR1-H of the sequences GFSLTSYH (SEQ ID NO: 265) or GFSLTSYS (SEQ ID NO: 248); and / or - At position 3 in CDR2-H, for example, at position 3 of CDR2-H of the sequences MWSDGD (SEQ ID NO: 265) or MWNDGD (SEQ ID NO: 248); and / or - At positions 4 and 9 in CDR3-H, for example, at positions 4 and 9 of CDR3-H of the sequences 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 positions 9 and 10 of CDR1-L of the sequences QSFLSSGDERNY (SEQ ID NO: 252) or QSFLSSGDGKNY (SEQ ID NO: 271) are substituted.

[0202] According to one embodiment, the antibody is a) a heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 227, CDR2-H of the sequences of SEQ ID NO: 228, SEQ ID NO: 353, SEQ ID NO: 279, and CDR3-H of the sequence of SEQ ID NO: 229, and a light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 231, CDR2-L of the sequence "RDD", and CDR3-L of the sequence of SEQ ID NO: 232; or n) a heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 199, CDR2-H of the sequence of SEQ ID NO: 200, and CDR3-H of the sequence of SEQ ID NO: 201, and a light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 203, CDR2-L of the sequence "DAN", and CDR3-L of the sequence of SEQ ID NO: 204; or o) a heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 206, CDR2-H of the sequence of SEQ ID NO: 207, and CDR3-H of the sequence of SEQ ID NO: 208, and a light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 210, CDR2-L of the sequence "ETS", and CDR3-L of the sequence of SEQ ID NO: 211; or p) a heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 213, CDR2-H of the sequence of SEQ ID NO: 214, and CDR3-H of the sequence of SEQ ID NO: 215, and a light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 217, CDR2-L of the sequence "NTN", and CDR3-L of the sequence of SEQ ID NO: 218; or q) a heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 220, CDR2-H of the sequence of SEQ ID NO: 221, and CDR3-H of the sequence of SEQ ID NO: 222, and a light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 224, CDR2-L of the sequence "RVS", and CDR3-L of the sequence of SEQ ID NO: 225; or ​​​​​​​​​r) A heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 234, CDR2-H of the sequence of SEQ ID NO: 235, and CDR3-H of the sequence of SEQ ID NO: 236, and a light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 238, CDR2-L of the sequence "GAS", and CDR3-L of the sequence of SEQ ID NO: 239; or s) A heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 241, CDR2-H of the sequence of SEQ ID NO: 242, and CDR3-H of the sequence of SEQ ID NO: 243, and a light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 245, CDR2-L of the sequence "YAS", and CDR3-L of the sequence of SEQ ID NO: 246; or t) A heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 248, CDR2-H of the sequence of SEQ ID NO: 249, and CDR3-H of the sequence of SEQ ID NO: 250, and a light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 252, CDR2-L of the sequence "WAS", and CDR3-L of the sequence of SEQ ID NO: 253; or u) A heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 255, CDR2-H of the sequence of SEQ ID NO: 200, and CDR3-H of the sequence of SEQ ID NO: 201, and a light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 257, CDR2-L of the sequence "DAN", and CDR3-L of the sequence of SEQ ID NO: 258; or v) A heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 199, CDR2-H of the sequence of SEQ ID NO: 260, and CDR3-H of the sequence of SEQ ID NO: 201, and a light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 262, CDR2-L of the sequence "DAS", and CDR3-L of the sequence of SEQ ID NO: 263; or w) A heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 265, CDR2-H of the sequence of SEQ ID NO: 266, and CDR3-H of the sequence of SEQ ID NO: 267, and a light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 268, CDR2-L of the sequence "DAS", and CDR3-L of the sequence of SEQ ID NO: 269; or u) A heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 255, CDR2-H of the sequence of SEQ ID NO: 200, and CDR3-H of the sequence of SEQ ID NO: 201, and a light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 257, CDR2-L of the sequence "DAN", and CDR3-L of the sequence of SEQ ID NO: 258; or v) A heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 199, CDR2-H of the sequence of SEQ ID NO: 260, and CDR3-H of the sequence of SEQ ID NO: 201, and a light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 262, CDR2-L of the sequence "DAS", and CDR3-L of the sequence of SEQ ID NO: 263; or v) A heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 199, CDR2-H of the sequence of SEQ ID NO: 260, and CDR3-H of the sequence of SEQ ID NO: 201, and a light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 262, CDR2-L of the sequence "DAS", and CDR3-L of the sequence of SEQ ID NO: 263; or w) A heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 265, CDR2-H of the sequence of SEQ ID NO: 266, and CDR3-H of the sequence of SEQ ID NO: 267, and a light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 268, CDR2-L of the sequence "DAS", and CDR3-L of the sequence of SEQ ID NO: 269; or w) A heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 265, CDR2-H of the sequence of SEQ ID NO: 266, and CDR3-H of the sequence of SEQ ID NO: 267, and A heavy chain variable domain comprising the CDR3-H thereof, and a light chain variable domain comprising the CDR1-L of the sequence of SEQ ID NO: 252, the CDR2-L of the sequence "WAS", and the CDR3-L of the sequence of SEQ ID NO: 253; or x) A heavy chain variable domain comprising the CDR1-H of the sequence of SEQ ID NO: 265, the CDR2-H of the sequence of SEQ ID NO: 266, and the CDR3-H of the sequence of SEQ ID NO: 269, and a light chain variable domain comprising the CDR1-L of the sequence of SEQ ID NO: 271, the CDR2-L of the sequence "WAS", and the CDR3-L of the sequence of SEQ ID NO: 253; or comprising a light chain variable domain; or y) A heavy chain variable domain comprising the CDR1-H of the sequence of SEQ ID NO: 273, the CDR2-H of the sequence of SEQ ID NO: 274, and the CDR3-H of the sequence of SEQ ID NO: 201, and a light chain variable domain comprising the CDR1-L of the sequence of SEQ ID NO: 276, the CDR2-L of the sequence "DAS", and the CDR3-L of the sequence of SEQ ID NO: 258; or comprising a light chain variable domain is included.

[0203] The anti-CD123 antibody according to the present invention is particularly a conventional antibody, particularly a conventional monoclonal antibody, or an antibody fragment, a bispecific or multispecific antibody.

[0204] The anti-CD123 antibody according to the present invention particularly comprises or consists of IgG or a fragment thereof.

[0205] The present 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. is provided.

[0206] Therefore, the present invention particularly relates to: a) The sequence of SEQ ID NO: 226 or SEQ ID NO: 277 or SEQ ID NO: 278 or at least one sequence thereof and A heavy chain variable domain with at least 85% identical sequence, and / or a light chain variable domain with a sequence of SEQ ID NO: 230 or a sequence at least 85% identical thereto; or b) A heavy chain variable domain with a sequence of SEQ ID NO: 198 or a sequence at least 85% identical thereto and / or a light chain variable domain with a sequence of SEQ ID NO: 202 or a sequence at least 85% identical thereto; or c) A heavy chain variable domain with a sequence of SEQ ID NO: 205 or a sequence at least 85% identical thereto and / or a light chain variable domain with a sequence of SEQ ID NO: 209 or a sequence at least 85% identical thereto; or d) A heavy chain variable domain with a sequence of SEQ ID NO: 212 or a sequence at least 85% identical thereto and / or a light chain variable domain with a sequence of SEQ ID NO: 216 or a sequence at least 85% identical thereto; or e) A heavy chain variable domain with a sequence of SEQ ID NO: 219 or a sequence at least 85% identical thereto and / or a light chain variable domain with a sequence of SEQ ID NO: 223 or a sequence at least 85% identical thereto; or f) A heavy chain variable domain with a sequence of SEQ ID NO: 233 or a sequence at least 85% identical thereto and / or a light chain variable domain with a sequence of SEQ ID NO: 237 or a sequence at least 85% identical thereto; or g) A heavy chain variable domain with a sequence of SEQ ID NO: 240 or a sequence at least 85% identical thereto and / or a light chain variable domain with a sequence of SEQ ID NO: 244 or a sequence at least 85% identical thereto; or h) A heavy chain variable domain with a sequence of SEQ ID NO: 247 or a sequence at least 85% identical thereto and / or a light chain variable domain with a sequence of SEQ ID NO: 251 or a sequence at least 85% identical thereto; or i) A heavy chain variable domain with a sequence of SEQ ID NO: 254 or a sequence at least 85% identical thereto and / or a light chain variable domain with a sequence of SEQ ID NO: 258 or a sequence at least 85% identical thereto; or j) A heavy chain variable domain with a sequence of SEQ ID NO: 261 or a sequence at least 85% identical thereto and / or a light chain variable domain with a sequence of SEQ ID NO: 265 or a sequence at least 85% identical thereto; or k) A heavy chain variable domain with a sequence of SEQ ID NO: 268 or a sequence at least 85% identical thereto and / or a light chain variable domain with a sequence of SEQ ID NO: 272 or a sequence at least 85% identical thereto; or l) A heavy chain variable domain with a sequence of SEQ ID NO: 275 or a sequence at least 85% identical thereto i) a heavy chain variable domain having the sequence of SEQ ID NO: 254 or a sequence at least 85% identical thereto; and / or a light chain of the sequence of SEQ ID NO: 256 or a sequence at least 85% identical thereto. Variable domain; or j) a heavy chain variable domain having the sequence of SEQ ID NO: 259 or a sequence at least 85% identical thereto; and / or a light chain of the sequence of SEQ ID NO: 261 or a sequence at least 85% identical thereto. Variable domain; or k) a heavy chain variable domain having the sequence of SEQ ID NO: 264 or a sequence at least 85% identical thereto; and / or a light chain of the sequence of SEQ ID NO: 251 or a sequence at least 85% identical thereto. Variable domain; 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 having the sequence of SEQ ID NO: 272 or a sequence at least 85% identical thereto; and / or a light chain of the sequence of SEQ ID NO: 275 or a sequence at least 85% identical thereto. Strange Domain The present invention relates to an anti-CD123 antibody comprising the compound

[0207] For example, the sequence of the heavy or light chain variable domain may be modified by one or more amino acid substitutions, in particular one or more conservative amino acid substitutions and / or standard amino acid substitutions, 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. It may vary depending on substitution at the residue. In particular, the sequences of the variable domains of the heavy or light chains may differ from the reference sequences SEQ ID NOs: 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 only by conservative amino acid substitutions.

[0208] Sequence changes compared to the sequences of SEQ ID NOs: 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 are present, in particular, 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.

[0209] In one embodiment, the anti-CD123 antibodies and fragments thereof of the present invention are, respectively, rat antibodies and fragments of rat antibodies.

[0210] The anti-CD123 antibodies of the present invention may also be chimeric antibodies, and in particular rat / human antibodies, for example, antibodies comprising rat variable domains of the heavy and light chains and CH domains and CL domains derived from human antibodies. 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 the 4D method (US20110027266).

[0211] Thus, in one embodiment, the anti-CD123 antibody of the present invention is: a) SEQ ID NO: 280, SEQ ID NO: 281, SEQ ID NO: 282, SEQ ID NO: 283, SEQ ID NO: 284, SEQ ID NO ​A heavy chain variable domain comprising an amino acid sequence selected from the group consisting of 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 a heavy chain variable domain; and b) a light chain variable domain comprising 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 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. The humanized antibody comprises a light chain variable domain.

[0212] In one embodiment, the anti-CD123 antibody according to the present invention comprises three CDR sequences or variable domains of the heavy chain, or six CDR sequences or variable domains of the heavy and light chains, of one of the so-called anti-CD123 antibodies listed above.

[0213] The present invention further refers to fragments of the humanized anti-CD123 antibody defined above. In one embodiment, the humanized anti-CD123 antibody described above is a chimeric antibody.

[0214] The anti-CD123 antibody according to the present invention may also be a single domain antibody or a fragment thereof. In particular, the single domain antibody fragment may consist of a variable heavy chain (VHH) comprising CDR1-H, CDR2-H and CDR3-H of one of the antibodies defined above. The CD123 antibody may also be a heavy chain antibody, i.e., an antibody lacking a light chain, which may or may not contain a CH1 domain. The single domain antibody or fragment thereof may also contain a framework region of a camelid single domain antibody and optionally a constant domain of a camelid single domain antibody.

[0215] The single domain antibody or fragment thereof may also contain a framework region of a camelid single domain antibody and optionally a constant domain of a camelid single domain antibody.

[0216] The anti-CD123 antibodies according to the invention may also be antibody fragments selected from the group consisting of Fv, Fab, F(ab’)2, Fab’, dsFv, (dsFv)2, scFv, sc(Fv)2, and bispecific antibodies, in particular humanized anti- body fragments.

[0217] The anti-CD123 antibodies may also be bispecific or multispecific antibodies formed from at least one antibody fragment or at least one variable domain of the anti-CD123 antibodies of the invention. Multispecific antibodies are multivalent protein complexes such as those described, for example, in EP 2 050 764 A1 or US 2005 / 0003403 A1.

[0218] The bispecific or multispecific CD123 antibodies according to the invention may be specific for (a) the extracellular domain of human or human and cynomolgus CD123 targeted by one of the above anti-CD123 antibodies, and (b) at least one other antigen.

[0219] In certain embodiments, the other antigen is CD3, and thus the resulting bispecific antibody is a CD3 / CD123 bispecific antibody. Conventional bispecific antibodies can be produced by techniques known to those skilled in the art.

[0220] The antibodies and fragments thereof according to the invention can be isolated (e.g., purified) from vectors such as membranes or lipid vesicles (e.g., liposomes) or used in a state contained therein.

[0221] In one further embodiment, the anti-CD123 antibodies of the invention are used in the production of the antibody-like binding proteins of the invention as further defined in the section “antibody-like binding proteins”.

[0222] Any combination of the above embodiments forms part of the invention.

[0223] Antibody-like binding protein The inventors have generated several antibody-like binding proteins, so-called "7G3x20G6", "7G3x4E7 ", "7G3x4B4", "7G3x18F5", "hz20G6x7G3", "7G3xhz4B4", "hz4B4x3E3", "hz20G6x7G3-TL4" and "hz20G6xhz7G3" antibody-like binding proteins, where the term "hz " refers to a humanized antibody. These antibody-like binding proteins have a CODV design, particularly a CODV-Fab or CODV-Ig design.

[0224] In the context of the present invention, " CODV format " refers to the cross-over dual variable (CODV) configuration of a bispecific or multispecific antibody. The CODV format allows for the interchangeability of variable domains while retaining folding and final binding affinity. The CODV format has been previously described in international patent application WO2012 / 135345 . Thus, in one embodiment, the antibody-like binding protein of the present invention is in a CODV format as previously described in international patent application WO2012 / 135345 (which is incorporated herein by reference) .

[0225] In one embodiment, the present invention refers to an antibody-like binding protein in the CODV-Fab format. Thus, in one embodiment, the present invention refers to an antibody-like binding protein comprising two polypeptide chains that form two antigen-binding sites, where the first polypeptide has the structure represented by formula [I]: V D1 -L 1 -V D2 -L 2 -C L [I] and the second polypeptide has the structure represented by formula [II]: V D3 -L 3 -V D4 -L 4 -C H1 [II] and has the structure represented by In the formula: V D1 is the variable domain of the heavy or light chain of the first immunoglobulin; V D2 is the variable domain of the heavy or light chain of the second immunoglobulin; V D3 is the variable domain of the heavy or light chain of said second immunoglobulin; V D4 is the variable domain of the heavy or light chain of said first immunoglobulin; C L is the constant domain of the light chain of the immunoglobulin; C H is the C H1 constant domain of the heavy chain of the immunoglobulin; L 1 L 2 L 3 and L 4 are amino acid linkers; and here, the first and second polypeptides form an interchain light-heavy chain pair, and here, V D1 and V D2 are both either the variable domain of the light chain or the variable domain of the heavy chain, and when V D1 and V D2 are the variable domains of the light chain, V D3 and V D4 are both the variable domains of the heavy chain, or when V D1 and V D2 are the variable domains of the heavy chain, V D3 and V D4 are both the variable domains of the light chain.

[0226] Adding an Fc domain to the antibody-like binding protein in CODV-Fab further stabilizes the antibody-like binding protein. More precisely, adding an Fc domain to the polypeptide of formula (II) of the antibody-like binding protein in CODV-Fab increases the half-life of the antibody-like binding protein In addition, the pharmacokinetic profile of the antibody-like binding protein is thus improved. Adding one Fc region to the CODV-Fab results in dimerization of the polypeptide containing the Fc domain, and the resulting antibody-like binding protein is an antibody-like binding protein in the form of CODV-Ig. Thus, the present invention further refers to an antibody-like binding protein in the form of CODV-Ig.

[0227] Accordingly, the present invention further refers to an antibody-like binding protein comprising four polypeptide chains forming four antigen-binding sites, wherein two polypeptide chains have the structure represented by formula [I]: V D1 -L 1 -V D2 -L 2 -C L [I] having the structure represented by and two polypeptide chains have the structure represented by formula [III]: V D3 -L 3 -V D4 -L 4 -C H1 -F c [III] having the structure represented by, wherein: V D1 is the variable domain of the heavy or light chain of the first immunoglobulin; V D2 is the variable domain of the heavy or light chain of the second immunoglobulin; V D3 is the variable domain of the heavy or light chain of the second immunoglobulin; V D4 is the variable domain of the heavy or light chain of the first immunoglobulin; C L is the light chain constant domain of the immunoglobulin; C H1 is the C H1 heavy chain constant domain of the immunoglobulin; F c is the immunoglobulin hinge region and CH 2 of the immunoglobulin, CH 3 an immunoglobulin heavy chain constant domain; L 1 , L 2 , L 3 , and L 4 is an amino acid linker; and wherein the polypeptide of formula I and the polypeptide of formula III form a cross-light-heavy chain pair; and Here, V D1 and V D2 are both either light chain variable domains or heavy chain variable domains, and V D1 and V D2 is a light chain variable domain, V D3 and V D4 are both heavy chain variable domains, or V D1 and V D2 is a heavy chain variable domain, V D3 and V D4 are both light chain variable domains.

[0228] In the above CODV-Ig format, two polypeptide chains having the structure represented by formula [III] are c Dimerizes through the domain.

[0229] In a further embodiment, the first F c The domain is added to the polypeptide of formula [II] of the antibody-like binding protein CODV-Fab, and a second F c Domain(F c2 The antibody-like binding site, called The polypeptide of formula [I] of the CODV-Fab fusion protein is added. Linker L 5 is the C of the polypeptide chain of formula [I] L and F c2 domain, resulting in a polypeptide chain of formula [IV].

[0230] Accordingly, the present invention further refers to an antibody-like binding protein comprising two polypeptide chains forming two antigen-binding sites, wherein one polypeptide chain has a structure represented by 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 formula [III]: and one polypeptide chain has a structure represented by formula [III]: V D3 -L 3 -V D4 -L 4 -C H1 -F c [III] In the formulas: wherein: V D1 is the variable domain of the heavy or light chain of a first immunoglobulin; V D2 is the variable domain of the heavy or light chain of a second immunoglobulin; V D3 is the variable domain of the heavy or light chain of said second immunoglobulin; V D4 is the variable domain of the heavy or light chain of said first immunoglobulin; C L is the light chain constant domain of an immunoglobulin; C H1 is the C H1 heavy chain constant domain of an immunoglobulin; F c is the immunoglobulin hinge region and CH 2 and CH 3 immunoglobulin heavy chain constant domains; F c2 is the immunoglobulin hinge region and CH 2 and CH 3 immunoglobulin heavy chain constant domains; L 1 L 2 , L 3 , L 4 and L 5 are amino acid linkers; And here, the polypeptide of formula [IV] and the polypeptide of formula [III] form a cross light chain - heavy chain pair and here, V D1 and V D2 are both either the variable domain of the light chain or the variable domain of the heavy chain, and when V D1 and V D2 are the variable domains of the light chain, V D3 and V D4 are both the variable domains of the heavy chain, or when V D1 and V D2 are the variable domains of the heavy chain, V D3 and V D4 are both the variable domains of the light chain.

[0231] This CODV form in which the polypeptide chains represented by formulas [III] and [IV] dimerize through their respective F c2 and F c regions is referred to herein as CODV - Fab - TL.

[0232] In another embodiment of CODV - Fab, the first F c domain is added to the polypeptide chain represented by formula [II] (resulting in formula [III]), and the antibody - like binding protein comprises a third polypeptide chain comprising or consisting of a second F c domain in (referred to as F c3 ).

[0233] The present invention further refers to an antibody - like binding protein comprising three polypeptide chains that form two antigen - binding sites, where the first polypeptide has the formula [I]: V D1 -L 1 -V D2 -L 2 -C L [I] has a structure represented by The second polypeptide has the formula [III]: V D3 -L 3 -V D4 -L 4 -C H1 -F c [III] has a structure represented by The third polypeptide F c3 (also referred to as the Fc stump) is the immunoglobulin hinge region and CH 2 of the immunoglobulin, CH 3 constant domains of the immunoglobulin heavy chain; where V D1 is the variable domain of the heavy or light chain of the first immunoglobulin; V D2 is the variable domain of the heavy or light chain of the second immunoglobulin; V D3 is the variable domain of the heavy or light chain of the second immunoglobulin; V D4 is the variable domain of the heavy or light chain of the first immunoglobulin; C L is the constant domain of the light chain of the immunoglobulin; C H1 is the C H1 constant domain of the heavy chain of the immunoglobulin; F c is the immunoglobulin hinge region and CH 2 of the immunoglobulin, CH 3 constant domains of the immunoglobulin heavy chain; L 1 、L 2 、L 3 、and L 4 are amino acid linkers; and here, the polypeptide of formula [I] and the polypeptide of formula [III] form an inter-chain light - heavy chain pair, and here, V D1 and V D2 is either the variable domain of the light chain or the variable domain of the heavy chain, and V D1 and V D2 are the variable domains of the light chain, V D3 and V D4 are both the variable domains of the heavy chain, or V D1 and V D2 are the variable domains of the heavy chain, V D3 and V D4 are both the variable domains of the light chain; and here, the polypeptide of formula [III] heterodimerizes with a third polypeptide c through its F domain.

[0234] Thus, in the above embodiment, the so-called "Fc stump" (F c3 ) heterodimerizes with the Fc region of the polypeptide according to formula III. This CODV format is referred to herein as CODV-Fab-OL. This construct avoids the formation of aggregates by CODV-Fab. In one embodiment of CODV-Fab-OL, F

[0235] and F c and F c3 are both immunoglobulin mutants in which the CH3 domain is modified : F c and F c3 are, respectively, so-called "Knob-into-Hole" techniques described in particular in patents US5731168 and US8216805 (which are incorporated herein by reference) to promote heteromultimer formation, genetically engineered at the CH3-CH3 boundary. Thus, in one embodiment, one of the CH3 domains of F

[0236] and F c and F c3 contains the mutations Y349C, T366S, L368A, and Y407V, while F c and F c3 The other of which has mutations S354C and T366W (Amino acid positions are shown with reference to the IgG1 sequence).

[0237] Suitable F c and F c3 Examples of pairs include paired SEQ ID NO: 396 (F c ) and SEQ ID NO: 397 (F c3 ), and paired SEQ ID NO: 394 (F c ) and SEQ ID NO: 398 (F c3 ).

[0238] In one embodiment of the invention, the first immunoglobulin or the second immunoglobulin is one anti-CD3 antibody as defined in the <<anti-CD3 antibody>> section above.

[0239] In another embodiment of the invention, the first immunoglobulin or the second immunoglobulin is one anti-CD123 antibody as defined in the <<anti-CD123 antibody>> section above.

[0240] According to one embodiment of the invention, the V D1 and V D2 of the polypeptide of formula I or formula [IV] are both , either the variable domain of the light chain or the variable domain of the heavy chain, and the V D3 and V D4 of polypeptide II or III are both the variable domain of the heavy chain or the light chain. This compatibility , also referred to as "swapability", thus determines the cross-over dual variable (CODV) configuration of the antibody-like binding protein of the invention.

[0241] According to the above definition, V D1 and V D4 are the variable domains of the heavy chain or the light chain of the first immunoglobulin, and V D2 and V D3 are the variable domains of the heavy chain or the light chain of the second immunoglobulin. Thus, V D1 and V D4 is considered to be a homologous domain, and V D2 and V D3 are the same.

[0242] Therefore, the term "interchange" refers to the interchange alignment of V D1 or V D2 of the polypeptide of formula [I] or formula [IV] with its homologous variable domain V or V D4 of the polypeptide of formula [II] or D3 is of formula [III].

[0243] 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.

[0244] The antibody-like binding proteins of the present invention can be produced using domains or sequences obtained from or derived from any human or non-human antibody, including, for example, human, rat, or humanized antibodies.

[0245] In one embodiment, the immunoglobulin is an IgG immunoglobulin.

[0246] Therefore, in one embodiment, C L is the light chain constant domain of an IgG immunoglobulin. In a further embodiment, C H1 is the C H1 heavy chain constant domain of an IgG immunoglobulin.

[0247] In one embodiment, the antibody-like binding proteins of the present invention can be produced using the domains or sequences of the anti-CD3 and anti-CD123 antibodies described herein.

[0248] As used herein, the term " linker " 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 a cross - double - variable - domain immunoglobulin. In some embodiments, The linker consists of 0 amino acids, meaning that there is no linker. The linker is inserted at the sequence level at the transition between variable domains or between a variable domain and a constant domain, respectively. Since the approximate size of the immunoglobulin domains is well understood, the transition between domains can be identified. The exact position of the domain transition can be determined by determining the position of the peptide chain that does not form secondary - structure elements such as beta - sheets or alpha - helices, as demonstrated by experimental data or estimated by techniques of modeling or secondary - structure prediction. The linkers described in the context of the present invention are linker L 1 、L 2 、L 3 、L 4 and L 5 are. L 1 is located between the N - terminal V D1 domain and the V D2 domain; L 2 is located between V D2 and the C - terminal C L domain. Linker L 3 and L 4 are located on the polypeptide as defined according to Formula II or III of the antibody - like protein. More precisely, L 3 is located between the N - terminal V D3 domain and the V D4 domain, and L 4 is located between V D4 and the C - terminal C H1 - Fc domain. L 5 is located between C L and the N - terminal F c2 domain. Linker L 1 、L 2 、L 3 、L 4 and L 5 Although they are independent, in some embodiments, they have the same sequence and / or length.

[0249] In some antibody-like binding proteins of the present invention, the length of L 3 is at least twice the length of L 1 . In other antibody-like binding proteins of the present invention, the length of L 4 is at least twice the length of L 2 . In some antibody-like binding proteins of the present invention, the length of L 1 is at least twice the length of L 3 . In other antibody-like binding proteins of the present invention, the length of L 2 is at least twice the length of L 4 .

[0250] In one embodiment, the linkers L 1 , L 2 , L 3 and L 4 contain 0 to 20 amino acids. In one embodiment, L 5 contains 0 to 10 amino acids.

[0251] In some antibody-like binding proteins of the present invention, L 1 is 3 to 12 amino acid residues long , L 2 is 3 to 14 amino acid residues long, L 3 is 1 to 8 amino acid residues long, and L 4 is 1 to 3 amino acid residues long. In other antibody-like binding proteins, L 1 is 5 to 10 amino acid residues long , L 2 is 5 to 8 amino acid residues long, L 3 is 1 to 5 amino acid residues long, and L 4 is 1 to 2 amino acid residues long. In preferred antibody-like binding proteins, L 1 is 7 amino acid residues long , L 2 is 5 amino acid residues long, L 3 is 1 amino acid residue in length and is L 4 is 2 amino acid residues in length.

[0252] In some antibody-like binding proteins of the present 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 preferred antibody-like binding proteins, 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.

[0253] In some antibody-like binding proteins of the present invention, L 1 , L 3 , or L 4 may be equal to zero. However, in antibody-like binding proteins where L 3 , or L 4 is equal to zero, the corresponding transition linker between the variable and constant regions or between the dual variable domains on other chains 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 zero, and L 2 is 3 or more amino acid residues.

[0254] In some of the antibody-like binding proteins of the present 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.

[0255] Examples of suitable linkers include a single glycine, threonine or serine residue; a dipeptide such as a diglycine peptide, a histidine-threonine peptide or a glycine-serine dipeptide; a tripeptide having three glycines, the tripeptide Thr-His-Thr, the tripe ptide Gly-Gly-Ser; a peptide having four glycine residues; a peptide having five glycine residues; a peptide having six glycine residues; a peptide having seven glycine residues; a peptide having eight glycine residues. Other combinations of amino acid residues 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 pep tide 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) can be used. Other suitable li nkers include a single Ser and Val residue; dipeptides 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. 3 60); Asp-Lys-Thr-His-Thr-Ser (SEQ ID No. 361); Asp-Lys-Thr-His-Thr-Ser-Pro (SEQ ID No. Ser-Asp-Lys-Thr-His-Thr-Ser-Pro (SEQ ID NO: 362); Ser-Asp-Lys-Thr-His-Thr-Ser-Pro-Pro (SEQ ID NO: 363); Ser-Asp-Lys-Thr-His-Thr-Ser-Pro-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, aspartic acid, glutamic acid, asparagine, glutamine, glycine, and proline have been shown to be suitable in the antibody-like binding proteins of the present invention.

[0256] The identity and sequence of the amino acid residues in the linker can vary depending on the type of secondary structure element to be achieved in the linker. For example, glycine, serine, and alanine are best for linkers with maximum flexibility. Some combinations of glycine, proline, threonine, and serine are useful when a more robust and extended linker is required. Any amino acid residue can be considered as a linker in combination with other amino acid residues for constructing larger peptide linkers as needed, depending on the desired properties.

[0257] In one embodiment, linker L 1 is the linker of the sequence Gly-Gln-Pro-Lys-Ala-Ala-Pro (SEQ ID NO: 307), linker L 2 is the linker of the sequence Thr-Lys-Gly-Pro-Ser (SEQ ID NO: 309), linker L 3 is the linker of the sequence "S", and linker L 4 is the linker of the sequence "RT". ​

[0258] In a further embodiment, linker L 1 , L 2 , L 3 , and L 4 The sequences of and are threonine; dipeptides such as histidine-threonine peptides; tripeptides 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 Column number 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 In the linker L 5 The sequence of is a single serine residue, a dipeptide such as glycine-serine Dipeptide; tripeptide Gly-Gly-Ser, peptide Gly-Gly-Gly-Ser (SEQ ID NO: 354), peptide Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 344), peptide Ser-Gly-Gly-Gly-Gly-Ser ( SEQ ID NO: 355), peptide Gly-Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 356), peptide Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 357), peptide Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 358), peptide Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 345), and peptide Gly-Gly-Ser-Gly-Ser-Ser-Gly-Ser-Gly-Gly (SEQ ID NO: 389) are selected from the group consisting of Selected.

[0259] As used herein, the term " Fc domain " includes native Fc and Fc variants and the sequences defined above. Similar to Fc variants and native Fc molecules, the term "Fc domain" includes molecules in monomeric or multimeric form, whether produced by digestion from whole antibodies or by other means.

[0260] As used herein, the term " native Fc " refers to a molecule comprising the sequence of a non-antigen-binding fragment obtained by digestion of an antibody or produced by other means, in monomeric or multimeric form, and may include the hinge region. The original immunoglobulin source of native Fc is, in particular, of human origin and may be any immunoglobulin, although IgGl and IgG2 are preferred. Native Fc molecules are composed of monomeric polypeptides that can be linked into dimeric or multimeric forms by covalent (i.e., disulfide bonds) and non-covalent bonds. The number of intermolecular disulfide bonds between the monomeric subunits of native Fc molecules ranges from 1 to 4 depending on the class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgGl, IgG2, IgG3, IgAl, and IgGA2). An example of native Fc is the disulfide-linked dimer obtained by papain digestion of IgG . As used herein, "native Fc" is inclusive of monomeric, dimeric, and multimeric forms.

[0261] As used herein, the term " Fc variant " refers to a molecule or sequence modified from native Fc, but which does not form a binding site for the salvage receptor, FcRn (neonatal Fc receptor) It includes. Exemplary Fc variants and their interactions with their salvage receptors are known in the art. Thus, the term "Fc variant" can include molecules or sequences humanized from non-human native Fc. Furthermore, native Fc includes regions that can be removed to provide structural features or biological activities not necessary for the antibody-like binding proteins of the present invention. Thus, the term "Fc variant" refers to a molecule or sequence lacking one or more native Fc sites or residues, or a molecule or sequence in which one or more Fc sites or residues have been modified, (1) disulfide bond formation, (2) incompatibility with a selected host cell, (3) N-terminal heterogeneity upon expression in a selected host cell or (4) glycosylation, (5) interaction with complement, (6) binding to Fc receptors other than salvage receptors, or (7) antibody-dependent cell-mediated cytotoxicity (ADCC) that affect or are involved in these molecules or sequences.

[0262] In some embodiments, when the antibody-like binding protein contains two F c domains, i.e., 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. Thus, in some embodiments, both the F and F c of CODV-Ig or both F c2 domains of CODV-Fab-TL, or the F c and F c of CODV-Fab-OL and F c3 are both of the IgG1 subclass, or of the IgG2 subclass, or of the IgG3 subclass, or of the IgG4 subclass.

[0263] All antibody-like binding proteins described herein do not have effector functions. This means that if the antibody-like binding protein contains one or more F c domains of the IgG1 subclass (i.e., F c in formula [III], F c3 in formula [IV][F c2 ), the one or more F c domains of the IgG1 backbone contain the double mutation L234A and L235A (so-called "LALA mutation") that abolishes the Fc effector function. The Fc double mutants L234A and L235A do not bind to either FcγR or C1q, and both the ADCC and CDC functions of the Fc domain of the IgG1 subclass are abolished (Hezareh, M. et al., J Virol. 2001 Dec;75(24):12161-12168).

[0264] In one example, the F c region contains the amino acid sequence of SEQ ID NO: 330, SEQ ID NO: 394, or SEQ ID NO: 396. In one embodiment, the F region contains two amino acid exchanges H435R and Y436F within the CH3 domain as described by Jendeberg, L. et al. (1997, J. Immunological Meth. 201:25-34). Thus, in one embodiment, the F C2 region contains the amino acid sequence of SEQ ID NO: 327. C2 In another embodiment, the F region contains the amino acid sequence of SEQ ID NO: 392. C2 In another embodiment, the F

[0265] The so-called CODV-Fab "7G3x20G6" antibody-like binding protein is: - V of the sequence of SEQ ID NO: 9 D1 , L of the sequence of SEQ ID NO: 307 1 , V of the sequence of SEQ ID NO: 308 D2 , and L of the sequence of SEQ ID NO: 309 2 ​and C of the sequence of SEQ ID NO: 310 L An amino acid sequence comprising

Chem.

Chem.

[0266] The so-called CODV-Fab "7G3x4E7" antibody-like binding protein is: - V of the sequence of SEQ ID NO: 21 D1 , L of the sequence of SEQ ID NO: 307 1 , V of the sequence of SEQ ID NO: 308 D2 , L of the sequence of SEQ ID NO: 309 2 and C of the sequence of SEQ ID NO: 310 L An amino acid sequence comprising

Chem.

Chem.

[0267] The so-called CODV-Fab "7G3x4B4" antibody-like binding protein is: - V of the sequence of SEQ ID NO: 16 D1 L of the sequence of SEQ ID NO: 307 1 V of the sequence of SEQ ID NO: 308 D2 L of the sequence of SEQ ID NO: 309 2 and C of the sequence of SEQ ID NO: 310 L An amino acid sequence containing

Chemical formula

Chemical formula

[0268] The so-called CODV-Fab "7G3x18F5" antibody-like binding protein is: - V of the sequence of SEQ ID NO: 26 D1 L of the sequence of SEQ ID NO: 307 1 V of the sequence of SEQ ID NO: 308 D2 L of the sequence of SEQ ID NO: 309 2 and C of the sequence of SEQ ID NO: 310 L An amino acid sequence containing

Chemical formula

Chemical formula

[0269] The so-called CODV-Fab "hz20G6x7G3" antibody-like binding protein is: - V of the sequence of SEQ ID NO: 308 D1 , L of the sequence of SEQ ID NO: 307 1 , V of the sequence of SEQ ID NO: 143 D2 , the sequence L of the sequence of SEQ ID NO: 309 2 and C of the sequence of SEQ ID NO: 310 L comprising the sequence

Chemical formula

Chemical formula

[0270] The so-called CODV-Fab "7G3xhz4B4" antibody-like binding protein is:[[]] - V of the sequence of SEQ ID NO: 158 D1 L of the sequence of SEQ ID NO: 307 1 V of the sequence of SEQ ID NO: 308 D2 sequence L of the sequence of SEQ ID NO: 309 2 and C of the sequence of SEQ ID NO: 310 L comprising the amino acid sequence

Chemical formula

Chemical formula

[0271] The so-called CODV-Fab "hz4B4x3E3" antibody-like binding protein is:[[]] - V of the sequence of SEQ ID NO: 230 D1 L of the sequence of SEQ ID NO: 307 1 V of the sequence of SEQ ID NO: 158 D2 sequence L of the sequence of SEQ ID NO: 309 2 and C of the sequence of SEQ ID NO: 310 L comprising the amino acid sequence

Chemical formula

Chemical formula

[0272] The so-called CODV-Fab "hz20G6xhz7G3" antibody-like binding protein is: - V of the sequence of SEQ ID NO: 385 D1 , L of the sequence of SEQ ID NO: 389 1 , V of the sequence of SEQ ID NO: 141 D2 , sequence number 389 of L of the sequence 2 and C of the sequence of SEQ ID NO: 310 L including the amino acid sequence:

Chemical formula

Chemical formula

[0273] In one example, so-called CODV-Fab "7G3x20G6", "7G3x4E7", "7G3x4B4", "7G3x18F5", "hz20G6x7G3", "7G3xhz4B4", "hz4B4x3E3" and "hz20G6xhz7G3" antibody-like The polypeptide according to formula II of the binding protein further comprises a hinge sequence and a sequence EPKSCDKTHTHHHHHH (Accession No. 352) corresponding to, for example, a Histag used for purification.

[0274] The so-called CODV-Fab "hz20G6x7G3-TL4" (also called CODV-Fab-TL4 "hz20G6x7G3") antibody-like binding protein is: - V of the sequence of Accession No. 308 D1 L of the sequence of Accession No. 307 1 V of the sequence of Accession No. 143 D2 sequence L of the sequence of Accession No. 309 2 C of the sequence of Accession No. 310 L F of the sequence of Accession No. 327 c2 (underlined), an amino acid sequence

Chemical formula

Chemical formula

[0275] In the above CODV-Fab-TL4 “hz20G6x7G3” antibody-like binding protein, the sequence of SEQ ID NO: 330 Fc and the F of the sequence of SEQ ID NO: 327 c2 are derived from the IgG4 backbone. The above antibody-like binding protein is in the CODV-Fab-TL form and comprises or consists of one polypeptide of formula III and one polypeptide of formula IV.

[0276] The so-called CODV-Fab-TL1 “hz20G6xhz7G3” antibody-like binding protein is:[[]] - V of the sequence of SEQ ID NO: 385 D1 L of the sequence of SEQ ID NO: 389 1 V of the sequence of SEQ ID NO: 141 D2 sequence L of the sequence of SEQ ID NO: 389 2 C of the sequence of SEQ ID NO: 310 L L containing 0 amino acids 5 and F of the sequence of SEQ ID NO: 392 c2 (underlined), an amino acid sequence

Chemical formula

Chemical formula

[0277] In the CODV-Fab-TL1 "hz20G6x7G3" antibody-like binding protein, the Fc of the sequence of SEQ ID NO: 394 and the F of the sequence of SEQ ID NO: 392 c2 is derived from the IgG1 backbone. The above antibody-like binding protein is in the CODV-Fab-TL format. It comprises or consists of one polypeptide of formula IV and one polypeptide of formula III.

[0278] The so-called CODV-Fab-OL1 "hz20G6xhz7G3" antibody-like binding protein is:[[]] - V of the sequence of SEQ ID NO: 385 D1 L of the sequence of SEQ ID NO: 389 1 V of the sequence of SEQ ID NO: 141 D2 arrangement L of the sequence of SEQ ID NO: 389 2 and C of the sequence of SEQ ID NO: 310 L including the amino acid sequence

Chemical formula

Chemical formula

[0279] The above antibody-like binding protein is in the CODV-Fab-OL format, i.e., it comprises or consists of one polypeptide of Formula I, one polypeptide of Formula III, and one Fc terminus. The F c and F c3 sequences are engineered according to the "knob-into-hole" technology and further contain the double mutation L234A and L235A. contains the double mutation L234A and L235A.

[0280] The F c sequence of the sequence of SEQ ID NO: 396 is designed to contain RF residues at positions 200-221 (bolded above) instead of the HY residues that would otherwise be present at these positions in the Fc region. The HY>RF mutation (i.e., H435R and Y436F in the CH3 domain as described in Jendeberg, L. et al. 1997, J. Immunological Meth., 201:25-34) is advantageous for purification purposes as it eliminates binding to Protein A. In the case of CODV-Fab-OL1 "hz20G6xhz7G3", the F terminus of the sequence of SEQ ID NO: 397 contains HY residues at positions 217-218 (bolded above). c terminus contains HY residues at positions 217-218 (bolded above).

[0281] The so-called CODV-Fab-OL1a "hz20G6xhz7G3" antibody-like binding protein is: - V D1 of the sequence of SEQ ID NO: 385, L 1 , V of the sequence of SEQ ID NO: 141 D2 , ar , L of the sequence of SEQ ID NO: 389 2 , and C of the sequence of SEQ ID NO: 310 L An amino acid sequence comprising [Chemical formula] (SEQ ID NO: 388) One polypeptide according to Formula I; - V of the sequence of SEQ ID NO: 138 D3 , L which is 0 amino acids 3 , V of the sequence of SEQ ID NO: 383 D4 (in italics and underlined), L which is 0 amino acids 4 , C of the sequence of SEQ ID NO: 313 H1 , and F of the sequence of SEQ ID NO: 400 c (underlined) An amino acid sequence comprising: [Chemical formula] (SEQ ID NO: 399) One polypeptide according to Formula III comprising; - And here, the so-called CODV-Fab-OL1a "hz20G6xhz7G3" antibody-like binding protein is , an amino acid sequence: [Chemical formula] (SEQ ID NO: 398) Fc fragment (F c3 ) further comprising, which heterodimerizes with the Fc region of the polypeptide according to Formula III.

[0282] F of the sequence of SEQ ID NO: 400 c contains HY residues at positions 200-221 (in bold above), while the F c fragment of the sequence of SEQ ID NO: 398 contains RF residues at positions 217-218 (in bold above).

[0283] The above antibody-like binding protein is in the form of CODV-Fab-OL, i.e., it comprises or consists of one polypeptide of Formula I, one polypeptide of Formula III, and one Fc fragment. Its F c and F c3 sequences are engineered according to the "knob-into-hole" technology and contain the double mutations L234A and L235A.

[0284] 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 hereinafter, for example, the anti-CD123 antibody "3E3-D3" or "7G3", or a humanized form thereof.

[0285] 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 example, those selected from the so-called "20G6-F3", "4B4-D7" anti-CD3 antibodies.

[0286] Therefore, V D1 and V D4 , or V D2 and V D3 is the variable domain of the heavy or light chain of an anti-CD3 antibody wherein the anti-CD3 antibody is: a) a heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 6, CDR2-H of the sequence of SEQ ID NO: 7, CDR3-H of the sequence of SEQ ID NO: 8, and a light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 10 or SEQ ID NO: 142, CDR2-L of the sequence "KVS ", and CDR3-L of the sequence of SEQ ID NO: 11; or b) a heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 13, CDR2-H of the sequence of SEQ ID NO: 14, CDR3-H of the sequence of SEQ ID NO: 15, and a light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 17 or SEQ ID NO: 184, CDR2-L of the sequence "KVS", and CDR3-L of the sequence of SEQ ID NO: 11; or c) a heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 13, CDR2-H of the sequence of SEQ ID NO: 19, CDR3-H of the sequence of SEQ ID NO: 20, and a light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 22, CDR2-L of the sequence "KVS", and CDR3-L of the sequence of SEQ ID NO: 11; or d) a heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 24, CDR2-H of the sequence of SEQ ID NO: 19, CDR3-H of the sequence of SEQ ID NO: 25, and a light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 27, CDR2-L of the sequence "KVS", and CDR3-L of the sequence of SEQ ID NO: 28 and wherein D4 when V D1 is the variable domain of the light chain, V D4 is the variable domain of the heavy chain as defined above, or when V D1 is the variable domain of the heavy chain, V V D3 when is the variable domain of the light chain, V D2 is the variable domain of the heavy chain as defined above, or V D3 is the variable domain of the heavy chain, V D2 is the variable domain of the light chain as defined above.

[0287] In a further embodiment, V D1 and V D4 or V D2 and V D3 are the variable domains of the heavy or light chain of an anti-CD3 antibody, where the anti-CD3 antibody is a humanized antibody, and: a) the heavy chain variable domain of the sequence of SEQ ID NO: 138, or a sequence at least 85% identical thereto and / or the light chain variable domain of the sequence of SEQ ID NO: 143, or a sequence at least 85% identical thereto ; or b) the heavy chain variable domain of the sequence of SEQ ID NO: 171, or a sequence at least 85% identical thereto and / or the light chain variable domain of the sequence of SEQ ID NO: 158, or a sequence at least 85% identical thereto ; or c) the heavy chain variable domain of the sequence of SEQ ID NO: 176, or a sequence at least 85% identical thereto and / or the light chain variable domain of the sequence of SEQ ID NO: 164, or a sequence at least 85% identical thereto ; or where, if V D4 is the variable domain of the light chain, V D1 is the variable domain of the heavy chain as defined above, or if V D4 is the variable domain of the heavy chain, V D1 is the variable domain of the light chain as defined above, or V D3 is the variable domain of the light chain, V D2 is the variable domain of the heavy chain as defined above, or if V D3 is the variable domain of the heavy chain, V D2 is the variable domain of the light chain as defined above.

[0288] In the above-mentioned sequence that is 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 six CDRs have not changed as compared with the six CDRs present in 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 of the reference sequence.

[0289] In one embodiment, the antibody-like binding protein of the present invention binds to human CD3. In another embodiment, the antibody-like binding protein of the present invention further binds to cynomolgus CD3. In particular, the antibody-like binding protein of the present invention binds to the extracellular domain of human CD3 or both human and cynomolgus CD3. More specifically, the antibody binds to CD3ε. Even more specifically, the antibody-like binding protein binds to the human or human and cynomolgus extracellular domain of CD3ε. The antibody-like binding protein binds to CD3ε whether expressed in isolated form, or in the form of a complex such as a CD3ε / δ complex whether present in the form of a soluble extracellular domain or in the full-length membrane-anchored CD3ε as present, for example, on T cells, or whether present as a single protein. The antibody-like binding protein according to the present invention is specific for surface human CD3 protein or for human and cynomolgus CD3 proteins, particularly for CD3ε.

[0290] The antibody-like binding according to the present invention has a ratio of affinity for cynomolgus CD3 to affinity for human CD3 (KD(cynomolgus) / KD(human)) that is ≤ 10, particularly ≤ 6, ≤ 5, ≤ 4, ≤ 3, ≤ 2, ≤ 1, or ≤ 0.5. Accordingly, the antibody-like binding protein according to the present invention can be used in toxicological studies conducted in monkeys, and the toxicity profile observed in monkeys is relevant to predicting possible adverse effects in humans.

[0291] ​​Furthermore, the antibody-like binding protein according to the present invention has an affinity (KD) for human CD3 or cynomolgus CD3, or both, which is ≦50 nM, ≦40 nM, or ≦30 nM, for example, ≦20 nM, for example, an affinity of 0.1 nM to 30 nM, particularly 0.4 nM to 20 nM, or 0.4 nM to 15 nM.

[0292] In one embodiment, the antibody-like binding protein of the present invention has a T cell activation 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. to have.

[0293] In one embodiment, the antibody-like binding protein of the present invention has a T cell activation 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.

[0294] " low T cell activation " in the context of the antibody-like binding protein of the present invention refers to a T cell activation of less than 20%, 18% less than, 16% less than, 14% less than, 12% less than, 10% less than.

[0295] " target cell " herein refers to a cell expressing a second antigen, and in one example, the target cell herein refers to a cell expressing CD123 such as a THP-1 cell.

[0296] " 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% .

[0297] In a further embodiment, the present invention relates to an antibody-like binding protein having biological and immunological specificity for at least one additional target antigen.

[0298] Accordingly, in one aspect of the invention, the antibody-like binding protein of the invention further binds to at least one other target antigen. Accordingly, in one embodiment, the antibody-like binding protein of the invention is bispecific and can bind to two different antigen targets or epitopes.

[0299] Accordingly, in one embodiment, if the second immunoglobulin is one anti-CD123 antibody as defined in the <<anti-CD123 antibody>> section above, the first immunoglobulin is an immunoglobulin specific for at least one additional target, or if the first immunoglobulin is one anti-CD123 antibody as defined in the <<anti-CD123 antibody>> section above, the second immunoglobulin is an immunoglobulin specific for at least one additional target.

[0300] In one further embodiment, if the second immunoglobulin is one anti-CD3 antibody as defined in the <<anti-CD3 antibody>> section above, the first immunoglobulin is an immunoglobulin specific for at least one additional target, or if the first immunoglobulin is one anti-CD3 antibody as defined in the <<anti-CD3 antibody>> section above, the second immunoglobulin is an immunoglobulin specific for at least one additional target.

[0301] The antibody-like binding protein of the invention has a T cell-engaging effect. This T cell-engaging effect induces cytotoxicity in target cells. In one embodiment, the target cells are CD123-expressing cells such as cancer cells expressing CD123, such as THP-1 or TF-1.

[0302] Accordingly, in one embodiment, the antibody-like binding protein according to the invention can bind to primary T cells in vitro and lyse target cells, where (EC 50 ) is ≦40 pM, ≦35 pM, for example ≦30 pM.

[0303] As used herein, " cytotoxicity " refers to the property of being toxic to cells of the antibody-like binding protein or anti-CD123 antibody or other compounds of the present invention. Cytotoxicity can be induced by various mechanisms of action and can thus be classified into cell-mediated cytotoxicity, apoptosis, antibody-dependent cell-mediated cytotoxicity (ADCC), or complement-dependent cytotoxicity (CDC).

[0304] " antibody-dependent cell-mediated cytotoxicity " or " ADCC " refers to the mechanism of cell-mediated immune defense by which effector cells of the immune system actively lyse target cells whose membrane surface antigens are bound to specific antibodies.

[0305] As used in the context of this specification, " complement-dependent cytotoxicity " or " CDC " refers to the lysis of target cells in the presence of complement system proteins.

[0306] " cell-mediated cytotoxicity " refers to the lysis of target cells 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.

[0307] In one embodiment, cytotoxicity as used herein refers to cell-mediated cytotoxicity, such as T cell mediated cytotoxicity.

[0308] Furthermore, in one embodiment, cell-mediated cytotoxicity refers to cell-mediated cytotoxicity by T cells.

[0309] Thus, the antibody-like binding protein of the present invention induces cell-mediated cytotoxicity in target cells mediated by T cells.

[0310] ​​​Methods for measuring cytotoxicity are known to those skilled in the art and include, among others, 51-chromium (Cr) release assays, viability / death cell staining of target cells including propidium iodide, 7-AAD, and other stains known to those skilled in the art, detection by flow cytometry or ELISA of soluble molecules released by T cells including granzyme and perforin, detection of lactate dehydrogenase (LDH) released into the medium from damaged cells as a biomarker for cytotoxicity and cytolysis, detection of cell surface mobilization of CD107a, annexin V (calcium-dependent phospholipid-binding protein) staining of apoptotic target cells, and the use of detection of, for example, activated caspase-3 (CASP3). Furthermore, those skilled in the art can distinguish between different mechanisms of cytotoxicity based on the selected assay and cytotoxicity based on the experimental setting. detection by flow cytometry or ELISA of soluble molecules released, and cell lysis, detection of lactate dehydrogenase (LDH) released into the medium from damaged cells as a biomarker for cytotoxicity and cytolysis, detection of cell surface mobilization of CD107a, annexin V (calcium-dependent phospholipid-binding protein) staining, and the use of detection of, for example, activated caspase-3 (CASP3). Furthermore, those skilled in the art can distinguish between different mechanisms of cytotoxicity based on the selected assay and cytotoxicity based on the experimental setting.

[0311] In one example, cell-mediated cytotoxicity can be measured, for example, as described in Example 3.2, using CFSE to label target cells and 7-AAD to label dead cells. using CFSE to label target cells and 7-AAD to label dead cells. can be measured.

[0312] In a further embodiment, the antibody-like binding protein can bind to CD3 and at least one additional antigen target, for example CD123. In a further embodiment, the antibody-like binding protein can bind to CD3 and at least one additional antigen target, for example CD123.

[0313] In one embodiment, the antibody-like binding protein can inhibit the function of this additional antigen target, for example CD123. In one embodiment, the antibody-like binding protein can inhibit the function of this additional antigen target, for example CD123.

[0314] 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 cynomolgus CD123. In particular, the present invention In another embodiment, the antibody-like binding protein further binds to cynomolgus CD123. In particular, the present invention The antibody-like binding protein of the invention binds to the extracellular domain of human CD123, or both human and cynomolgus CD123. More specifically, the antibody-like binding protein binds to the distal portion of CD123, for example, amino acids starting from position 19 to 49 of human CD123 of amino acid sequence SEQ ID NO: 104 and binds thereto. The antibody-like binding protein is expressed in isolated form or is present in cells expressing CD123, such as AML cells or CD123-transfected cells, and binds to CD123 regardless of whether it is present in the soluble extracellular domain or the full-length membrane-anchored CD123. The antibody-like binding proteins according to the invention are specific for cells expressing human or human and cynomolgus CD123 proteins on their surface, such as cancer cells expressing CD123.

[0315] Thus, the antibody-like binding proteins according to the invention have an affinity (KD) for human CD123 or cynomolgus CD123, or both, of ≤20 nM, ≤15 nM, or ≤10 nM, for example ≤5 nM, for example having an affinity of 0.01 nM to 5 nM, particularly 0.1 nM to 5 nM.

[0316] Thus, in one embodiment, the first immunoglobulin is 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 humanized forms thereof, such as the so-called "20G6-F3" , one anti-CD3 antibody selected from the group consisting of "4B4-D7", "4E7-C9", "18F5-H10", "hz4B4" and "hz20G6" anti-CD3 antibodies, and the second immunoglobulin is 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 antibody, one anti-CD123 antibody selected from the group consisting of antibodies.

[0317] Thus, in a further embodiment, the second immunoglobulin is 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 humanized forms thereof, such as one anti-CD3 antibody selected from the group consisting of the so-called "20G6-F3", "4B4-D7", "4E7-C9", "18F5-H10", "hz4B4" and "hz20G6" anti-CD3 antibodies, and the first immunoglobulin is 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 antibody, one anti-CD123 antibody selected from the group consisting of antibodies.

[0318] Thus, in one embodiment, V D1 and V D4 or V D2 and V D3 comprises a heavy chain variable domain and a light chain variable domain, each of which is defined by three CDR sequences or by one of 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 either the heavy chain and light chain variable domain sequences of one of the "9F6-G3" anti-CD123 antibodies as defined above, where V D2 and V D3 both contain the three CDR sequences of the heavy chain and light chain variable domain sequences of one of the anti-CD3 antibodies defined above, then V and V D1 both contain the three CDR sequences of the heavy chain and light chain variable domain sequences of one of the anti-CD123 antibodies defined above, or D4 where V and V where V D1 and V D4 contain the CDR sequences of the heavy chain and light chain variable domain sequences of one of the anti-CD3 antibodies defined above, then V and V D2 both contain the three CDRs of the heavy chain and light chain variable domain sequences of one of the anti-CD123 antibodies defined above. D3 Accordingly, in one embodiment, V

[0319] and V D1 and V D4 are the variable domains of the heavy or light chain of an anti-CD3 antibody, where the anti-CD3 antibody comprises a heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 6, CDR2-H of the sequence of SEQ ID NO: 7, CDR3-H of the sequence of SEQ ID NO: 8, and a light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 10 or SEQ ID NO: 142, CDR2-L of the sequence "KVS" and CDR3-L of the sequence of SEQ ID NO: 11, and V and V D2 and V D3 is the variable domain of the 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, wherein when both V D3 and V D4 are variable domains of the heavy chain, V D1 and V D2 are both variable domains of the light chain, or when both V D3 and V D4 are variable domains of the light chain, V D1 and V D2 are both variable domains of the heavy chain.

[0320] Thus, in a further embodiment, V D2 and V D3 are variable domains of the heavy or light chain of an anti-CD3 antibody, wherein the anti-CD3 antibody comprises a heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 6, CDR2-H of the sequence of SEQ ID NO: 7, CDR3-H of the sequence of SEQ ID NO: 8, and a light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 10 or SEQ ID NO: 142, CDR2-L of the sequence "KVS", and CDR3-L of the sequence of SEQ ID NO: 11, and V D1 and V D4 are variable domains of the 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, wherein V D3 and V D4 When both are variable domains of the heavy chain, V D1 and V D2 are both variable domains of the light chain, or V D3 and V D4 When both are variable domains of the light chain, V D 1 and V D2 are both variable domains of the heavy chain.

[0321] Thus, in a further embodiment, V D1 and V D4 are variable domains of the heavy or light chain of an anti-CD3 antibody, wherein the anti-CD3 antibody comprises a heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 13, CDR2-H of the sequence of SEQ ID NO: 14, CDR3-H of the sequence of SEQ ID NO: 15, and a light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 17 or SEQ ID NO: 184, CDR2-L of the sequence "KVS", and CDR3-L of the sequence of SEQ ID NO: 11, and V and V D2 are variable domains of the heavy or light chain of an anti-CD123 antibody selected from the group consisting of the "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 in the section on "anti-CD123 antibody", D3 wherein, when both V and V D3 are both variable domains of the heavy chain, V D4 and V D1 are both variable domains of the light chain, or when both V D2 and V D3 are both variable domains of the light chain, D4 V and V D1 are both variable domains of the heavy chain. D2

[0322] Thus, in a further embodiment, V D1 and V D4 are the variable domains of the heavy or light chain of an anti-CD3 antibody, wherein the anti-CD3 antibody comprises a heavy chain variable domain comprising CDR1-H of the sequence of SEQ ID NO: 13, CDR2-H of the sequence of SEQ ID NO: 14, CDR3-H of the sequence of SEQ ID NO: 15, and a light chain variable domain comprising CDR1-L of the sequence of SEQ ID NO: 17 or SEQ ID NO: 184, CDR2-L of the sequence "KVS", and CDR3-L of the sequence of SEQ ID NO: 11, and V D2 and V D3 are the variable domains of the 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 in the section "anti-CD123 antibody", wherein, when both V D3 and V D4 are variable domains of the heavy chain, V D1 and V D2 are both variable domains of the light chain, or when both V D3 and V D4 are variable domains of the light chain, V D1 and V D2 are both variable domains of the heavy chain.

[0323] In a further embodiment, V D1 and V D4 are the variable domains of the heavy or light chain of a humanized anti-CD3 antibody, wherein the anti-CD3 antibody comprises a heavy chain variable heavy chain variable domain of the sequence of SEQ ID NO: 138 and / or a light chain variable domain of the sequence of SEQ ID NO: 143, and V D2 and V D3 is the variable domain of the heavy or light chain of an anti-CD123 antibody selected from the group consisting of the anti-CD123 antibodies "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" described above in the section "Anti-CD123 Antibodies", wherein, when both V D3 and V D4 are variable domains of the heavy chain, V D1 and V D2 are both variable domains of the light chain, or when both V D3 and V D4 are variable domains of the light chain, V D1 and V D2 are both variable domains of the heavy chain.

[0324] In a further embodiment, V D2 and V D3 are the variable domains of the heavy or light chain of a humanized anti-CD3 antibody, wherein the anti-CD3 antibody comprises the variable domain of the heavy chain of the sequence of SEQ ID NO: 138 and / or the variable domain of the light chain of the sequence of SEQ ID NO: 143, and V D1 and V D4 are the variable domains of the heavy or light chain of an anti-CD123 antibody selected from the group consisting of the anti-CD123 antibodies "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" described above in the section "Anti-CD123 Antibodies", wherein when both V D3 and V D4 are variable domains of the heavy chain, V D1 and V D2 are both variable domains of the light chain, and when both V D3 and V D4 are variable domains of the light chain, V D1 and V D2 Both are variable domains of the heavy chain.

[0325] In a further embodiment, V D1 and V D4 are the variable domains of the heavy or light chain of a humanized anti-CD3 antibody, where the anti-CD3 antibody comprises the heavy chain variable domain of the sequence of SEQ ID NO: 171 and / or the light chain variable domain of the sequence of SEQ ID NO: 158, and V and V D2 and V D3 are the variable domains of the heavy or light chain of an anti-CD123 antibody selected from the group consisting of the anti-CD123 antibodies "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" described above in the section "Anti-CD123 antibody", where, when V D3 and V D4 are both variable domains of the heavy chain, V D1 and V D2 are both variable domains of the light chain, or when V D3 and V D4 are both variable domains of the light chain, V D1 and V D2 are both variable domains of the heavy chain.

[0326] In a further embodiment, V D2 and V D3 are the variable domains of the heavy or light chain of a humanized anti-CD3 antibody, where the anti-CD3 antibody comprises the heavy chain variable domain of the sequence of SEQ ID NO: 171 and / or the light chain variable domain of the sequence of SEQ ID NO: 158, and V and V D1 and V D4 is the variable domain of the heavy or light chain of an anti-CD123 antibody selected from the group consisting of the anti-CD123 antibodies "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" as described above in the section on "anti-CD123 antibodies", where V D3 and V D4 are both variable domains of the heavy chain, V D1 and V D2 are both variable domains of the light chain, or where V D3 and V D4 are both variable domains of the light chain, V D1 and V D2 are both variable domains of the heavy chain.

[0327] In a further embodiment, V D1 and V D4 are the variable domains of the heavy or light chain of a humanized anti-CD3 antibody, where the anti-CD3 antibody comprises the heavy chain variable domain of the sequence of SEQ ID NO: 176 and / or the light chain variable domain of the sequence of SEQ ID NO: 164, and V D2 and V D3 are the variable domains of the heavy or light chain of an anti-CD123 antibody selected from the group consisting of the anti-CD123 antibodies "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" as described above in the section on "anti-CD123 antibodies", where V D3 and V D4 are both variable domains of the heavy chain, V D1 and V D2 are both variable domains of the light chain, or where V D3 and V D4 are both variable domains of the light chain, V D1 and V D2 Both are variable domains of the heavy chain.

[0328] In a further embodiment, V D2 and V D3 are the variable domains of the heavy or light chain of a humanized anti-CD3 antibody, where the anti-CD3 antibody comprises the heavy chain variable domain of the sequence of SEQ ID NO: 176 and / or the light chain variable domain of the sequence of SEQ ID NO: 164, and V and V D1 and V D4 are the variable domains of the heavy or light chain of an anti-CD123 antibody selected from the group consisting of the anti-CD123 antibodies "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" described above in the section on "anti-CD123 antibodies", where when V D3 and V D4 are both variable domains of the heavy chain, V D1 and V D2 are both variable domains of the light chain, or when V D3 and V D4 are both variable domains of the light chain, V D1 and V D2 are both variable domains of the heavy chain.

[0329] According to a further aspect of the invention, the first or second immunoglobulin is the anti-CD123 antibody 7G3. Thus, in one embodiment, V D1 and V D4 or V D2 and V D3 comprise the variable domains of the heavy and light chains defined by the CDR sequences of the heavy and light chain variable domain sequences of the antibody 7G3 as defined hereinafter in this specification. In one embodiment, V D1 and V D 4, or V D2 and and V D3 It includes the heavy-chain variable domain and the light-chain variable domain of the antibody 7G3 described in patent application WO2013 / 173820 (incorporated herein by reference).

[0330] Therefore, the so-called "7G3" anti-CD123 antibody used herein is: - The heavy-chain variable domain consisting of CDR1-H of the sequence of SEQ ID NO: 375, CDR2-H of the sequence of SEQ ID NO: 376, and CDR3-H of the sequence of SEQ ID NO: 377, and the sequence

Chemical Structure

Chemical Structure

[0331] In a further aspect of the invention, the antibody 7G3 may also be a humanized antibody or a fragment of a humanized antibody. Therefore, in one embodiment, the antibody 7G3 of the invention is - The heavy-chain variable domain consisting of CDR1-H of the sequence of SEQ ID NO: 381, CDR2-H of the sequence of SEQ ID NO: 377, and CDR3-H of the sequence of SEQ ID NO: 382, and the sequence the sequence

Chemical Structure

Chemical Structure

[0332] In one embodiment, the antibody-like binding protein that specifically binds to human CD3ε and human CD123 is a) The light chain variable domain of a first immunoglobulin (V D1 ) consisting of the amino acid sequence of Accession No. 9 or a sequence that is at least 85% identical thereto, the light chain variable domain of a second immunoglobulin (V ) consisting of the amino acid sequence of Accession No. 308 or a sequence that is D2 at least 85% identical thereto, the heavy chain variable domain of a second immunoglobulin (V ) consisting of the amino acid sequence of Accession No. 312 or a sequence that is at least 85% identical thereto, and the heavy chain variable domain of a first immunoglobulin (V D3 ) consisting of the amino acid sequence of Accession No. 5 or a sequence that is at least also 85% identical thereto, or D4 ) b) The light chain variable domain of a first immunoglobulin (V ) consisting of the amino acid sequence of Accession No. 21 or a sequence that is at least 85% identical thereto, the light chain variable domain of a second immunoglobulin (V D1 ) consisting of the amino acid sequence of Accession No. 308 or a sequence that is at least 85% identical thereto, D2 ) The heavy chain variable domain of a second immunoglobulin (V D3 ) consisting of the amino acid sequence of Accession No. 312 or a sequence that is at least 85% identical thereto, and the heavy chain variable domain of a first immunoglobulin (V D4 ) heavy chain variable domain, or c) an amino acid sequence consisting of SEQ ID NO: 16 or a sequence at least 85% identical thereto, the first immunoglobulin (V D1 ) light chain variable domain, amino acid sequence SEQ ID NO: 308 or a second immunoglobulin (V D2 ) light chain variable domain consisting of a sequence at least 85% identical thereto, a second immunoglobulin (V D3 ) heavy chain variable domain consisting of amino acid sequence SEQ ID NO: 312 or a sequence at least 85% identical thereto, and a first immunoglobulin (V D4 ) heavy chain variable domain consisting of amino acid sequence SEQ ID NO: 12 or a sequence at least 85% identical thereto, or d) an amino acid sequence consisting of SEQ ID NO: 26 or a sequence at least 85% identical thereto, the first immunoglobulin (V D1 ) light chain variable domain, amino acid sequence SEQ ID NO: 308 or a second immunoglobulin (V D2 ) light chain variable domain consisting of a sequence at least 85% identical thereto, a second immunoglobulin (V D3 ) heavy chain variable domain consisting of amino acid sequence SEQ ID NO: 312 or a sequence at least 85% identical thereto, and a first immunoglobulin (V D4 ) heavy chain variable domain consisting of amino acid sequence SEQ ID NO: 23 or a sequence at least 85% identical thereto, or e) a first immunoglobulin (V D1 ) light chain variable domain consisting of amino acid sequence SEQ ID NO: 308 or a sequence at least 85% identical thereto, amino acid sequence SEQ ID NO: 143 or a second immunoglobulin (V D2 ) light chain variable domain consisting of a sequence at least 85% identical thereto, a second immunoglobulin (V D3 ) heavy chain variable domain and amino acid sequence SEQ ID NO: 312 or at least 85% identical sequence consisting of a first immunoglobulin (V D4 ) heavy chain variable domain, or f) amino acid sequence SEQ ID NO: 158 or at least 85% identical sequence consisting of a first immunoglobulin (V D1 ) light chain variable domain, amino acid sequence SEQ ID NO: 308 or that at least 85% identical sequence consisting of a second immunoglobulin (V D2 ) light chain variable domain, amino acid sequence SEQ ID NO: 312 or at least 85% identical sequence consisting of a second immunoglobulin (V D3 ) heavy chain variable domain and amino acid sequence SEQ ID NO: 171 or at least 85% identical sequence consisting of a first immunoglobulin (V D4 ) heavy chain variable domain, g) amino acid sequence SEQ ID NO: 230 or at least 85% identical sequence consisting of a first immunoglobulin (V D1 ) light chain variable domain, amino acid sequence SEQ ID NO: 158 or that at least 85% identical sequence consisting of a second immunoglobulin (V D2 ) light chain variable domain, amino acid sequence SEQ ID NO: 171 or at least 85% identical sequence consisting of a second immunoglobulin (V D3 ) heavy chain variable domain and amino acid sequence SEQ ID NO: 226 or at least 85% identical sequence consisting of a first immunoglobulin (V D4 ) heavy chain variable domain, h) amino acid sequence SEQ ID NO: 385 or at least 85% identical sequence consisting of a first immunoglobulin (V D1 ) light chain variable domain, amino acid sequence SEQ ID NO: 141 or that at least 85% identical sequence consisting of a second immunoglobulin (V D2 ) light chain variable domain, A second immunoglobulin (V D3 ) heavy chain variable domain consisting of the amino acid sequence SEQ ID NO: 138 or a sequence that is at least 85% identical thereto, and an amino acid sequence SEQ ID NO: 383 or a sequence that is at least 85% identical thereto, of a first immunoglobulin (V D4 ) heavy chain variable domain .

[0333] In the above sequence that is at least 85% identical to the reference sequence (for example, a sequence that is at least 85% identical to SEQ ID NO: 383 or SEQ ID NO: 385), the sequences of the six CDRs have not changed compared to the six CDRs present in the reference sequence.

[0334] In one embodiment, the antibody-binding protein according to any of definitions a) to g) further comprises the linker L of the sequence of SEQ ID NO: 307 1 , the linker L of the sequence of SEQ ID NO: 309 2 , the linker L of the amino acid sequence "S" 3 , the linker L of the amino acid sequence "RT" 4 and the linker C of the sequence of SEQ ID NO: 313 H1 .

[0335] In one embodiment, the antibody-binding protein according to any of definitions a) to g) further comprises the F of the sequence of SEQ ID NO: 327 c2 .

[0336] In one embodiment, the antibody-binding protein according to definition h) comprises the linker L of the sequence of SEQ ID NO: 389 , the linker L of the sequence of SEQ ID NO: 389 1 , the linker L consisting of 0 amino acids 2 and L 3 and L 4 , and the C of the sequence of SEQ ID NO: 313 in the sequence H1 .

[0337] In one embodiment, the antibody-binding protein according to definition h) further comprises the F of the sequence of SEQ ID NO: 392 c2 .

[0338] ​ In one embodiment, the linker L of the antibody-binding protein according to any one of definitions a) to h) 5 contains 0 amino acids.

[0339] In a further embodiment, the antibody-binding protein according to any one of definitions a) to g) has a linker L of the sequence of SEQ ID NO: 307 1 , an L of the sequence of SEQ ID NO: 309 2 , an L of the amino acid sequence "S" 3 , amino acid sequence "RT" of L 4 , a C of the sequence of SEQ ID NO: 329 H1 and an F of the sequence of SEQ ID NO: 330 c and further comprises.

[0340] In a further embodiment, the antibody-binding protein according to definition h) has a linker L of the sequence of SEQ ID NO: 389 , an L of the sequence of SEQ ID NO: 389 1 , an L consisting of 0 amino acids 2 and L 3 and L 4 , a C of the sequence of SEQ ID NO: 313 H1 and an F of the sequence of SEQ ID NO: 394 c and further comprises.

[0341] In a further embodiment, the antibody-binding protein according to definition h) further comprises an Fc fragment of the sequence of SEQ ID NO: 397 or SEQ ID NO: 398, or a sequence that is at least 85% identical to SEQ ID NO: 397 or SEQ ID NO: 398.

[0342] In one embodiment, an antibody-binding protein that specifically binds to human CD3ε and human CD123 is: a) the sequence of SEQ ID NO: 388 (V of the sequence of SEQ ID NO: 385 D1 , L of the sequence of SEQ ID NO: 389 1 , V of the sequence of SEQ ID NO: 141 , L of the sequence of SEQ ID NO: 389 D2 and C of the sequence of SEQ ID NO: 310 2 ), L ) or a sequence that is at least 85% identical to SEQ ID NO: 388 [wherein the sequences of the three CDRs of the hz7G3 light chain variable domain (V of the sequence of SEQ ID NO: 385 ), SEQ ID NO: 378, "WAS", and SEQ ID NO: 379, and D1 similarly, the sequences of the three CDRs of the hz20G6 light chain variable domain (V of the sequence of SEQ ID NO: 141 ), SEQ ID NO: 142, "KVS", and SEQ ID NO: 11 have not been changed]; a polypeptide of formula [I] consisting of; and D2 b) the sequence of SEQ ID NO: 390 (V of the sequence of SEQ ID NO: 138 ), L which is 0 amino acids ), V of the sequence of SEQ ID NO: 383 D3 ), L which is 0 amino acids 3 ), and C of the sequence of SEQ ID NO: 313 D4 ), L which is 0 amino acids 4 ), or H1 ) or a sequence that is at least 85% identical to SEQ ID NO: 390 [wherein the sequences of the three CDRs of the hz7G3 heavy chain variable domain (V of the sequence of SEQ ID NO: 383 D4 ), SEQ ID NO: 381, SEQ ID NO: 384, and SEQ ID NO: 382, and similarly, the sequences of the three CDRs of the hz20G6 heavy chain variable domain (V of the sequence of SEQ ID NO: 138 D3 ), SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8 have not been changed]; a polypeptide of formula [II] consisting of; comprising or consisting of these, and wherein the polypeptide of formula [I] and the polypeptide of formula [II] form a cross light chain - heavy chain pair.

[0343] In one embodiment, the antibody - binding protein that specifically binds to human CD3ε and human CD123 is: a) the amino acid sequence SEQ ID NO: 391 (V of the sequence of SEQ ID NO: 385 D1 ), L of the sequence of SEQ ID NO: 389 1 ), V of the sequence of SEQ ID NO: 141 D2 ), L of the sequence of SEQ ID NO: 389 2 ), C of the sequence of SEQ ID NO: 310 L ), 0 amino acids ​L containing 5 , and F of the sequence of SEQ ID NO: 392 c2 ), or a polypeptide according to formula [IV] having a sequence that is at least 85% identical to SEQ ID NO: 391 [wherein the three CDRs of the sequence of SEQ ID NO: 378, "WAS", and the sequence of SEQ ID NO: 379 of the hz7G3 light chain variable domain (V of the sequence of SEQ ID NO: 385) D1 ), and the three CDRs of the sequence of SEQ ID NO: 142, "KVS", and the sequence of SEQ ID NO: 11 of the hz20G6 light chain variable domain (V of the sequence of SEQ ID NO: 141) D2 ) are not modified]; and b) a polypeptide according to formula [III] having the amino acid sequence SEQ ID NO: 393 (V of the sequence of SEQ ID NO: 138 , L which is 0 amino acids D3 , V of the sequence of SEQ ID NO: 383 3 , L which is 0 amino acids D4 , C of the sequence of SEQ ID NO: 313 4 , and F of the sequence of SEQ ID NO: 394 H1 ), or c ) a polypeptide according to formula [III] having a sequence that is at least 85% identical to SEQ ID NO: 393 [wherein the three CDRs of the sequence of SEQ ID NO: 381, SEQ ID NO: 384, and the sequence of SEQ ID NO: 382 of the hz7G3 heavy chain variable domain (V of the sequence of SEQ ID NO: 383) , and the three CDRs of the sequence of SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8 of the hz20G6 heavy chain variable domain (V of the sequence of SEQ ID NO: 138) D4 ) are not modified]; D3 including or consisting essentially of these, and wherein the polypeptide of formula [IV] and the polypeptide of formula [III] form a cross light chain - heavy chain pair . In the above antibody - like binding protein, the polypeptide chains represented by formulas [III] and [IV] dimerize through their respective F

[0344] regions. c2 and F c

[0345] ​​ In one embodiment, an antibody-binding protein that specifically binds to human CD3ε and human CD123 is: a) an amino acid sequence SEQ ID NO: 388 (V of the sequence of SEQ ID NO: 385 D1 , L of the sequence of SEQ ID NO: 389 1 , V of the sequence of SEQ ID NO: 141 D2 , L of the sequence of SEQ ID NO: 389 2 and C of the sequence of SEQ ID NO: 310 L ), or a polypeptide according to formula [I] consisting of a sequence that is at least 85% identical to SEQ ID NO: 388 [wherein the three CDRs of the sequence numbers 378, "WAS", and 379 of the hz7G3 light chain variable domain (V of the sequence of SEQ ID NO: 385 ), and the three CDRs of the sequence numbers 142, "KVS", and 11 of the hz20G6 light chain variable domain (V of the sequence of SEQ ID NO: 141 D1 ) are not altered]; and D2 ) are not altered], and b) an amino acid sequence SEQ ID NO: 395 (V of the sequence of SEQ ID NO: 138 D3 , L which is 0 amino acids 3 , V of the sequence of SEQ ID NO: 383 D4 , L which is 0 amino acids 4 , C of the sequence of SEQ ID NO: 313 H1 , and F of the sequence of SEQ ID NO: 396 c ), or a polypeptide according to formula [III] consisting of a sequence that is at least 85% identical to SEQ ID NO: 395 [wherein the three CDRs of the sequence numbers 381, 384, and 382 of the hz7G3 heavy chain variable domain (V of the sequence of SEQ ID NO: 383 D4 ), and the three CDRs of the sequence numbers 6, 7, 8 D3 of the sequence of the hz20G6 heavy chain variable domain (V of the sequence of SEQ ID NO: 138 ) are not altered]; and c) one Fc fragment (polypeptide F c3 ) having the amino acid sequence SEQ ID NO: 397, or a sequence that is at least 85% identical thereto, or consisting essentially of these, wherein the above F c3 The stump or a sequence that is at least 85% identical thereto heterodimerizes with the F region of the polypeptide according to formula [III]; c and herein, the polypeptide of formula [I] and the polypeptide of formula [III] form an interchain light chain-heavy chain pair. In one embodiment, the antibody-binding protein that specifically binds to human CD3ε and human CD123 is:

[0346] a) one polypeptide according to formula [I] consisting of the amino acid sequence (V of the sequence of SEQ ID NO: 388 (SEQ ID NO: 385), L of the sequence of SEQ ID NO: 389, V of the sequence of SEQ ID NO: 141, L of the sequence of SEQ ID NO: 389, and C of the sequence of SEQ ID NO: 310) or a sequence that is at least 85% identical to SEQ ID NO: 388 [wherein the three CDRs of the hz7G3 light chain variable domain (V of the sequence of SEQ ID NO: 385), "WAS", and the sequence of SEQ ID NO: 379, and the three CDRs of the hz20G6 light chain variable domain (V of the sequence of SEQ ID NO: 141) are unchanged]; D1 b) a polypeptide according to formula III of the amino acid sequence (V of the sequence of SEQ ID NO: 399 (SEQ ID NO: 138), L which is 0 amino acids, V of the sequence of SEQ ID NO: 383, L which is 0 amino acids, C of the sequence of SEQ ID NO: 313, and F of the sequence of SEQ ID NO: 400), or 1 a sequence that is at least 85% identical thereto [wherein the three CDRs of the shz7G3 heavy chain variable domain (V of the sequence of SEQ ID NO: 383) are the sequences of SEQ ID NO: 381, SEQ ID NO: 384, and SEQ ID NO: 382]; D2 2 L D1 D2 D3 3 D4 4 H1 c D4 ​​​​​​​​​​​​​​​​​​, and the three CDRs of the sequences of SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8 of the hz20G6 heavy chain variable domain (V of the sequence of SEQ ID NO: 138) D3 ) are not changed]; and c) one Fc fragment (polypeptide F c3 ) of an amino acid sequence of SEQ ID NO: 398 or a sequence that is at least 85% identical thereto, wherein the above F c3 fragment or a sequence that is at least 85% identical thereto heterodimerizes 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 an interchain light-heavy chain pair.

[0347] Immune complex In one embodiment, the anti-CD123 antibody of the present invention is bound or linked to a growth inhibitor, a cytotoxic agent, or a prodrug activating enzyme. In particular, the anti-CD123 antibody of the present invention is actually useful for targeting the above growth inhibitor , cytotoxic agent, or prodrug to cancerous cells that overexpress CD123 on their surfaces.

[0348] Nucleic acids, vectors, and recombinant host cells A further object of the present invention relates to a nucleic acid sequence comprising or consisting of a sequence encoding an anti-CD3 antibody, an anti-CD123 antibody, or an antibody-like binding protein as defined above.

[0349] Typically, the nucleic acid is a DNA or RNA molecule, which can be contained in any suitable vector such as a plasmid, cosmid, episome, artificial chromosome, phage, or viral vector.

[0350] The terms " vector ", " cloning vector " and " expression vector " refers to a mediator that allows a DNA or RNA sequence (e.g., a foreign gene) to be introduced into a host cell, transform the host, and promote the expression (e.g., transcription and translation) of the introduced sequence.

[0351] Accordingly, a further object of the present invention relates to a vector containing the nucleic acid of the present invention.

[0352] Such vectors may contain regulatory elements such as promoters, enhancers, terminators, etc. to cause or induce the expression of the above polypeptide upon administration to a subject. Examples of promoters and enhancers used in expression vectors for animal cells include the early promoter and enhancer of SV40 (Mizukami T. et al. 1987), the LTR promoter and enhancer of Moloney murine leukemia virus (Kuwana Y et al. 1987), the promoter of immunoglobulin H chain (Mason JO et al. 1985) and en hancer (Gillies SD et al. 1983), etc.

[0353] Any expression vector for animal cells can be used as long as the 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), etc. Other examples of plasmids include replicating plasmids containing an origin of replication, or integrating plasmids such as pUC , pcDNA, pBR, etc. Examples of integrating plasmids include pUC, pcDNA, pBR, etc.

[0354] Other examples of viral vectors include adenovirus, retrovirus, herpes virus, and AAV vectors. Such recombinant viruses can be produced by techniques known in the art, for example, by transfecting packaging cells or by transient transfection with a helper plasmid or virus. Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc. Detailed protocols for producing such replication-deficient recombinant viruses can be found, for example, 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. By techniques known in the art, for example, by transfecting packaging cells or by transient transfection with a helper plasmid or virus. Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc. Detailed protocols for producing such replication-deficient recombinant viruses can be found, for example, 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. Detailed protocols for producing such replication-deficient recombinant viruses can be found, for example, 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.

[0355] A further object of the present invention relates to cells transfected, infected or transformed by the nucleic acid and / or vector according to the present invention.

[0356] The term " transformation " means introducing a "foreign" (i.e., exogenous) gene, DNA or RNA sequence into a host cell such that the host cell expresses the introduced gene or sequence to produce the desired substance, typically a protein or enzyme, encoded by the introduced gene or sequence. A host cell that receives and expresses the introduced DNA or RNA is a " transformed ".

[0357] The nucleic acid of the present invention can be used to produce the recombinant antibody of the present invention in a suitable expression system. The term " expression system " means, for example, a host cell and a compatible vector that are under appropriate conditions for the expression of a protein encoded by foreign DNA carried by the vector and introduced into the host cell.

[0358] 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 (e.g., bacteria) and eukaryotic cells ( e.g., yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include E. coli, Kluyveromyces or Saccharomyces yeast, 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., those produced from lymphoblasts, fibroblasts, embryonic cells, epithelial cells, nerve cells, adipocytes, etc.). Examples include mouse SP2 / 0-Ag14 cells (ATCC CRL1581), mouse P3X63-Ag8.653 cells (ATCC CRL1580), CHO cells deficient in the dihydro folate reductase gene (hereinafter referred to as the "DHFR gene") (Urlaub G et al; 1980), rat YB2 / 3HL.P2.G11.16Ag.20 cells (ATCC CRL1662, hereinafter referred to as "YB2 / 0 cells"), etc. YB2 / 0 cells are preferred because the ADCC activity of chimeric or humanized antibodies is enhanced when expressed in these cells.

[0359] In particular, for the expression of a humanized antibody or antibody-like binding protein, the expression vector may be of a type in which the gene encoding the antibody heavy chain and the gene encoding the antibody light chain are present on separate vectors, or of a type (tandem type) in which both genes are present on the same vector. In terms of the ease of construction of humanized antibody and antibody-like binding protein expression vectors, the ease of introduction into animal cells, and the balance between the expression levels of antibody H and L chains in animal cells, a tandem-type humanized antibody expression vector is preferred (Shitara K et al. J Immunol Methods. 1994 Jan. 3;167(1-2):271-8). Examples of tandem-type humanized antibody expression vectors include pKANTEX93 (WO97 / 10354), pEE18, and the like.

[0360] The present invention also relates to a method for producing a recombinant host cell that expresses an anti-CD3 antibody, an anti-CD123 antibody, or an antibody-like binding protein according to the present invention. The method comprises: (i) introducing the above-described recombinant nucleic acid or vector into a transformation-receptive host cell in vitro or ex vivo; (ii) culturing the obtained recombinant host cell in vitro or ex vivo; and (iii) selecting cells that express and / or secrete the above antibody. The method includes the steps consisting of the above steps.

[0361] Such a recombinant host cell can be used for the production of the anti-CD3 antibody, at least one anti-CD123 antibody, or at least one antibody-like binding protein of the present invention.

[0362] Method for producing the antibody and antibody-like binding protein of the present invention One embodiment of the present invention provides a method for producing an antibody-like binding protein comprising two polypeptide chains that form two antigen-binding sites. Here, the first polypeptide has the formula [I]: V D1 -L 1 -V D2 -L 2 -C L [I] has a structure represented by and the second polypeptide has a structure represented by formula [II]: V D3 -L 3 -V D4 -L 4 -C H1 [II] has a structure represented by wherein: V D1 is the variable domain of the heavy or light chain of the first immunoglobulin; V D2 is the variable domain of the heavy or light chain of the second immunoglobulin; V D3 is the variable domain of the heavy or light chain of said second immunoglobulin; V D4 is the variable domain of the heavy or light chain of said first immunoglobulin; C L is the constant domain of the light chain of an immunoglobulin; C H1 is the C H1 constant domain of the heavy chain of an immunoglobulin; L 1 、L 2 、L 3 、and L 4 are amino acid linkers; and here, the first and second polypeptides form a cross light chain - heavy chain pair, and here, V D1 and V D2 are both either the variable domain of the light chain or the variable domain of the heavy chain, and when V D1 and V D2 are the variable domains of the light chain, V D3 and V D4 are both the variable domains of the heavy chain, and when V D1 and V D2 are the variable domains of the heavy chain, V D3 and V D4 are both the variable domains of the light chain.

[0363] In a further embodiment, the present invention provides a method for producing an antibody-like binding protein comprising four polypeptide chains forming four antigen-binding sites, wherein two of the polypeptide chains have the structure represented by formula [I]: V D1 -L 1 -V D2 -L 2 -C L [I] and two of the polypeptide chains have the structure represented by formula (III): and two of the polypeptide chains have the structure represented by formula (III): V D3 -L 3 -V D4 -L 4 -C H1 -F c [III] wherein: wherein: V D1 is the variable domain of the heavy or light chain of a first immunoglobulin; V D2 is the variable domain of the heavy or light chain of a second immunoglobulin; V D3 is the variable domain of the heavy or light chain of said second immunoglobulin; V D4 is the variable domain of the heavy or light chain of said first immunoglobulin; C L is the light chain constant domain of an immunoglobulin; C H1 is the 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 domain 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 an inter-chain light-heavy chain pair, and where V D1 and V D2 are both either the variable domain of the light chain or the variable domain of the heavy chain and where V D1 and V D2 are the variable domains of the light chain, V D3 and V D4 are both the variable domains of the heavy chain, or where V D1 and V D2 are the variable domains of the heavy chain, V D3 and V D4 are both the variable domains of the light chain.

[0364] In a further embodiment, the present invention provides a method for producing an antibody-like binding protein comprising four polypeptide chains forming four antigen-binding sites, wherein two of the polypeptide chains have the structure represented by formula [IV]: V D1 -L 1 -V D2 -L 2 -C L -L 5 -F c2 [IV] and two of the polypeptide chains have the structure represented by formula [III]: wherein: V D3 -L 3 -V D4 -L 4 -C H1 -F c [III] wherein: In the formula: V D1 is the variable domain of the heavy or light chain of a first immunoglobulin; V D2 is the variable domain of the heavy or light chain of a second immunoglobulin; V D3 is the variable domain of the heavy or light chain of said second immunoglobulin; V D4 is the variable domain of the heavy or light chain of said first immunoglobulin; C L is the constant domain of the light chain of an immunoglobulin; C H1 is the C heavy chain constant domain of an immunoglobulin; H1 and F c is the immunoglobulin hinge region and CH of an immunoglobulin; 2 CH 3 is the immunoglobulin heavy chain constant domain; F c2 is the immunoglobulin hinge region and CH of an immunoglobulin; 2 CH 3 is the immunoglobulin heavy chain constant domain; L 1 L 2 L 3 L 4 and L 5 are amino acid linkers; and here, the polypeptide of formula I and the polypeptide of formula III form an inter - light - chain / heavy - chain pair, and here V D1 and V D2 are each either the variable domain of the light chain or the variable domain of the heavy chain, and when V D1 and V D2 are the variable domains of the light chain, V D3 and V D4 are both the variable domains of the heavy chain, or when V D1 and V D2 are the variable domains of the heavy chain, V D3 and V D4 are both the variable domains of the light chain.

[0365] In one embodiment of the invention, the first immunoglobulin or the second immunoglobulin is one anti - CD3 antibody as defined in the <<anti - CD3 antibody>> section above.

[0366] In another embodiment of the invention, the first immunoglobulin or the second immunoglobulin is one anti - CD123 antibody as defined in the <<anti - CD123 antibody>> section above.

[0367] The anti-CD3 antibody, anti-CD123 antibody and / or antibody-like binding protein of the present invention can be produced by any technique known in the art, for example, without limitation, by any scientific, biological, genetic or enzymatic technique, alone or in combination.

[0368] Once the amino acid sequence of the desired sequence is known, those skilled in the art can readily produce the above antibodies or immunoglobulin chains by standard techniques for polypeptide production. For example, using well-known solid-phase methods, particularly using a commercially available peptide synthesizer (e.g., one manufactured by Applied Biosystems, Foster City, California), they can be synthesized according to the manufacturer's instructions. This can be done. Alternatively, the antibodies, immunoglobulin chains and antibody-like binding proteins of the present invention can be synthesized by recombinant DNA techniques well known in the art. For example, these fragments can be obtained as DNA expression products after incorporation into an expression vector of a DNA sequence encoding the desired (poly)peptide and introduction of such a vector into a suitable eukaryotic or prokaryotic host expressing the desired polypeptide, and can be isolated later using well-known techniques from these. Once the amino acid sequence of the desired sequence is known, those skilled in the art can readily produce the above antibodies or immunoglobulin chains by standard techniques for polypeptide production. For example, using well-known solid-phase methods, particularly using a commercially available peptide synthesizer (e.g., one manufactured by Applied Biosystems, Foster City, California), they can be synthesized according to the manufacturer's instructions. This can be done. Alternatively, the antibodies, immunoglobulin chains and antibody-like binding proteins of the present invention can be synthesized by recombinant DNA techniques well known in the art. For example, these

[0369] In particular, the present invention further relates to a method for producing the anti-CD3 antibody, anti-CD123 antibody and / or antibody-like binding protein of the present invention, the method comprising: (i) culturing a transformed host cell according to the present invention; (ii) expressing the antibody or polypeptide; and (iii) recovering the expressed antibody or polypeptide.

[0370] The anti-CD3 antibody, anti-CD123 antibody and / or antibody-like binding protein of the present invention are appropriately separated from the medium by conventional immunoglobulin purification procedures such as, for example, protein A-sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.

[0371] In a particular embodiment, the humanized chimeric anti-CD3 antibody and / or anti-CD123 of the present invention can be produced by obtaining nucleic acid sequences encoding the humanized VL and VH domains as previously described, constructing a human chimeric antibody expression vector by inserting them into an expression vector for an animal cell having genes encoding a human antibody CH and a human antibody CL, and expressing the coding sequences by introducing the expression vector into an animal cell. Similarly, humanized antibody-like binding proteins can be produced by expressing the heavy or light chain variable domains (VL and VH) of a first humanized immunoglobulin. D1 ) the heavy or light chain variable domain of the second humanized immunoglobulin N(V D2 ) for the variable domain (V D3 ) and for the heavy or light chain variable domain of said first immunoglobulin, it can be obtained by using the heavy and light chain variable domains of two humanized antibodies.

[0372] The CH domain of the human chimeric antibody or the antibody-like binding protein of the present invention may be any region belonging to a human immunoglobulin heavy chain, but is preferably of the IgG class. and any of the subclasses within the IgG class, such as IgG1, IgG2, IgG3, and IgG4. In addition, any region belonging to a human immunoglobulin light chain may be used as the CL of the human chimeric antibody or antibody-like binding protein of the present invention, and either the kappa class or the lambda class may be used.

[0373] Methods for producing humanized or chimeric antibodies include conventional recombinant DNA and gene transcription. These techniques include transfection techniques and are well known in the art (see Morrison SL. et al. (1984) and patent documents US 5,202,238; and US 5,204,244).

[0374] Production of humanized antibodies based on conventional recombinant DNA and gene transfection techniques Methods for doing so are well known in the art (see, for example, Riechmann L. et al. 1988; Neuberger MS. et al. 1985). Antibodies can be humanized using various techniques known in the art, including, for example, the techniques disclosed in application WO2009 / 032661, CDR-grafting (EP 239,400; PCT publication WO91 / 09967; US Patent 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 (US Patent No. 5,565,332). General recombinant DNA techniques for the production of such antibodies are also known (see European patent application EP 125023 and international patent application WO 96 / 02576).

[0375] The Fab of the present invention can be obtained by treating an antibody that specifically reacts with CD3 or CD123 with a protease such as papain. Also, Fab can be produced by inserting a DNA sequence encoding both chains of the Fab of the antibody into a vector for prokaryotic expression or eukaryotic expression, and introducing the vector into prokaryotic cells or eukaryotic cells (suitable for each) to express Fab.

[0376] The F(ab’)2 of the present invention can be obtained by treating an antibody that specifically reacts with CD3 or CD123 with a protease, pepsin. Also, F(ab’)2 is the Fab’ described below ​​​​It can be produced by binding via a thioether bond or a disulfide bond.

[0377] The Fab’ of the present invention can be obtained by treating F(ab’)2 that specifically reacts with CD3 or CD123 with a reducing agent such as dithiothreitol. In addition, Fab’ can be produced by inserting a DNA sequence encoding the Fab’ chain of an antibody into a vector for prokaryotic expression or a vector for eukaryotic expression, and introducing the vector into prokaryotic or eukaryotic cells (suitable for each) to effect its expression.

[0378] The scFv of the present invention can be produced by selecting previously described CDRs or VH and VL domains, constructing DNA encoding the scFv fragment, inserting the DNA into a prokaryotic or eukaryotic expression vector, and then introducing the expression vector into prokaryotic or eukaryotic cells (suitable for each) to express the scFv. To generate a humanized scFv fragment, a well-known technique called CDR grafting can be used, which involves selecting the complementarity-determining regions (CDRs) according to the present invention and grafting them onto the framework of a human scFv fragment with a known three-dimensional structure (see, for example, W098 / 45322; WO 87 / 02671; US5,859,205; US5,585,089; US4,816,567; EP0173494).

[0379] Modification of the antibody of the present invention Amino acid sequence modifications of the antibodies or antibody-like binding proteins described herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody or antibody-like binding protein. For example, a humanized antibody simply grafts only the CDRs in the FRs of the VH and VL of a human antibody in the VH and VL of an antibody derived from a non-human animal. When produced from, it is known that the antigen-binding activity can be decreased compared to the activity of the original antibody derived from a non-human animal. Some amino acid residues of the VH and VL of a non-human antibody are thought to be directly or indirectly related to the antigen-binding activity, not only in the CDR but also in the FR. Therefore, substitution of these amino acid residues with different amino acid residues from the FR of the VH and VL of a human antibody would decrease the binding activity. To solve the problem, in a human antibody with non-human CDRs grafted, attempts must be made to identify amino acid residues that are directly related to antibody binding, or amino acid residues that interact with the amino acid residues of the CDR, or amino acid residues that maintain the three-dimensional structure of the antibody and are directly related to binding to the antigen, between the amino acid sequences of the FR of the VH and VL of the human antibody. The decreased antigen-binding activity can be increased by replacing the identified amino acids with the amino acid residues of the original antibody derived from a non-human animal. The antibody-like binding protein of the present invention may contain the variable region of a humanized antibody, and thus the considerations described herein apply equally to the antibody-like binding protein of the present invention. When replacing these amino acid residues with different amino acid residues from the FR of the VH and VL of a human antibody, the binding activity will be decreased. To solve the problem, in a human antibody with non-human CDRs grafted, attempts must be made to identify amino acid residues that are directly related to antibody binding, or amino acid residues that interact with the amino acid residues of the CDR, or amino acid residues that maintain the three-dimensional structure of the antibody and are directly related to binding to the antigen, between the amino acid sequences of the FR of the VH and VL of the human antibody. In a human antibody with non-human CDRs grafted, between the amino acid sequences of the FR of the VH and VL of the human antibody, amino acid residues that are directly related to antibody binding, or amino acid residues that interact with the amino acid residues of the CDR, or amino acid residues that maintain the three-dimensional structure of the antibody and are directly related to binding to the antigen must be identified. The decreased antigen-binding activity can be increased by replacing the identified amino acids with the amino acid residues of the original antibody derived from a non-human animal. The antibody-like binding protein of the present invention may contain the variable region of a humanized antibody, and thus the considerations described herein apply equally to the antibody-like binding protein of the present invention. The antibody-like binding protein of the present invention may contain the variable region of a humanized antibody, and thus the considerations described herein apply equally to the antibody-like binding protein of the present invention. Modifications and changes can be made in the structure of the antibodies of the present invention, and in the DNA sequences encoding them, and still result in functional antibodies, antibody-like binding proteins or polypeptides having the desired characteristics.

[0380] When making changes in the amino acid sequence of a polypeptide, the hydropathic index of the amino acids can be considered. The importance of the hydrophobic amino acid index in conferring an interactive biological function on a protein is generally understood in the art. The relative hydrophobic characteristics of the amino acids contribute to the secondary structure of the resulting protein, which in turn interacts with other molecules of the protein, such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc.

[0381] When making changes in the amino acid sequence of a polypeptide, the hydropathic index of the amino acids can be considered. The importance of the hydrophobic amino acid index in conferring an interactive biological function on a protein is generally understood in the art. The relative hydrophobic characteristics of the amino acids contribute to the secondary structure of the resulting protein, which in turn contributes to the interaction of the protein with other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc. contributes to the interaction of the protein with other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc. It is accepted that it defines. Each amino acid is assigned a hydrophobicity index based on their hydrophobicity and charge, and these are as follows: 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); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lys ine (-3.9); and arginine (-4.5).

[0382] A further object of the present invention also encompasses functionally conserved variants of the polypeptides of the anti-CD3 antibody, anti-CD123 antibody and antibody-like binding protein of the present invention.

[0383] For example, certain amino acids can be substituted by other amino acids in the protein structure without much loss of activity. Since the interaction ability and properties of a protein define its biological functional activity, certain amino acid substitutions can be made in the protein sequence and, of course, in the DNA encoding that sequence, and yet a similar tan protein can be obtained. Therefore, it is contemplated that various modifications that do not much lose their biological activity can be made in the antibody sequence of the present invention, or in the corresponding DNA sequence encoding its polypeptide be carried out.

[0384] ​Certain amino acids can be substituted by other amino acids having similar hydrophobicity indices or scores and still result in a protein having similar biological activity, i.e., it is known in the art that a protein with equivalent biological function can still be obtained. Using well-established techniques such as alanine scanning approaches, it is also possible to identify all amino acids that can be substituted in the antibodies or polypeptides of the present invention without significantly losing binding to the antigen. Since such residues are not involved in antigen binding or maintaining the structure of the antibody, they can be considered neutral. One or more of these neutral positions above can be substituted with alanine or another amino acid without changing the major characteristics of the antibodies or polypeptides of the present invention.

[0385] Thus, as outlined above, amino acid substitutions generally are based on the relative similarity of the amino acid side-chain substituents, e.g., their hydrophobicity, hydrophilicity, charge, size, and the like. Examples of substitutions taking into account the various characteristics described above are well known to those of skill in the art and include: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine.

[0386] It may also be desirable to modify the anti-CD3 antibodies, anti-CD123 antibodies, and antibody-like binding proteins of the present invention with respect to effector function, e.g., to enhance or decrease antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) of the antibody. This can be achieved by introducing one or more amino acid substitutions in the Fc region of the antibody (this specification In the specification, it is also referred to as an Fc variant in the context of the antibody-like binding proteins of the present invention. Alternatively, or additionally, cysteine residues can be introduced into the Fc region, thereby enabling the formation of interchain disulfide bonds in this region. Accordingly, the resulting homodimeric antibodies can have improved or reduced internalization ability and / or increased complement-mediated cell killing and / or antibody-dependent cell cytotoxicity (ADCC) (Caron PC. et al. 1992; and Shopes B. 1992).

[0387] Another type of amino acid modification of the anti-CD3 antibody, anti-CD123 antibody and antibody-like binding proteins of the present invention may be useful for altering the original glycosylation pattern of the anti-CD3 antibody, anti-CD123 antibody and antibody-like binding proteins, i.e., by deleting one or more carbohydrate moieties found in the anti-CD3 antibody, anti-CD123 antibody and antibody-like binding proteins, and / or by adding one or more glycosylation sites not present in the anti-CD3 antibody, anti-CD123 antibody and antibody-like binding proteins. The presence of either the tripeptide sequence asparagine- X-serine, and asparagine-X-threonine [where X is any amino acid except proline] gives rise to potential glycosylation sites. The addition or deletion of glycosylation sites to the anti-CD3 antibody, anti-CD123 antibody and antibody-like binding proteins is conveniently achieved by altering the amino acid sequence to include one or more of the above tripeptide sequences (for N-linked glycosylation sites).

[0388] Another type of modification involves the removal of sequences identified either computationally or experimentally as potentially giving rise to degradation products or heterogeneity in the anti-CD3 antibody, anti-CD123 antibody and antibody-like binding protein preparations. By way of example, deamidation of asparagine and glutamine residues can occur depending on factors such as pH and surface exposure. Asparagine residues are present mainly in the sequence Asn-Gly when present in, and to a lesser extent in other dipeptide sequences such as Asn-Ala When done, it is particularly susceptible to deamidation. Such deamidation sites, especially Asn-Gly, are present in the anti-CD3 antibodies, anti-CD123 antibodies and antibody-like binding proteins of the present invention. Therefore, it may be desirable to remove the site, typically by removing one of the residues that is believed to be the cause by conservative substitution. Such substitutions in the sequence to remove one or more of the residues that are believed to be the cause are also intended to be encompassed by the present invention. Another type of covalent modification involves chemically or enzymatically coupling a glycoside to an anti-CD3 antibody, an anti-CD123 antibody and an antibody-like binding protein. These procedures are advantageous in that they do not require the production of anti-CD3 antibodies, anti-CD123 antibodies or antibody-like binding proteins in host cells having glycosylation ability for N- or O-linked glycosylation. Depending on the coupling mode used, the sugar can be coupled to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyl groups such as serine, threonine or hydroxyproline, (e) aromatic residues such as phenylalanine, tyrosine or tryptophan, or (f) the amide group of glutamine. For example, such methods are described in WO87 / 05330. Removal of any carbohydrate moieties present in an anti-CD3 antibody, an anti-CD123 antibody or an antibody-like binding protein can be achieved 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 cleavage of most or all of the sugars except the linked sugars (N-acetylglucosamine or N-acetylgalactosamine), but the antibody remains intact.

[0389] When present in the anti-CD3 antibody, anti-CD123 antibody and antibody-like binding protein or polypeptide, it is thus desirable to remove the site, typically by removing one of the residues that is believed to be the cause by conservative substitution. Such substitutions in the sequence to remove one or more of the residues that are believed to be the cause are also intended to be encompassed by the present invention. Another type of covalent modification involves chemically or enzymatically coupling a glycoside to an anti-CD3 antibody, an anti-CD123 antibody and an antibody-like binding protein. These procedures are advantageous in that they do not require the production of anti-CD3 antibodies, anti-CD123 antibodies or antibody-like binding proteins in host cells having glycosylation ability for N- or O-linked glycosylation. Depending on the coupling mode used, the sugar can be coupled to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such ...

Claims

**Claim 1** An antibody-like binding protein that specifically binds to human CD123 and one other target antigen, comprising two polypeptide chains that form two antigen-binding sites, wherein the first polypeptide chain has the formula [IV]: V D1 -L 1 -V D2 -L 2 -C L -L 5 -Fc 2 [IV] has a structure represented by, and the second polypeptide chain has the formula [III]: V D3 -L 3 -V D4 -L 4 -C H1 -Fc [III] has a structure represented by, wherein: V D1 is a variable domain of the heavy or light chain of a first immunoglobulin; V D2 is the variable domain of the heavy or light chain of a second immunoglobulin; V D3 is the variable domain of the heavy or light chain of the second immunoglobulin; V D4 is the variable domain of the heavy or light chain of the above first immunoglobulin; C L is the constant domain of the light chain of an immunoglobulin; C H1 is the constant domain of the heavy chain of an immunoglobulin; H1 ​ Fc is the immunoglobulin hinge region and CH of an immunoglobulin 2 , CH 3 and is the constant domain of an immunoglobulin heavy chain; Fc 2 is the immunoglobulin hinge region and CH of an immunoglobulin 2 , CH 3 is the immunoglobulin heavy chain constant domain; L 1 、L 2 、L 3 、L 4 and L 5 is an amino acid linker; Here, the first polypeptide and the second polypeptide form a cross light chain - heavy chain pair; Here, V D1 and V D2 are both either the variable domain of the light chain or the variable domain of the heavy chain, and when V D1 and V D2 are the variable domains of the light chain, V D3 and V D4 are both the variable domains of the heavy chain, or when V D1 and V D2 are the variable domains of the heavy chain, V D3 and V D4 are both the variable domains of the light chain, Here, the first immunoglobulin or the second immunoglobulin is (a) a heavy chain variable domain comprising CDR1-H having the amino acid sequence of SEQ ID NO: 227, CDR2-H having the amino acid sequence of SEQ ID NO: 228 or SEQ ID NO: 353 or SEQ ID NO: 279, and CDR3-H having the amino acid sequence of SEQ ID NO: 229, and a light chain variable domain comprising CDR1-L having the amino acid sequence of SEQ ID NO: 231, CDR2-L having the amino acid sequence "RDD", and CDR3-L having the amino acid sequence of SEQ ID NO: 232, or (b) a heavy chain variable domain comprising CDR1-H having the amino acid sequence of SEQ ID NO: 199 or SEQ ID NO: 273 or SEQ ID NO: 255, CDR2-H having the amino acid sequence of SEQ ID NO: 200 or SEQ ID NO: 274 or SEQ ID NO: 260, and CDR3-H having the amino acid sequence of SEQ ID NO: 201, and a light chain variable domain comprising CDR1-L having the amino acid sequence of SEQ ID NO: 203 or SEQ ID NO: 276 or SEQ ID NO: 262 or SEQ ID NO: 257, CDR2-L having the amino acid sequence "DAN" or "DAS", and CDR3-L having the amino acid sequence of SEQ ID NO: 204 or SEQ ID NO: 258 or SEQ ID NO: 263, or (c) a heavy chain variable domain comprising CDR1-H having the amino acid sequence of SEQ ID NO: 248 or SEQ ID NO: 265, CDR2-H having the amino acid sequence of SEQ ID NO: 249 or SEQ ID NO: 266, and CDR3-H having the amino acid sequence of SEQ ID NO: 250 or SEQ ID NO: 267 or SEQ ID NO: 269, and a light chain variable domain comprising CDR1-L having the amino acid sequence of SEQ ID NO: 252 or SEQ ID NO: 271, CDR2-L having the amino acid sequence "WAS", and CDR3-L having the amino acid sequence of SEQ ID NO: 253, or (d) A heavy chain variable domain comprising CDR1-H having the amino acid sequence of SEQ ID NO: 206, CDR2-H having the amino acid sequence of SEQ ID NO: 207, and CDR3-H having the amino acid sequence of SEQ ID NO: 208, and a light chain variable domain comprising CDR1-L having the amino acid sequence of SEQ ID NO: 210, CDR2-L having the amino acid sequence "ETS", and CDR3-L having the amino acid sequence of SEQ ID NO: 211, or (e) A heavy chain variable domain comprising CDR1-H having the amino acid sequence of SEQ ID NO: 213, CDR2-H having the amino acid sequence of SEQ ID NO: 214, and CDR3-H having the amino acid sequence of SEQ ID NO: 215, and a light chain variable domain comprising CDR1-L having the amino acid sequence of SEQ ID NO: 217, CDR2-L having the amino acid sequence "NTN", and CDR3-L having the amino acid sequence of SEQ ID NO: 218, or (f) A heavy chain variable domain comprising CDR1-H having the amino acid sequence of SEQ ID NO: 220, CDR2-H having the amino acid sequence of SEQ ID NO: 221, and CDR3-H having the amino acid sequence of SEQ ID NO: 222, and a light chain variable domain comprising CDR1-L having the amino acid sequence of SEQ ID NO: 224, CDR2-L having the amino acid sequence "RVS", and CDR3-L having the amino acid sequence of SEQ ID NO: 225, or (g) A heavy chain variable domain comprising CDR1-H having the amino acid sequence of SEQ ID NO: 234, CDR2-H having the amino acid sequence of SEQ ID NO: 235, and CDR3-H having the amino acid sequence of SEQ ID NO: 236, and a light chain variable domain comprising CDR1-L having the amino acid sequence of SEQ ID NO: 238, CDR2-L having the amino acid sequence "GAS", and CDR3-L having the amino acid sequence of SEQ ID NO: 239, or (h) A heavy chain variable domain comprising CDR1-H having the amino acid sequence of SEQ ID NO: 241, CDR2-H having the amino acid sequence of SEQ ID NO: 242, and CDR3-H having the amino acid sequence of SEQ ID NO: 243, and a light chain variable domain comprising CDR1-L having the amino acid sequence of SEQ ID NO: 245, CDR2-L having the amino acid sequence "YAS", and CDR3-L having the amino acid sequence of SEQ ID NO: 246, or (i) A heavy chain variable domain comprising CDR1-H having the amino acid sequence of SEQ ID NO: 381, CDR2-H having the amino acid sequence of SEQ ID NO: 384, and CDR3-H having the amino acid sequence of SEQ ID NO: 382, and A light chain variable domain comprising CDR1-L having the amino acid sequence of SEQ ID NO: 378, CDR2-L having the amino acid sequence "WAS", and CDR3-L having the amino acid sequence of SEQ ID NO: 379, An antibody-like binding protein that is an anti-CD123 antibody comprising the same. **Claim 2** An antibody-like binding protein that specifically binds to human CD123 and one other target antigen, comprising three polypeptide chains that form two antigen-binding sites, wherein the first polypeptide has the formula [I]: V D1 -L 1 -V D2 -L 2 -C L [I] has a structure represented by, and the second polypeptide has the formula [III]: V D3 -L 3 -V D4 -L 4 -C H1 -F c [III] has a structure represented by, The third polypeptide Fc 3 is the immunoglobulin hinge region and CH of an immunoglobulin 2 , CH 3 is the immunoglobulin heavy chain constant domain; wherein V D1 is the variable domain of the heavy or light chain of a first immunoglobulin; V D2 is the variable domain of the heavy or light chain of a second immunoglobulin; V D3 is the variable domain of the heavy or light chain of the second immunoglobulin; V D4 is the variable domain of the heavy or light chain of the above first immunoglobulin; C L is the constant domain of the light chain of an immunoglobulin; C H1 is the constant domain of the heavy chain of an immunoglobulin; H1 ​ Fc is the immunoglobulin hinge region and CH of an immunoglobulin 2 , CH 3 and is the constant domain of an immunoglobulin heavy chain; L 1 、 L 2 、 L 3 、 and L 4 is an amino acid linker; wherein the first polypeptide and the second polypeptide form an interchanged light chain-heavy chain pair; Here, V D1 and V D2 are both either the variable domain of the light chain or the variable domain of the heavy chain, and when V D1 and V D2 are the variable domains of the light chain, V D3 and V D4 are both the variable domains of the heavy chain, or when V D1 and V D2 are the variable domains of the heavy chain, V D3 and V D4 are both the variable domains of the light chain; wherein the second polypeptide heterodimerizes with a third polypeptide through its Fc domain, wherein the first immunoglobulin or the second immunoglobulin is (a) a heavy chain variable domain comprising CDR1-H having the amino acid sequence of SEQ ID NO: 227, CDR2-H having the amino acid sequence of SEQ ID NO: 228 or SEQ ID NO: 353 or SEQ ID NO: 279, and CDR3-H having the amino acid sequence of SEQ ID NO: 229, and a light chain variable domain comprising CDR1-L having the amino acid sequence of SEQ ID NO: 231, CDR2-L having the amino acid sequence "RDD", and CDR3-L having the amino acid sequence of SEQ ID NO: 232, or (b) a heavy chain variable domain comprising CDR1-H having the amino acid sequence of SEQ ID NO: 199 or SEQ ID NO: 273 or SEQ ID NO: 255, CDR2-H having the amino acid sequence of SEQ ID NO: 200 or SEQ ID NO: 274 or SEQ ID NO: 260, and CDR3-H having the amino acid sequence of SEQ ID NO: 201, and a light chain variable domain comprising CDR1-L having the amino acid sequence of SEQ ID NO: 203 or SEQ ID NO: 276 or SEQ ID NO: 262 or SEQ ID NO: 257, CDR2-L having the amino acid sequence "DAN" or "DAS", and CDR3-L having the amino acid sequence of SEQ ID NO: 204 or SEQ ID NO: 258 or SEQ ID NO: 263, or (c) a heavy chain variable domain comprising CDR1-H having the amino acid sequence of SEQ ID NO: 248 or SEQ ID NO: 265, CDR2-H having the amino acid sequence of SEQ ID NO: 249 or SEQ ID NO: 266, and CDR3-H having the amino acid sequence of SEQ ID NO: 250 or SEQ ID NO: 267 or SEQ ID NO: 269, and A light chain variable domain comprising CDR1-L having the amino acid sequence of SEQ ID NO: 252 or SEQ ID NO: 271, CDR2-L having the amino acid sequence "WAS", and CDR3-L having the amino acid sequence of SEQ ID NO: 253, or (d) A heavy chain variable domain comprising CDR1-H having the amino acid sequence of SEQ ID NO: 206, CDR2-H having the amino acid sequence of SEQ ID NO: 207, and CDR3-H having the amino acid sequence of SEQ ID NO: 208, and A light chain variable domain comprising CDR1-L having the amino acid sequence of SEQ ID NO: 210, CDR2-L having the amino acid sequence "ETS", and CDR3-L having the amino acid sequence of SEQ ID NO: 211, or (e) A heavy chain variable domain comprising CDR1-H having the amino acid sequence of SEQ ID NO: 213, CDR2-H having the amino acid sequence of SEQ ID NO: 214, and CDR3-H having the amino acid sequence of SEQ ID NO: 215, and A light chain variable domain comprising CDR1-L having the amino acid sequence of SEQ ID NO: 217, CDR2-L having the amino acid sequence "NTN", and CDR3-L having the amino acid sequence of SEQ ID NO: 218, or (f) A heavy chain variable domain comprising CDR1-H having the amino acid sequence of SEQ ID NO: 220, CDR2-H having the amino acid sequence of SEQ ID NO: 221, and CDR3-H having the amino acid sequence of SEQ ID NO: 222, and A light chain variable domain comprising CDR1-L having the amino acid sequence of SEQ ID NO: 224, CDR2-L having the amino acid sequence "RVS", and CDR3-L having the amino acid sequence of SEQ ID NO: 225, or (g) A heavy chain variable domain comprising CDR1-H having the amino acid sequence of SEQ ID NO: 234, CDR2-H having the amino acid sequence of SEQ ID NO: 235, and CDR3-H having the amino acid sequence of SEQ ID NO: 236, and A light chain variable domain comprising CDR1-L having the amino acid sequence of SEQ ID NO: 238, CDR2-L having the amino acid sequence "GAS", and CDR3-L having the amino acid sequence of SEQ ID NO: 239, or (h) A heavy chain variable domain comprising CDR1-H having the amino acid sequence of SEQ ID NO: 241, CDR2-H having the amino acid sequence of SEQ ID NO: 242, and CDR3-H having the amino acid sequence of SEQ ID NO: 243, and A light chain variable domain comprising CDR1-L having the amino acid sequence of SEQ ID NO: 245, CDR2-L having the amino acid sequence "YAS", and CDR3-L having the amino acid sequence of SEQ ID NO: 246, or (i) A heavy chain variable domain comprising CDR1-H having the amino acid sequence of SEQ ID NO: 381, CDR2-H having the amino acid sequence of SEQ ID NO: 384, and CDR3-H having the amino acid sequence of SEQ ID NO: 382, and A light chain variable domain comprising CDR1-L having the amino acid sequence of SEQ ID NO: 378, CDR2-L having the amino acid sequence "WAS", and CDR3-L having the amino acid sequence of SEQ ID NO: 379, An antibody-like binding protein which is an anti-CD123 antibody comprising the same.

3. The antibody-like binding protein according to any one of Claims 1 or 2, wherein the first and second immunoglobulins are IgG immunoglobulins.

4. C L is the constant domain of the light chain of IgG immunoglobulin, and / or C H1 is IgG immunity The C of globulin H1 The antibody-like binding protein according to any one of claims 1 or 2, which is a heavy chain constant domain.

5. (a) The length of L 3 is at least twice the length of L 1 ; or (b) L 4 The length of 2 is at least twice the length of The antibody-like binding protein according to any one of Claims 1 or 2.

6. (a) The length of L 1 is at least twice the length of L 3 ; or (b) L 2 whose length is L 4 is at least twice the length of The antibody-like binding protein according to any one of Claims 1 or 2.

7. 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 which is 1 to 3 amino acid residues in length, The antibody-like binding protein according to any one of Claims 1 or 2.

8. 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 which is 1 to 2 amino acid residues in length, The antibody-like binding protein according to Claim 7.

9. L 1 is 7 amino acid residues in length; L 2 is 5 amino acid residues in length; L 3 is one amino acid residue in length; and L 4 which is two amino acid residues in length, The antibody-like binding protein according to Claim 8.

10. 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 which is 2 to 15 amino acid residues in length, The antibody-like binding protein according to any one of Claims 1 or 2.

11. L 1 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 which is 2 to 12 amino acid residues in length, The antibody-like binding protein according to Claim 10.

12. L 1 is one amino acid residue in length; L 2 is two amino acid residues in length; L 3 is 7 amino acid residues in length; and L 4 which is 5 amino acid residues in length, The antibody-like binding protein according to Claim 11.

13. L 1 、 L 2 、 L 3 、 and L 4 The antibody-like binding protein according to any one of claims 1 or 2, wherein at least one of the linkers selected from the group consisting of is included with at least one cysteine residue.

14. (a) L 1 has a length of 0 amino acid residues, and L 3 has a length of at least 1 amino acid residue; (b) L 3 has a length of 0 amino acid residues, and L 1 has a length of at least 1 amino acid residue; or (c) L 4 is 0 amino acid residues in length and L 2 is at least 1 amino acid residue in length; The antibody-like binding protein according to any one of Claims 1 or 2.

15. L 1 is 0 amino acid residues in length, and L 3 is 2 amino acid residues in length or more, the antibody-like binding protein according to claim 14.

16. L 4 is 0 amino acid residues in length, and L 2 is 3 or more amino acid residues in length, the antibody-like binding protein according to claim 14.

17. L 5 The antibody-like binding protein according to claim 1, wherein L has a length of 0 to 10 amino acid residues.

18. Fc and Fc 3 wherein one of the C H3 domains has the amino acid mutations Y349C, T366S , including L368A and Y407V, and the other C of Fc and Fc 3 domain H3 is The antibody-like binding protein according to Claim 2, comprising amino acid mutations S354C and T366W.

19. A pharmaceutical composition comprising the antibody-like binding protein according to any one of Claims 1 to 18 and a pharmaceutically acceptable carrier.

20. An isolated nucleic acid comprising a sequence encoding the antibody-like binding protein according to any one of Claims 1 to 18.

21. An expression vector comprising the nucleic acid molecule according to Claim 20.

22. A host cell transfected, infected or transformed with the nucleic acid according to Claim 20.

23. A host cell transfected, infected or transformed with the expression vector according to Claim 21.

24. A kit comprising at least one antibody-like binding protein according to any one of claims 1 to 18.

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