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

Abstract

The present invention relates to anti-CD3 antibodies, anti-CD123 antibodies, and bispecific antibodies that specifically bind to CD3 and / or CD123, and more particularly to antibody-like binding proteins that specifically bind to CD3 and specifically bind to at least one other antigen such as CD123. The present invention also relates to antibody-like binding proteins that specifically bind to CD123 and specifically bind to at least one other antigen. The present invention further relates to anti-CD3 antibodies and anti-CD123 antibodies. 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 in treating cancer. The present invention further relates to isolated nucleic acids, vectors, and host cells comprising sequences encoding the antibody-like binding proteins, anti-CD3, or anti-CD123 antibodies, and the use of the anti-CD123 antibodies as diagnostic tools.

Description

[0001] This divisional application of the present invention is based on the patent application with the filing date of January 22, 2016, application number 201680017477.8 (international application number PCT / EP2016 / 051386), and title "Anti-CD3 Antibodies, Anti-CD123 Antibodies, and Bispecific Antibodies Specifically Binding to CD3 and / or CD123". Technical Field

[0002] The present invention relates to antibody-like binding proteins that specifically bind to CD3 and specifically bind to at least one other antigen, such as CD123. The present invention further relates to anti-CD3 antibodies and anti-CD123 antibodies. The present invention also relates to antibody-like binding proteins that specifically bind to CD123 and specifically bind to at least one other 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 in the treatment of cancer. The present invention further relates to isolated nucleic acids, vectors and host cells comprising sequences encoding the antibody-like binding proteins, anti-CD3 or anti-CD123 antibodies and the use of the anti-CD123 antibodies as diagnostic tools. Background Art

[0003] The development of first-generation bispecific antibodies has been more than 20 years. Since then, multiple clinical studies have tested bispecific antibodies engineered to target cancer cell surface antigens. Such anti-cancer fusion proteins contain two or more functional domains that localize immune effector cells near the targeted cancer cells to obtain anti-cancer activity.

[0004] With the development of bispecific antibody technology, another group of fusion proteins called bispecific T-cell engagers (BiTE) are generated only by using a flexible linker to connect two antibody single-chain variable regions (scFv) (excluding the Fc amino acid segment), one scFv binds to the targeted cell and the other binds to CD3 on the surface of T cells. A BiTE with CD19xCD3 bispecific binding activity, blinatumomab, has shown promising results in a phase II clinical trial for patients with minimal residual disease in B-line acute lymphoblastic leukemia.

[0005] CD123 (interleukin-3 receptor alpha chain IL-3Rα) is a tumor antigen overexpressed in a variety of hematological malignancies. A large number of AML blasts express surface CD123 and this expression does not vary with the subtype of AML. Higher expression of CD123 on AML at diagnosis has been reported to be associated with a poor prognosis. CD123 has been reported to be expressed on leukemia stem cells (LSC). Increasing evidence suggests that AML originates from leukemia stem cells that have always shown to be quiescent and relatively resistant to DNA-damaging chemotherapy.

[0006] Increased CD123 expression on LSCs compared to hematopoietic stem cells (HSCs) thus provides a therapeutic opportunity for targeting.

[0007] The monoclonal antibody (MAb) 7G3 raised against CD123 has previously been shown to inhibit IL-3-mediated proliferation and activation in both leukemia cell lines and primary cells (U.S. Patent No. 6,177,078). However, whether targeting CD123 can functionally ablate AML-LSCs is unclear.

[0008] The use of CD123xCD3 antibody-like binding proteins results in tumor cell killing, as shown by the inventors herein.

[0009] The concept of generating bispecific antibody-like binding proteins with CD123xCD3 bispecific binding activity has been proposed and described in International Patent Application WO2013 / 173820.

[0010] In addition, molecules of a CD123 x CD3 bispecific affinity retargeting (DART) bispecific antibody from MacroGenics entered Phase I clinical trials in 2014.

[0011] However, as shown by the inventors, such molecules of the CD123xCD3 bispecific affinity retargeting (DART) bispecific antibody from MacroGenics, for example, have 82% CD4+-expressing T-cell activation and 83% CD8+-expressing T-cell activation in the absence of target cells. Inappropriate activation of T cells can lead to severe side effects such as cytokine release syndrome. Cytokine release syndrome refers to the release of cytokines by activated T cells that produce a systemic inflammatory response similar to that found in severe infections and is characterized by hypotension, fever, and rigors. Deaths due to cytokine release syndrome have been reported, for example, for OKT3.

[0012] Accordingly, despite these advances in bispecific antibody technology, there is a need for other cancer therapies, particularly those that effectively target and kill cancer cells directly or indirectly.

[0013] The inventors have successfully generated, screened, and selected specific rat anti-CD3 antibodies that exhibit high affinity for both human and cynomolgus macaque (Macaca fascicularis) CD3 proteins.

[0014] The inventors have developed antibody-like binding proteins that are bi- and immunospecific for antigen CD3 and at least one other target antigen. In one example, to demonstrate the use of these anti-CD3 antibodies in the generation of bispecific antibody-like binding proteins, the inventors generated anti-CD3 / anti-CD123 antibody-like binding proteins and demonstrated their therapeutic use. Those bispecific anti-CD3 / anti-CD123 antibody-like binding proteins have low T-cell activation, as observed for anti-CD3 antibodies alone. However, once target cells expressing CD12, such as THP-1 cells, are present, the bispecific anti-CD3 / anti-CD123 antibody-like binding proteins exhibit high T-cell activation. Accordingly, the anti-CD3 antibodies of the invention as defined above are particularly effective for the preparation of the antibody-like binding proteins of the invention. SUMMARY OF THE INVENTION

[0015] Specifically, the present invention relates to the following:

[0016] 1. An isolated antibody that binds to the extracellular domain of the human CD3ε protein, comprising:

[0017] a) A heavy chain variable domain that contains CDR1-H consisting of the sequence GFX1X2X3X4AW (SEQ ID NO: 331), where X1 is T or S, X2 is F or V, X3 is S or T and X4 is N, K, L or Y, or any combination thereof; and

[0018] CDR2-H consisting of the sequence IKX1X2X3NX4YX5T (SEQ ID NO: 332), where X1 is A or D, X2 is K or R, X3 is S or A, X4 is N or S and X5 is A or E, or any combination thereof; and

[0019] CDR3-H consisting of the sequence TWRHYYSSHTMDA (SEQ ID NO: 69) or RALTYYGYKRDAMDG (SEQ ID NO: 129) or RX1X2X3YX4X5X6X7X8X9X 10 X 11 DX 12 (SEQ ID NO: 333), where X1 is Y, G or A, X2 is V, T or L, X3 is H, N, Y or Q, X4 is G, R or A, X5 is F or V or no amino acid, X6 is R or no amino acid, X7 is F, S or I or no amino acid, X8 is F, L, N, M, Y, S, A or G, X9 is Y, A, K, S, N, T, F or L, X 10 is A, P, G or T, X 11 is M, L, F or S and X 12 is A, V or Y, or any combination thereof, and

[0020] A light chain variable domain containing CDR1-L consisting of the sequence QX1LX2HX3NGX4TY (SEQ ID NO: 334), where X1 is R or S, X2 is V or E, X3 is N, D or T and X4 is N or Y, or any combination thereof; and

[0021] CDR2-L consisting of the sequence 'KVS'; and

[0022] CDR3-L consisting of the sequence GQGX1X2YPFT (SEQ ID NO: 335), where X1 is T, A or S and X2 is H, E or Q, or any combination thereof; or

[0023] a) A heavy chain variable domain containing CDR1-H of the sequence SEQ ID NO: 30 or a sequence differing from SEQ ID NO: 30 by one amino acid substitution; CDR2-H of the sequence 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 SEQ ID NO: 32 or a sequence differing from SEQ ID NO: 32 by one amino acid substitution; and

[0024] A light chain variable domain containing CDR1-L of the sequence SEQ ID NO: 34 or a sequence differing from 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 SEQ ID NO: 35 or a sequence differing from SEQ ID NO: 35 by amino acid substitution; or

[0025] b) A heavy chain variable domain containing CDR1-H of the sequence SEQ ID NO: 50 or a sequence differing from SEQ ID NO: 50 by one amino acid substitution; CDR2-H of the sequence SEQ ID NO: 51 or a sequence differing from SEQ ID NO: 51 by one or more amino acid substitutions; CDR3-H of the sequence SEQ ID NO: 52 or a sequence differing from SEQ ID NO: 52 by one amino acid substitution; and

[0026] A light chain variable domain containing CDR1-L of the sequence SEQ ID NO: 54 or a sequence differing from SEQ ID NO: 54 by one amino acid substitution; 'NAN' or a sequence differing from the sequence 'NAN' by one amino acid substitution and CDR3-L of the sequence SEQ ID NO: 55 or a sequence differing from SEQ ID NO: 55 by one amino acid substitution; or

[0027] c) A heavy chain variable domain comprising CDR1-H having the sequence SEQ ID NO: 90 or a sequence differing from SEQ ID NO: 90 by one amino acid substitution; CDR2-H having the sequence SEQ ID NO: 91 or a sequence differing from SEQ ID NO: 91 by one or more amino acid substitutions; CDR3-H having the sequence SEQ ID NO: 32 or a sequence differing from SEQ ID NO: 32 by one amino acid substitution; and

[0028] a light chain variable domain comprising CDR1-L having the sequence SEQ ID NO: 93 or a sequence differing from SEQ ID NO: 93 by one amino acid substitution; CDR2-L having the sequence 'GAS' or a sequence differing from 'GAS' by one amino acid substitution; and CDR3-L having the sequence SEQ ID NO: 94 or a sequence differing from SEQ ID NO: 94 by one amino acid substitution; or

[0029] d) A heavy chain variable domain comprising CDR1-H having the sequence SEQ ID NO: 96 or a sequence differing from SEQ ID NO: 96 by one amino acid substitution; CDR2-H having the sequence SEQ ID NO: 97 or a sequence differing from SEQ ID NO: 97 by one or more amino acid substitutions; CDR3-H having the sequence SEQ ID NO: 98 and differing from SEQ ID NO: 98 by one amino acid substitution; and

[0030] a light chain variable domain comprising CDR1-L having the sequence SEQ ID NO: 100 or a sequence differing from SEQ ID NO: 100 by one amino acid substitution; CDR2-L having the sequence 'NTN' or a sequence differing from the sequence 'NTN' by one amino acid substitution; and CDR3-L having the sequence SEQ ID NO: 101 or a sequence differing from SEQ ID NO: 101 by one amino acid substitution; or

[0031] e) A heavy chain variable domain comprising CDR1-H having the sequence SEQ ID NO: 103 or a sequence differing from SEQ ID NO: 103 by one amino acid substitution; CDR2-H having the sequence SEQ ID NO: 104 or a sequence differing from SEQ ID NO: 104 by one or more amino acid substitutions; CDR3-H having the sequence SEQ ID NO: 105 or a sequence differing from SEQ ID NO: 105 by one amino acid substitution; and

[0032] A light chain variable domain comprising CDR1-L having the sequence SEQ ID NO: 10 or a sequence differing from SEQ ID NO: 10 by one amino acid substitution; CDR2-L having the sequence 'KVS' or a sequence differing from the sequence 'KVS' by one amino acid substitution; and CDR3-L having the sequence SEQ ID NO: 11 or a sequence differing from SEQ ID NO: 11 by one amino acid substitution; or

[0033] f) A heavy chain variable domain comprising CDR1-H having the sequence SEQ ID NO: 116 or a sequence differing from SEQ ID NO: 116 by one amino acid substitution; CDR2-H having the sequence SEQ ID NO: 117 or a sequence differing from SEQ ID NO: 117 by one or more amino acid substitutions; CDR3-H having the sequence SEQ ID NO: 118 or a sequence differing from SEQ ID NO: 118 by one amino acid substitution; and

[0034] A light chain variable domain comprising CDR1-L having the sequence SEQ ID NO: 100 or a sequence differing from SEQ ID NO: 100 by one amino acid substitution; CDR2-L having the sequence 'VTN' or a sequence differing from the sequence 'VTN' by one amino acid substitution; and CDR3-L having the sequence SEQ ID NO: 120 or a sequence differing from SEQ ID NO: 120 by one amino acid substitution; or

[0035] g) A heavy chain variable domain comprising CDR1-H having the sequence SEQ ID NO: 122 or a sequence differing from SEQ ID NO: 122 by one amino acid substitution; CDR2-H having the sequence SEQ ID NO: 123 or a sequence differing from SEQ ID NO: 123 by one or more amino acid substitutions; CDR3-H having the sequence SEQ ID NO: 124 or a sequence differing from SEQ ID NO: 124 by one amino acid substitution; and

[0036] A light chain variable domain comprising CDR1-L having the sequence SEQ ID NO: 126 or a sequence differing from SEQ ID NO: 126 by one amino acid substitution; CDR2-L having the sequence 'RDD' or a sequence differing from the sequence 'RDD' by one amino acid substitution; and CDR3-L having the sequence SEQ ID NO: 127 or a sequence differing from SEQ ID NO: 127 by one amino acid substitution.

[0037] 2. The isolated antibody according to item 1, comprising:

[0038] a) A heavy chain variable domain that contains CDR1-H of SEQ ID NO: 6, CDR2-H of SEQ ID NO: 7, CDR3-H of SEQ ID NO: 8, and

[0039] a light chain variable domain that contains CDR1-L of SEQ ID NO: 10 or SEQ ID NO: 142, CDR2-L of the sequence 'KVS', and CDR3-L of SEQ ID NO: 11; or

[0040] b) A heavy chain variable domain that contains CDR1-H of SEQ ID NO: 13, CDR2-H of SEQ ID NO: 14, CDR3-H of SEQ ID NO: 15, and

[0041] a light chain variable domain that contains CDR1-L of SEQ ID NO: 17 or SEQ ID NO: 184, CDR2-L of the sequence 'KVS', and CDR3-L of SEQ ID NO: 11; or

[0042] c) A heavy chain variable domain that contains CDR1-H of SEQ ID NO: 13, CDR2-H of SEQ ID NO: 19, CDR3-H of SEQ ID NO: 20, and

[0043] a light chain variable domain that contains CDR1-L of SEQ ID NO: 22, CDR2-L of the sequence 'KVS', and CDR3-L of SEQ ID NO: 11; or

[0044] d) A heavy chain variable domain that contains CDR1-H of SEQ ID NO: 24, CDR2-H of SEQ ID NO: 19, CDR3-H of SEQ ID NO: 25, and

[0045] a light chain variable domain that contains CDR1-L of SEQ ID NO: 27, CDR2-L of the sequence 'KVS', and CDR3-L of SEQ ID NO: 28; or

[0046] e) A heavy chain variable domain that contains CDR1-H of SEQ ID NO: 30, CDR2-H of SEQ ID NO: 31, CDR3-H of SEQ ID NO: 32, and

[0047] A light chain variable domain comprising CDR1-L of SEQ ID NO: 34, CDR2-L of the sequence 'RDD', and CDR3-L of SEQ ID NO: 35; or

[0048] f) A heavy chain variable domain comprising CDR1-H of SEQ ID NO: 13, CDR2-H of SEQ ID NO: 37, CDR3-H of SEQ ID NO: 38, and

[0049] A light chain variable domain comprising CDR1-L of SEQ ID NO: 10, CDR2-L of the sequence 'KVS', and CDR3-L of SEQ ID NO: 28; or

[0050] g) A heavy chain variable domain comprising CDR1-H of SEQ ID NO: 13, CDR2-H of SEQ ID NO: 37, CDR3-H of SEQ ID NO: 41, and

[0051] A light chain variable domain comprising CDR1-L of SEQ ID NO: 17, CDR2-L of the sequence 'KVS', and CDR3-L of SEQ ID NO: 11; or

[0052] h) A heavy chain variable domain comprising CDR1-H of SEQ ID NO: 13, CDR2-H of SEQ ID NO: 37, CDR3-H of SEQ ID NO: 44, and

[0053] A light chain variable domain comprising CDR1-L of SEQ ID NO: 10, CDR2-L of the sequence 'KVS', and CDR3-L of SEQ ID NO: 11; or

[0054] i) A heavy chain variable domain comprising CDR1-H of SEQ ID NO: 13, CDR2-H of SEQ ID NO: 37, CDR3-H of SEQ ID NO: 47, and

[0055] A light chain variable domain comprising CDR1-L of SEQ ID NO: 10, CDR2-L of the sequence 'KVS', and CDR3-L of SEQ ID NO: 11; or

[0056] j) A heavy chain variable domain comprising CDR1-H of SEQ ID NO: 50, CDR2-H of SEQ ID NO: 51, CDR3-H of SEQ ID NO: 52, and

[0057] A light chain variable domain comprising CDR1-L of SEQ ID NO: 54, CDR2-L of the sequence 'NAN', and CDR3-L of SEQ ID NO: 55; or

[0058] k) A heavy chain variable domain comprising CDR1-H of SEQ ID NO: 57, CDR2-H of SEQ ID NO: 37, CDR3-H of SEQ ID NO: 58, and

[0059] A light chain variable domain comprising CDR1-L of SEQ ID NO: 10, CDR2-L of the sequence 'KVS', and CDR3-L of SEQ ID NO: 28; or

[0060] l) A heavy chain variable domain comprising CDR1-H of SEQ ID NO: 13, CDR2-H of SEQ ID NO: 37, CDR3-H of SEQ ID NO: 61, and

[0061] A light chain variable domain comprising CDR1-L of SEQ ID NO: 10, CDR2-L of the sequence 'KVS', and CDR3-L of SEQ ID NO: 11; or

[0062] m) A heavy chain variable domain comprising CDR1-H of SEQ ID NO: 64, CDR2-H of SEQ ID NO: 65, CDR3-H of SEQ ID NO: 47, and

[0063] A light chain variable domain comprising CDR1-L of SEQ ID NO: 67, CDR2-L of the sequence 'KVS', and CDR3-L of SEQ ID NO: 28; or

[0064] n) A heavy chain variable domain comprising CDR1-H of SEQ ID NO: 13, CDR2-H of SEQ ID NO: 37, CDR3-H of SEQ ID NO: 69, and

[0065] A light chain variable domain comprising CDR1-L of SEQ ID NO: 10, CDR2-L of the sequence 'KVS', and CDR3-L of SEQ ID NO: 71; or

[0066] o) A heavy chain variable domain that contains CDR1-H of SEQ ID NO: 13, CDR2-H of SEQ ID NO: 37, CDR3-H of SEQ ID NO: 84, and

[0067] A light chain variable domain that contains CDR1-L of SEQ ID NO: 17, CDR2-L of the sequence 'KVS', and CDR3-L of SEQ ID NO: 11; or

[0068] p) A heavy chain variable domain that contains CDR1-H of SEQ ID NO: 75, CDR2-H of SEQ ID NO: 76, CDR3-H of SEQ ID NO: 77, and

[0069] A light chain variable domain that contains CDR1-L of SEQ ID NO: 10, CDR2-L of the sequence 'KVS', and CDR3-L of SEQ ID NO: 11; or

[0070] q) A heavy chain variable domain that contains CDR1-H of SEQ ID NO: 80, CDR2-H of SEQ ID NO: 76, CDR3-H of SEQ ID NO: 81, and

[0071] A light chain variable domain that contains CDR1-L of SEQ ID NO: 10, CDR2-L of the sequence 'KVS', and CDR3-L of SEQ ID NO: 11; or

[0072] r) A heavy chain variable domain that contains CDR1-H of SEQ ID NO: 13, CDR2-H of SEQ ID NO: 37, CDR3-H of SEQ ID NO: 84, and

[0073] A light chain variable domain that contains CDR1-L of SEQ ID NO: 10, CDR2-L of the sequence 'KVS', and CDR3-L of SEQ ID NO: 11; or

[0074] s) A heavy chain variable domain that contains CDR1-H of SEQ ID NO: 13, CDR2-H of SEQ ID NO: 37, CDR3-H of SEQ ID NO: 47, and

[0075] A light chain variable domain that contains CDR1-L of SEQ ID NO: 10, CDR2-L of the sequence 'KVS', and CDR3-L of SEQ ID NO: 88; or

[0076] (t) A heavy chain variable domain that contains CDR1-H of SEQ ID NO: 90, CDR2-H of SEQ ID NO: 91, CDR3-H of SEQ ID NO: 32, and

[0077] A light chain variable domain that contains CDR1-L of SEQ ID NO: 93, CDR2-L of the sequence 'GAS', and CDR3-L of SEQ ID NO: 94; or

[0078] (u) A heavy chain variable domain that contains CDR1-H of SEQ ID NO: 96, CDR2-H of SEQ ID NO: 97, CDR3-H of SEQ ID NO: 98, and

[0079] A light chain variable domain that contains CDR1-L of SEQ ID NO: 100, CDR2-L of the sequence 'NTN', and CDR3-L of SEQ ID NO: 101; or

[0080] v) A heavy chain variable domain that contains CDR1-H of SEQ ID NO: 103, CDR2-H of SEQ ID NO: 104, CDR3-H of SEQ ID NO: 105, and

[0081] A light chain variable domain that contains CDR1-L of SEQ ID NO: 10, CDR2-L of the sequence 'KVS', and CDR3-L of SEQ ID NO: 11; or

[0082] w) A heavy chain variable domain that contains CDR1-H of SEQ ID NO: 80, CDR2-H of SEQ ID NO: 19, CDR3-H of SEQ ID NO: 108, and

[0083] A light chain variable domain that contains CDR1-L of SEQ ID NO: 10, CDR2-L of the sequence 'KVS', and CDR3-L of SEQ ID NO: 11; or

[0084] x) A heavy chain variable domain that contains CDR1-H of SEQ ID NO: 13, CDR2-H of SEQ ID NO: 37, CDR3-H of SEQ ID NO: 111, and

[0085] A light chain variable domain comprising CDR1-L of SEQ ID NO: 113, CDR2-L of the sequence 'KVS', and CDR3-L of SEQ ID NO: 114; or

[0086] y) A heavy chain variable domain comprising CDR1-H of SEQ ID NO: 116, CDR2-H of SEQ ID NO: 117, CDR3-H of SEQ ID NO: 118, and

[0087] A light chain variable domain comprising CDR1-L of SEQ ID NO: 100, CDR2-L of the sequence 'VTN', and CDR3-L of SEQ ID NO: 120; or

[0088] z) A heavy chain variable domain comprising CDR1-H of SEQ ID NO: 122, CDR2-H of SEQ ID NO: 123, CDR3-H of SEQ ID NO: 124, and

[0089] A light chain variable domain comprising CDR1-L of SEQ ID NO: 126, CDR2-L of the sequence 'RDD', and CDR3-L of SEQ ID NO: 127; or

[0090] aa) A heavy chain variable domain comprising CDR1-H of SEQ ID NO: 13, CDR2-H of SEQ ID NO: 19, CDR3-H of SEQ ID NO: 129, and

[0091] A light chain variable domain comprising CDR1-L of SEQ ID NO: 10, CDR2-L of the sequence 'KVS', and CDR3-L of SEQ ID NO: 11; or

[0092] bb) A heavy chain variable domain comprising CDR1-H of SEQ ID NO: 103, CDR2-H of SEQ ID NO: 104, CDR3-H of SEQ ID NO: 105; and

[0093] A light chain variable domain comprising CDR1-L of SEQ ID NO: 133, CDR2-L of the sequence 'LVS', and CDR3-L of SEQ ID NO: 134.

[0094] 3. An antibody-like binding protein that specifically binds to human CD3ε, comprising two polypeptide chains that form two antigen-binding sites, wherein the first polypeptide has the structure shown by formula [I]:

[0095] V D1 -L1-V D2 -L2-C L [I]

[0096] and the second polypeptide has a structure represented by formula [II]:

[0097] V D3 -L3-V D4 -L4-C H1 [II]

[0098] Wherein:

[0099] V D1 is the variable domain of the first immunoglobulin heavy or light chain;

[0100] V D2 is the variable domain of the second immunoglobulin heavy or light chain;

[0101] V D3 is the variable domain of the second immunoglobulin heavy or light chain;

[0102] V D4 is the variable domain of the first immunoglobulin heavy or light chain;

[0103] C L is the light chain constant domain of the immunoglobulin;

[0104] C H1 is the C H1 heavy chain constant domain of the immunoglobulin;

[0105] L1, L2, L3 and L4 are amino acid linkers;

[0106] and wherein the first and second polypeptides form a cross light chain - heavy chain pair, and

[0107] wherein said V D1 and V D4 , or V D2 and V D3 comprise the heavy chain variable domain and the light chain variable domain of the antibody as defined in item 1.

[0108] 4. The antibody-like binding protein according to item 3, wherein the polypeptide represented by formula [II] further comprises an F c domain.

[0109] 5. The antibody-like binding protein according to item 3 or 4, which comprises two polypeptide chains forming two antigen binding sites, wherein one polypeptide chain specifically has a structure represented by formula [I]:

[0110] V D1 -L1-V D2 -L2-C L [I]

[0111] and a polypeptide chain has a structure shown by formula [III]:

[0112] V D3 -L3-V D4 -L4-C H1 -F c [III]

[0113] Wherein:

[0114] V D1 is the variable domain of the first immunoglobulin heavy or light chain;

[0115] V D2 is the variable domain of the second immunoglobulin heavy or light chain;

[0116] V D3 is the variable domain of the second immunoglobulin heavy or light chain;

[0117] V D4 is the variable domain of the first immunoglobulin heavy or light chain;

[0118] C L is the constant domain of the immunoglobulin light chain;

[0119] C H1 is the C H1 constant domain of the immunoglobulin heavy chain;

[0120] F c is the hinge region of the immunoglobulin and the CH2, CH3 constant domains of the immunoglobulin heavy chain;

[0121] L1, L2, L3 and L4 are amino acid linkers;

[0122] and the polypeptide of formula I and the polypeptide of formula III form a cross light chain-heavy chain pair, and

[0123] wherein said V D1 and V D4 , or V D2 and V D3 comprise the heavy chain variable domain and the light chain variable domain of the antibody as defined in item 1.

[0124] 6. The antibody-like binding protein according to item 4 or 5, wherein the polypeptide of formula [I] further comprises an F c domain (F c2 ).

[0125] 7. An antibody-like binding protein according to any one of items 4 - 6, comprising two polypeptide chains forming two antigen-binding sites, wherein one polypeptide chain has a structure represented by formula [IV]:

[0126] V D1 -L1-V D2 -L2-C L -L5-F c2 [IV]

[0127] and one polypeptide chain has a structure represented by formula [III]:

[0128] V D3 -L3-V D4 -L4-C H1 -F c [III]

[0129] Wherein:

[0130] V D1 is the variable domain of the first immunoglobulin heavy or light chain;

[0131] V D2 is the variable domain of the second immunoglobulin heavy or light chain;

[0132] V D3 is the variable domain of the second immunoglobulin heavy or light chain;

[0133] V D4 is the variable domain of the first immunoglobulin heavy or light chain;

[0134] C L is the light chain constant domain of the immunoglobulin;

[0135] C H1 is the C H1 heavy chain constant domain of the immunoglobulin;

[0136] F c is the immunoglobulin hinge region and the CH2 and CH3 immunoglobulin heavy chain constant domains of the immunoglobulin;

[0137] F c2 is the immunoglobulin hinge region and the CH2 and CH3 immunoglobulin heavy chain constant domains of the immunoglobulin;

[0138] L1, L2, L3, L4, and L5 are amino acid linkers;

[0139] wherein the polypeptide of formula [IV] and the polypeptide of formula [III] form a crossed light chain.

[0140] 8. The antibody-like binding protein according to item 4 or 5, which comprises a third polypeptide chain containing an F c domain (F c3 ).

[0141] 9. The antibody-like binding protein according to any one of items 4, 5, and 8, which comprises three polypeptide chains forming two antigen-binding sites, wherein:

[0142] The first polypeptide has a structure represented by formula [I]:

[0143] V D1 -L1-V D2 -L2-C L [I]

[0144] The second polypeptide has a structure represented by formula [III]:

[0145] V D3 -L3-V D4 -L4-C H1 -F c [III]

[0146] The third polypeptide F c3 , which is the immunoglobulin hinge region and the CH2 and CH3 immunoglobulin heavy chain constant domains of an immunoglobulin;

[0147] wherein

[0148] V D1 is the variable domain of the first immunoglobulin heavy or light chain;

[0149] V D2 is the variable domain of the second immunoglobulin heavy or light chain;

[0150] V D3 is the variable domain of the second immunoglobulin heavy or light chain;

[0151] V D4 is the variable domain of the first immunoglobulin heavy or light chain;

[0152] C L is the light chain constant domain of an immunoglobulin;

[0153] C H1 is the C H1 heavy chain constant domain of an immunoglobulin;

[0154] F c is the immunoglobulin hinge region and the CH2 and CH3 immunoglobulin heavy chain constant domains of an immunoglobulin;

[0155] L1, L2, L3, and L4 are amino acid linkers;

[0156] and wherein the polypeptide of formula [I] and the polypeptide of formula [III] form a cross light chain - heavy chain pair,

[0157] wherein the polypeptide of formula [III] heterodimerizes with a third polypeptide through its F c domain; and

[0158] wherein said V D1 and V D4 , or V D2 and V D3 comprise a heavy chain variable domain and a light chain variable domain as defined in item 1.

[0159] 10. The antibody - like binding protein according to any one of items 3 - 9, wherein said antibody - like binding protein binds to at least one other target antigen.

[0160] 11. The antibody - like binding protein according to item 10, wherein said at least one other antigen target is CD123.

[0161] 12. The antibody - like binding protein according to item 11, wherein said antibody - like binding protein comprises:

[0162] a) A light chain variable domain (V D1 ) of a first immunoglobulin, which is composed of the amino acid sequence SEQ ID NO: 9 or a sequence at least 85% identical thereto, a light chain variable domain (V D2 ) of a second immunoglobulin, which is composed of the amino acid sequence SEQ ID NO: 308 or a sequence at least 85% identical thereto, a heavy chain variable domain (V D3 ) of a second immunoglobulin, which is composed of the amino acid sequence SEQ ID NO: 312 or a sequence at least 85% identical thereto, and a heavy chain variable domain (V D4 ) of a first immunoglobulin, which is composed of the amino acid sequence SEQ ID NO: 5 or a sequence at least 85% identical thereto, or

[0163] b) A light chain variable domain (V D1 ) of a first immunoglobulin, which is composed of the amino acid sequence SEQ ID NO: 21 or a sequence at least 85% identical thereto, a light chain variable domain (V D2 ) of a second immunoglobulin, which is composed of the amino acid sequence SEQ ID NO: 308 or a sequence at least 85% identical thereto, a heavy chain variable domain (V D3 ) of a second immunoglobulin, which is composed of the amino acid sequence SEQ ID NO: 312 or a sequence at least 85% identical thereto, and a heavy chain variable domain (VD4 ), which consists of the amino acid sequence SEQ ID NO: 18 or a sequence at least 85% identical thereto, or

[0164] c) The light chain variable domain of the first immunoglobulin (V D1 ), which consists of the amino acid sequence SEQ ID NO: 16 or a sequence at least 85% identical thereto, the light chain variable domain of the second immunoglobulin (V D2 ), which consists of the amino acid sequence SEQ ID NO: 308 or a sequence at least 85% identical thereto, the heavy chain variable domain of the second immunoglobulin (V D3 ), which consists of the amino acid sequence SEQ ID NO: 312 or a sequence at least 85% identical thereto, and the heavy chain variable domain of the first immunoglobulin (V D4 ), which consists of the amino acid sequence SEQ ID NO: 12 or a sequence at least 85% identical thereto, or

[0165] d) The light chain variable domain of the first immunoglobulin, which consists of the amino acid sequence SEQ ID NO: 26 or a sequence at least 85% identical thereto (V D1 ), the light chain variable domain of the second immunoglobulin (V D2 ), which consists of the amino acid sequence SEQ ID NO: 308 or a sequence at least 85% identical thereto, the heavy chain variable domain of the second immunoglobulin (V D3 ), which consists of the amino acid sequence SEQ ID NO: 312 or a sequence at least 85% identical thereto, and the heavy chain variable domain of the first immunoglobulin (V D4 ), which consists of the amino acid sequence SEQ ID NO: 23 or a sequence at least 85% identical thereto, or

[0166] e) The light chain variable domain of the first immunoglobulin (V D1 ), which consists of the amino acid sequence SEQ ID NO: 308 or a sequence at least 85% identical thereto, the light chain variable domain of the second immunoglobulin (V D2 ), which consists of the amino acid sequence SEQ ID NO: 143 or a sequence at least 85% identical thereto, the heavy chain variable domain of the second immunoglobulin (V D3 ), which consists of the amino acid sequence SEQ ID NO: 138 or a sequence at least 85% identical thereto, and the heavy chain variable domain of the first immunoglobulin (V D4 ), which consists of the amino acid sequence SEQ ID NO: 312 or a sequence at least 85% identical thereto, or

[0167] f) The light chain variable domain of the first immunoglobulin (VD1 ) which is composed of the amino acid sequence SEQ ID NO: 158 or a sequence that is at least 85% identical thereto, the light chain variable domain (V D2 ) of the second immunoglobulin, which is composed of the amino acid sequence SEQ ID NO: 308 or a sequence that is at least 85% identical thereto, the heavy chain variable domain (V D3 ) of the second immunoglobulin, which is composed of the amino acid sequence SEQ ID NO: 312 or a sequence that is at least 85% identical thereto, and the heavy chain variable domain (V D4 ) of the first immunoglobulin, which is composed of the amino acid sequence SEQ ID NO: 171 or a sequence that is at least 85% identical thereto,

[0168] g) The light chain variable domain (V D1 ) of the first immunoglobulin, which is composed of the amino acid sequence SEQ ID NO: 230 or a sequence that is at least 85% identical thereto, the light chain variable domain (V D2 ) of the second immunoglobulin, which is composed of the amino acid sequence SEQ ID NO: 158 or a sequence that is at least 85% identical thereto, the heavy chain variable domain (V D3 ) of the second immunoglobulin, which is composed of the amino acid sequence SEQ ID NO: 171 or a sequence that is at least 85% identical thereto, and the heavy chain variable domain (V D4 ) of the first immunoglobulin, which is composed of the amino acid sequence SEQ ID NO: 226 or a sequence that is at least 85% identical thereto,

[0169] h) The light chain variable domain (V D1 ) of the first immunoglobulin, which is composed of the amino acid sequence SEQ ID NO: 385 or a sequence that is at least 85% identical thereto, the light chain variable domain (V D2 ) of the second immunoglobulin, which is composed of the amino acid sequence SEQ ID NO: 141 or a sequence that is at least 85% identical thereto, the heavy chain variable domain (V D3 ) of the second immunoglobulin, which is composed of the amino acid sequence SEQ ID NO: 138 or a sequence that is at least 85% identical thereto, and the heavy chain variable domain (V D4 ) of the first immunoglobulin, which is composed of the amino acid sequence SEQ ID NO: 383 or a sequence that is at least 85% identical thereto.

[0170] 13. The antibody-like binding molecule according to item 3, which comprises:

[0171] a) A polypeptide according to formula [I], which is composed of a V containing the sequence SEQ ID NO: 385 D1, L1 of SEQ ID NO: 389, V of SEQ ID NO: 141 D2 , L2 of SEQ ID NO: 389 and C of SEQ ID NO: 310 L composed of the amino acid sequence SEQ ID NO: 388, or

[0172] composed of a sequence that is at least 85% identical to SEQ ID NO: 388, wherein the three CDRs of the sequence SEQ ID NO: 378, 'WAS', and SEQ ID NO: 379 of V D1 and the three CDRs of the sequence SEQ ID NO: 142, 'KVS', and SEQ ID NO: 11 of V D2 remain unchanged, and

[0173] b) A polypeptide according to formula [II], which is composed of V containing the sequence SEQ ID NO: 138 D3 , L3 is 0 amino acids, V of SEQ ID NO: 383 D4 , L4 is 0 amino acids and C of SEQ ID NO: 313 H1 composed of the amino acid sequence SEQ ID NO: 390, or

[0174] composed of a sequence that is at least 85% identical to SEQ ID NO: 390, wherein the three CDRs of the sequence SEQ ID NO: 381, SEQ ID NO: 384, and SEQ ID NO: 382 of V D4 and the three CDRs of the sequence SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8 of V D3 remain unchanged;

[0175] and wherein the polypeptide of formula [I] and the polypeptide of formula [II] form a cross light chain - heavy chain pair.

[0176] 14. The antibody - like binding molecule according to item 6 or 7, which comprises:

[0177] a) A polypeptide according to formula [IV], which is composed of V containing the sequence SEQ ID NO: 385 D1 , L1 of SEQ ID NO: 389, V of SEQ ID NO: 141 D2 , L2 of SEQ ID NO: 389, C of SEQ ID NO: 310 L , L5 containing 0 amino acids and F of SEQ ID NO: 392 c2composed of the amino acid sequence SEQ ID NO: 391, or

[0178] a sequence that is at least 85% identical to SEQ ID NO: 391, wherein V D1 the three CDRs of the sequences SEQ ID NO: 378, 'WAS', and SEQ ID NO: 379 and V D2 the three CDRs of the sequences SEQ ID NO: 142, 'KVS', and SEQ ID NO: 11 are unchanged; and

[0179] b) A polypeptide according to formula [III], which is composed of a V containing the sequence SEQ ID NO: 138 D3 where L3 is 0 amino acids, a V of the sequence SEQ ID NO: 383 D4 where L4 is 0 amino acids and a C of the sequence SEQ ID NO: 313 H1 and an F of the sequence SEQ ID NO: 394 c composed of the amino acid sequence SEQ ID NO: 393, or

[0180] a sequence that is at least 85% identical to SEQ ID NO: 393, wherein V D4 the three CDRs of the sequences SEQ ID NO: 381, SEQ ID NO: 384, and SEQ ID NO: 382, and V D3 the three CDRs of the sequences SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 are unchanged;

[0181] and wherein the polypeptide of formula [IV] and the polypeptide of formula [III] form a cross light chain - heavy chain pair.

[0182] 15. The antibody - like binding molecule according to item 8 or 9, which comprises:

[0183] a) A polypeptide according to formula [I], which is composed of a V containing the sequence SEQ ID NO: 385 D1 the L1 of the sequence SEQ ID NO: 389, a V of the sequence SEQ ID NO: 141 D2 the L2 of the sequence SEQ ID NO: 389, and a C of the sequence SEQ ID NO: 310 L composed of the amino acid sequence SEQ ID NO: 388, or

[0184] a sequence that is at least 85% identical to SEQ ID NO: 388, wherein V D1The three CDRs of SEQ ID NO: 378, 'WAS' and SEQ ID NO: 379 and V D2 The sequences of the three CDRs of SEQ ID NO: 142, 'KVS' and SEQ ID NO: 11 remain unchanged; and

[0185] b) A polypeptide according to formula [III], which consists of a V comprising the sequence SEQ ID NO: 138 D3 , L3 is 0 amino acids, a V of SEQ ID NO: 383 D4 , L4 is 0 amino acids and a C of SEQ ID NO: 313 H1 and an F of SEQ ID NO: 396 c to form the amino acid sequence SEQ ID NO: 395, or

[0186] a sequence that is at least 85% identical to SEQ ID NO: 395, wherein the three CDRs of the sequence SEQ ID NO: 381, SEQ ID NO: 384 and SEQ ID NO: 382 of V D4 , and the three CDRs of the sequence SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 of V D3 remain unchanged; and

[0187] c) A polypeptide F c3 , which consists of the sequence SEQ ID NO: 397 or a sequence that is at least 85% identical thereto, wherein the F c3 or a sequence that is at least 85% identical thereto heterodimerizes with the F of the polypeptide according to formula [III c region; and

[0188] wherein the polypeptide of formula [I] and the polypeptide of formula [III] form a cross light chain - heavy chain pair.

[0189] 16. The antibody - like binding molecule according to item 8 or 9, which comprises:

[0190] a) A polypeptide according to formula [I], which consists of a V comprising the sequence SEQ ID NO: 385 D1 , the L1 of SEQ ID NO: 389, a V of SEQ ID NO: 141 D2 , the L2 of SEQ ID NO: 389 and a C of SEQ ID NO: 310 L to form the amino acid sequence SEQ ID NO: 388, or

[0191] a sequence that is at least 85% identical to SEQ ID NO: 388, wherein VD1 three CDRs of the sequences SEQ ID NO: 378, ‘WAS’, and SEQ ID NO: 379, and V D2 the three CDRs of the sequences SEQ ID NO: 142, ‘KVS’, and SEQ ID NO: 11 are unchanged; and

[0192] b) A polypeptide according to formula [III] which consists of a V comprising the sequence SEQ ID NO: 138 D3 , L3 is 0 amino acids, a V of the sequence SEQ ID NO: 383 D4 , L4 is 0 amino acids and a C of the sequence SEQ ID NO: 313 H1 and an F of the sequence SEQ ID NO: 400 c and consists of the amino acid sequence SEQ ID NO: 399, or

[0193] a sequence that is at least 85% identical to SEQ ID NO: 399, wherein the three CDRs of the sequence SEQ ID NO: 381, SEQ ID NO: 384, and SEQ ID NO: 382 of V D4 , and the three CDRs of the sequence SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 of V D3 are unchanged; and

[0194] c) A polypeptide F c3 which consists of the sequence SEQ ID NO: 398 or a sequence that is at least 85% identical thereto, wherein the F c3 or a sequence that is at least 85% identical thereto heterodimerizes with the F of the polypeptide according to formula [III]; and c wherein the polypeptide of formula [I] and the polypeptide of formula [III] form a cross light chain - heavy chain pair.

[0195] wherein the polypeptide of formula [I] and the polypeptide of formula [III] form a cross light chain - heavy chain pair.

[0196] 17. An isolated antibody that binds to the extracellular domain of the human CD123 protein and comprises:

[0197] a) A heavy chain variable domain comprising a CDR1-H having the sequence SEQ ID NO: 227 or a sequence that differs from SEQ ID NO: 227 by one amino acid substitution; a CDR2-H having the sequence SEQ ID NO: 228 or SEQ ID NO: 353 or SEQ ID NO: 279, or a sequence that differs from SEQ ID NO: 228 or SEQ ID NO: 353 or SEQ ID NO: 279 by one or more amino acid substitutions; a CDR3-H having the sequence SEQ ID NO: 229 or a sequence that differs from SEQ ID NO: 229 by one amino acid substitution; and

[0198] a light chain variable domain comprising a CDR1-L having the sequence SEQ ID NO: 231 or a sequence that differs from SEQ ID NO: 231 by one amino acid substitution; a CDR2-L having the sequence 'RDD' or a sequence that differs from 'RDD' by one amino acid substitution; and a CDR3-L having the sequence SEQ ID NO: 232 or a sequence that differs from SEQ ID NO: 232 by one amino acid substitution; or

[0199] b) A heavy chain variable domain comprising a CDR1-H consisting of the sequence X1YTFTDX2I (SEQ ID NO: 336), where X1 is G or A and X2 is H, Y or N, or any combination thereof; and

[0200] a CDR2-H consisting of the sequence INPYSX1GX2 (SEQ ID NO: 337), where X1 is G or D and X2 is T or A, or any combination thereof; and

[0201] a CDR3-H consisting of the sequence ALNYGSYYAMDA (SEQ ID NO201), and

[0202] a light chain variable domain comprising a CDR1-L consisting of the sequence X1DIX2X3N (SEQ ID NO: 338), where X1 is E or K, X2 is F, H or Y and X3 is N or S, or any combination thereof; and

[0203] a CDR2-L consisting of the sequence 'DAN' or 'DAS'; and

[0204] a CDR3-L consisting of the sequence X1QYNX2YPYT (SEQ ID NO: 339), where X1 is H or Q and X2 is I, K or N, or any combination thereof; or

[0205] c) A heavy chain variable domain comprising a CDR1-H consisting of the sequence GFSLTSYX1 (SEQ ID NO: 340), where X1 is H or S; and

[0206] a CDR2-H consisting of the sequence MWX1DGDT (SEQ ID NO: 341), where X1 is S or N; and

[0207] a CDR3-H consisting of the sequence ARGX1X2X3X4X5X6X7X8X9FX 10 Y (SEQ ID NO: 342), where X1 is D, Y or H, X2 is Y or R, X3 is S or T, X4 is S or P, X5 is Y or no amino acid, X6 is L, I or no amino acid, X7 is Y or no amino acid, X8 is L or no amino acid, X9 is W or no amino acid, X 10 is A or D, or any combination thereof, and

[0208] a light chain variable domain comprising a CDR1-L consisting of the sequence QSFLSSGDX1X2NY (SEQ ID NO: 343), where X1 is E or G and X2 is R or K, or any combination thereof; and

[0209] a CDR2-L consisting of the sequence 'WAS'; and

[0210] a CDR3-L consisting of the sequence QQYYDTPLT (SEQ ID NO: 253), or

[0211] d) A heavy chain variable domain comprising a CDR1-H of the sequence SEQ ID NO: 206 or a sequence differing from SEQ ID NO: 206 by one amino acid substitution; a CDR2-H of the sequence SEQ ID NO: 207 or a sequence differing from SEQ ID NO: 207 by one or more amino acid substitutions; a CDR3-H of the sequence SEQ ID NO: 208 or a sequence differing from SEQ ID NO: 208 by one amino acid substitution; and

[0212] a light chain variable domain comprising a CDR1-L of the sequence SEQ ID NO: 210 or a sequence differing from SEQ ID NO: 210 by one amino acid substitution; a CDR2-L of the sequence 'ETS' or a sequence differing from 'ETS' by one amino acid substitution and a CDR3-L of the sequence SEQ ID NO: 211 or a sequence differing from SEQ ID NO: 211 by one amino acid substitution; or

[0213] e) A heavy chain variable domain comprising CDR1-H of the sequence SEQ ID NO: 213 or a sequence differing from SEQ ID NO: 213 by one amino acid substitution; CDR2-H of the sequence SEQ ID NO: 214 or a sequence differing from SEQ ID NO: 214 by one or more amino acid substitutions; CDR3-H of the sequence SEQ ID NO: 215 or a sequence differing from SEQ ID NO: 215 by one amino acid substitution; and

[0214] A light chain variable domain comprising CDR1-L of the sequence SEQ ID NO: 217 or a sequence differing from SEQ ID NO: 217 by one amino acid substitution; CDR2-L of the sequence 'NTN' or a sequence differing from 'NTN' by one amino acid substitution and CDR3-L of the sequence SEQ ID NO: 218 or a sequence differing from SEQ ID NO: 218 by one amino acid substitution; or

[0215] f) A heavy chain variable domain comprising CDR1-H of the sequence SEQ ID NO: 220 or a sequence differing from SEQ ID NO: 220 by one amino acid substitution; CDR2-H of the sequence SEQ ID NO: 221 or a sequence differing from SEQ ID NO: 221 by one or more amino acid substitutions; CDR3-H of the sequence SEQ ID NO: 222 or a sequence differing from SEQ ID NO: 222 by one amino acid substitution; and

[0216] A light chain variable domain comprising CDR1-L of the sequence SEQ ID NO: 224 or a sequence differing from SEQ ID NO: 224 by one amino acid substitution; CDR2-L of the sequence 'RVS' or a sequence differing from 'RVS' by one amino acid substitution and CDR3-L of the sequence SEQ ID NO: 225 or a sequence differing from SEQ ID NO: 225 by one amino acid substitution; or

[0217] g) A heavy chain variable domain comprising CDR1-H of the sequence SEQ ID NO: 234 or a sequence differing from SEQ ID NO: 234 by one amino acid substitution; CDR2-H of the sequence SEQ ID NO: 235 or a sequence differing from SEQ ID NO: 235 by one or more amino acid substitutions; CDR3-H of the sequence SEQ ID NO: 236 or a sequence differing from SEQ ID NO: 236 by one amino acid substitution; and

[0218] A light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 238 or a sequence differing from SEQ ID NO: 238 by one amino acid substitution; CDR2-L of sequence 'GAS' or a sequence differing from 'GAS' by one amino acid substitution; and CDR3-L of sequence SEQ ID NO: 239 or a sequence differing from SEQ ID NO: 239 by one amino acid substitution; or

[0219] h) A heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 241 or a sequence differing from SEQ ID NO: 241 by one amino acid substitution; CDR2-H of sequence SEQ ID NO: 242 or a sequence differing from SEQ ID NO: 242 by one or more amino acid substitutions; CDR3-H of sequence SEQ ID NO: 243 or a sequence differing from SEQ ID NO: 243 by one amino acid substitution; and

[0220] A light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 245 or a sequence differing from SEQ ID NO: 245 by one amino acid substitution; CDR2-L of sequence 'YAS' or a sequence differing from 'YAS' by one amino acid substitution; and CDR3-L of sequence SEQ ID NO: 246 or a sequence differing from SEQ ID NO: 246 by one amino acid substitution.

[0221] 18. An antibody-like binding protein that specifically binds to human CD123, comprising two polypeptide chains that form two antigen-binding sites, wherein the first polypeptide has the structure shown by formula [I]:

[0222] V D1 -L1-V D2 -L2-C L [I]

[0223] and the second polypeptide has the structure shown by formula [II]:

[0224] V D3 -L3-V D4 -L4-C H1 [II]

[0225] wherein:

[0226] V D1 is the variable domain of the first immunoglobulin heavy or light chain;

[0227] V D2 is the variable domain of the second immunoglobulin heavy or light chain;

[0228] V D3 is the variable domain of the second immunoglobulin heavy or light chain;

[0229] V D4 is the variable domain of the first immunoglobulin heavy or light chain;

[0230] C L is the light chain constant domain of the immunoglobulin;

[0231] C H1 is the C H1 heavy chain constant domain of the immunoglobulin;

[0232] L1, L2, L3 and L4 are amino acid linkers;

[0233] and wherein the first and the second polypeptides form a cross light chain - heavy chain pair; and

[0234] wherein said V D1 and V D4 or V D2 and V D3 comprise the heavy chain variable domain and the light chain variable domain of an antibody as defined in item 17.

[0235] 19. A pharmaceutical composition comprising an antibody - like binding protein according to any one of items 3 - 16 or 18, or an anti - CD123 antibody according to item 17, or an anti - CD3 antibody according to item 1 or 2, and a pharmaceutically acceptable carrier.

[0236] 20. Use of an antibody - like binding protein according to any one of items 3 - 16 or 18, or an anti - CD123 antibody according to item 17, or an anti - CD3 antibody according to item 1 or 2, or a pharmaceutical composition according to claim 19 as a medicament.

[0237] 21. Use of an antibody - like binding protein according to any one of items 3 - 16 or 18, or an anti - CD123 antibody according to item 17 or a pharmaceutical composition thereof for the treatment of cancer.

[0238] 22. Use of an anti - CD3 antibody according to item 1 or 2 or a pharmaceutical composition thereof for the prevention or treatment of pathological immune responses.

[0239] 23. An antibody - like binding protein, an antibody or a pharmaceutical composition for use according to the use of item 21, wherein the cancer is a hematological cancer.

[0240] 24. A method of treating or preventing a disease or disorder, comprising: administering a therapeutically effective amount of an antibody-like binding protein according to any one of items 3-16 or 18, or an anti-CD123 antibody according to item 17, or an anti-CD3 antibody according to item 1 or 2, or a pharmaceutical composition according to item 19, to a subject in need thereof.

[0241] 25. An isolated nucleic acid comprising a sequence encoding an antibody-like binding protein according to any one of items 3-16 or 18, or an anti-CD123 antibody according to item 17, or an anti-CD3 antibody according to item 1 or 2.

[0242] 26. A host cell transformed with the nucleic acid according to item 25.

[0243] 27. A kit comprising at least one antibody according to any one of items 1, 2 or 17, and at least one antibody-like binding protein according to any one of items 3-16.

[0244] 28. Use of the antibody according to item 17 for detecting CD123 expression in a biological sample of a subject, in vitro or in vivo.

[0245] 29. Use of the antibody according to item 28, wherein the antibody is labeled with a detectable molecule or substance.

[0246] 30. Use of the antibody according to any one of items 28-29, wherein the use is for diagnosing the presence of cancer in a subject, determining the susceptibility of a patient with cancer to a therapeutic agent targeting CD123, or monitoring the effectiveness of an anti-CD123 cancer therapy or detecting cancer recurrence after an anti-CD123 cancer therapy.

[0247] 31. Use of the antibody according to item 30, wherein the anti-CD123 cancer therapy uses the antibody-like binding protein according to any one of items 3-16 or 18 or the anti-CD123 antibody according to item 17. Detailed Description of the Invention

[0248] Definitions

[0249] Throughout the present invention, the term " and / or " is grammatically combined and should thus be understood to cover one or more situations that may occur. For example, the statement "such a native sequence protein can be prepared using standard recombinant and / or synthetic methods" indicates that the native sequence protein can be prepared using standard recombinant and synthetic methods, or the native sequence protein can be prepared using standard recombinant methods, or the native sequence protein can be prepared using synthetic methods.

[0250] Furthermore, throughout the present invention, the term " comprise” is to be understood as covering all specifically recited features as well as any optional, additional, unrecited ones. As used herein, the term “ comprise ”s use also discloses embodiments in which there are no other features than the specifically recited features (i.e., “ consist of ”). In addition, the indefinite articles “a” or “an” do not exclude a plurality. The fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used advantageously.

[0251] The term “ gene ” means a DNA sequence encoding or corresponding to a specific amino acid sequence that includes one or more proteins or enzymes, and may or may not include regulatory DNA sequences such as promoter sequences (which, for example, determine under what conditions a gene is expressed). Some genes that are not structural genes can be transcribed from DNA into RNA but not translated into an amino acid sequence. Other genes can function as regulators of structural genes or as regulators of DNA transcription. Specifically, the term gene can denote a genomic sequence encoding a protein, i.e., a sequence that includes regulator, promoter, intron, and exon sequences.

[0252] “ at least 85% identical to the reference sequence ”s sequence has 85% or more sequence identity over its full length with the full length of a reference sequence, specifically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0253] In the context of the present invention, “ percent identity ” is calculated using global pairwise sequence alignment (i.e., comparing two sequences over their full lengths). Methods for aligning the identity of two or more sequences are well known in the art. When considering their full lengths, for example, the 《needle》 program applying the Needleman - Wunsch global alignment algorithm (Needleman and Wunsch, 1970 J. Mol. Biol. 48:443 - 453) can be used to find the best alignment (including gaps) of two sequences. The Needle program is available, for example, on the ebi.ac.uk global website. The percentage of 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.

[0254] A protein consisting of an amino acid sequence that is “at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical” to a reference sequence may contain mutations such as deletions, insertions and / or substitutions compared to the reference sequence. In the case of substitutions, a protein consisting of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a reference sequence may correspond to a homologous sequence from a species different from the reference sequence.

[0255] “ amino acid substitution ” may be conservative or non-conservative. Preferably, the substitution is a conservative substitution, where one amino acid is replaced by another amino acid having similar structure and / or chemical properties. The substitutions preferably correspond to conservative substitutions as shown in the following table.

[0256] conservative substitution amino acid type Ala, Val, Leu, Ile, Met, Pro, Phe, Trp amino acids with aliphatic hydrophobic side chains Ser, Tyr, Asn, Gln, Cys amino acids with uncharged but polar side chains Asp, Glu amino acids with acidic side chains Lys, Arg, His amino acids with basic side chains Gly neutral side chain

[0257] “ antibody ” is also referred to as “ immunoglobulin ” which may be a natural or conventional antibody, where 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, λ (lambda) and κ (kappa). There are five major heavy chain classes (or isotypes), which determine the functional activity of the antibody molecule: IgM, IgD, IgG, IgA and IgE. Each chain contains distinct sequence domains. The light chain includes two domains or regions, a variable domain (VL) and a constant domain (CL). The heavy chain includes four domains, a variable domain (VH) and three constant domains (CH1, CH2 and CH3, collectively referred to as CH). The variable regions of the light chain (VL) and the heavy chain (VH) both determine the binding recognition and specificity against an antigen. The constant domain of the light chain (CL) and the constant domains of 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 immunoglobulin Fab fragment and consists of the variable portions of one light chain and one heavy chain. The specificity of an antibody depends on the structural complementarity between the antibody binding site and the antigen determinant. The antibody binding site is composed of residues mainly from the highly variable regions or complementarity-determining regions (CDR). Occasionally, residues from non-highly variable or framework regions (FR) affect the overall domain structure and thus the binding site. Complementarity-determining regions or CDRs refer to the amino acid sequences that together define the binding affinity and the specificity of the native immunoglobulin binding site of the native Fv region. Each of the light and heavy chains of an immunoglobulin has three CDRs, called CDR1-L, CDR2-L, CDR3-L and CDR1-H, CDR2-H, CDR3-H, respectively. The conventional antibody antigen binding site thus consists of six CDRs, comprising the CDR sets from each of the heavy and light chain V regions.

[0258] In the context of the present invention, an antibody or immunoglobulin is IgM, IgD, IgG, IgA and IgE.

[0259] “ framework region ” (FR) refers to the amino acid sequences inserted between the CDRs, i.e., those parts of the variable regions of the light and heavy chains of immunoglobulins that are relatively conserved between different immunoglobulins in a single species. Each of the light and heavy chains of an immunoglobulin has four FRs, called FR1-L, FR2-L, FR3-L, FR4-L and FR1-H, FR2-H, FR3-H, FR4-H, respectively. Accordingly, the variable domain of the light chain can thus be designated as (FR1-L)-(CDR1-L)-(FR2-L)-(CDR2-L)-(FR3-L)-(CDR3-L)-(FR4-L) and the variable domain of the heavy chain can thus be represented as (FR1-H)-(CDR1-n)-(FR2-H)-(CDR2-H)-(FR3-H)-(CDR3-H)-(FR4-H).

[0260] Given the amino acid sequences of the CDRs, those 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.

[0261] As used herein, “ human framework region ” is a framework region that is substantially the same as (about 85% or more, specifically 90%, 95%, 97%, 99% or 100%) the framework region of a naturally occurring human antibody.

[0262] In the context of the present invention, the definition of CDR / FR in an immunoglobulin light or heavy chain is determined based on the definition of IMGT (Lefranc et al. Dev. Comp. Immunol., 2003, 27(1): 55-77; www.imgt.org).

[0263] As used in this experiment, the term “ antibody ” refers to conventional antibodies and their fragments, as well as single domain antibodies and their fragments, specifically the variable heavy chain of a single domain antibody and chimeric, humanized, bispecific or multispecific antibodies.

[0264] As used herein, an antibody or immunoglobulin also includes those more recently described and whose complementarity determining regions are single domain polypeptide moieties “ single domain antibody”. Examples of single-domain antibodies include heavy-chain antibodies, antibodies that are naturally lacking a light chain, 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 mice, humans, camels, llamas, sheep, rabbits, cows. A single-domain antibody can be a single-domain antibody of a naturally occurring heavy-chain antibody known to lack a light chain. Specifically, Camelidae species such as camels, dromedaries, llamas, alpacas, and guanacos produce heavy-chain antibodies that are naturally lacking a light chain. The heavy-chain antibodies of camels also lack the CH1 domain.

[0265] The variable heavy chains of these single-domain antibodies lacking a light chain are known in the art as “ VHH ” or “ nanobody body ”. Similar to a conventional VH domain, VHH contains four FRs and three CDRs. Nanobodies have advantages over conventional antibodies: they are approximately ten times smaller than IgG molecules, and as a result, properly folded functional nanobodies can be produced by in vitro expression and obtained in high yields. In addition, nanobodies are very stable and can resist the action of proteases. Harmsen and De Haard HJ (Appl. Microbiol. Biotechnol. 2007 Nov; 77(1): 13-22) have reviewed the properties and production of nanobodies.

[0266] As used herein, the term “ monoclonal antibody ” or “ mAb ” refers to an antibody molecule having a single amino acid composition against a specific antigen and should not be construed as requiring the antibody to be produced by any specific method. Monoclonal antibodies can be produced by a single clone of B cells or hybridomas, but can also be recombinant, i.e., produced by protein engineering.

[0267] The term “ chimeric antibody ” refers to an engineered antibody that, in its broadest sense, contains one or more regions from one antibody and one or more regions from one or more other antibodies. Specifically, a chimeric antibody contains a VH domain and a VL domain derived from an antibody of a non-human animal, which are combined with a CH domain and a CL domain of another antibody (specifically a human antibody). As the non-human animal, any animal can be used, such as mice, rats, hamsters, rabbits, etc. A chimeric antibody can also refer to a multispecific antibody that is specific for at least two different antigens.

[0268] The term “ humanized antibody"An antibody that is initially fully or partially of non-human origin and has been modified to replace certain amino acids (specifically, amino acids in the framework regions of the heavy and light chains) to avoid or minimize an immune response in humans. The constant domains of a humanized antibody are mostly human CH and CL domains.

[0269] Multiple methods for humanizing antibody sequences are known in the art; see, for example, Almagro and Fransson (2008) Front Biosci. 13:1619-1633. A commonly used method is CDR grafting or antibody resurfacing, which involves grafting the CDR sequences of a donor antibody, usually a murine antibody, into the framework scaffold of a human antibody with a different specificity. Since CDR grafting can reduce the binding specificity and affinity of the non-human antibody being grafted, and thus reduce biological activity, back mutations can be introduced at selected positions in the CDR-grafted antibody to retain the binding specificity and affinity of the parental antibody. Site identification for possible restoring mutations can be performed using information available in the literature and antibody databases. The amino acid residues that are candidates for back mutations are typically those that are located on the surface of the antibody molecule, while residues that are buried or have low surface exposure generally will not be altered. An alternative humanization technique to CDR grafting and back mutations is surface remodeling, in which non-surface-exposed residues from the non-human source are retained while surface residues are changed to human residues. Another alternative technique is called "guided selection" (Jespers et al, (1994) Biotechnology 12, 899) and can be used to derive fully human antibodies from murine or rat antibodies that retain the epitope and binding characteristics of the parental antibody. Another method of humanization is called 4D humanization. The 4D humanization protocol is described in patent application US20110027266 A1 (WO2009032661A1) and is exemplified in the following 4D humanization of the variable light chain (VL) and variable heavy chain (VH) domains of a rat antibody. In one example, a homology model of the rat antibody was completed using typical MOE software (v.2011.10 - Chemical Computing Group, Quebec, Canada) using a PDB structure (Berman et al, Nucleic Acids Research, 2000, 28:235-242) as a template and then the energy was minimized using standard procedures implemented in MOE. Subsequently, molecular dynamics (MD) simulations were performed on the 3D homology model (completed with MOE software) of the rat antibody and compared with, for example, 49 human models available within MOE that were derived from 7 representative light chains (vk1, vk2, vk3, vk4, vλ1, vλ2, vλ3) and 7 representative heavy chains (vh1a, vh1b, vh2, vh3, vh4, vh5, vh6) designed by LGCR / SDI. For example, a model (Vkx-Vhx) of a chain pair with the best combination of hydrophobicity, electrostatic component, and sequence identity outside the CDRs was selected for "4D humanization". For the paired association between the rat antibody variable domains and the selected model, the sequences were typically aligned based on the best 3D superposition of the alpha carbons of the corresponding homology models. Subsequently, unwanted motifs were considered and mutated.Finally, the obtained humanized sequences are aligned for sequence similarity using, for example, the IEDB database (http: / / www.immuneepitope.org; locally available as of January 30, 2012) to ensure that no sequence contains any of the listed known B- or T-cell epitopes.

[0270] For chimeric antibodies, humanization typically involves modification of the framework regions of the variable region sequences.

[0271] The amino acid residues of the CDR portions generally will not be altered in humanization, although in some instances, it may be desirable to alter an individual CDR amino acid residue, for example, to remove glycosylation sites, deamidation sites, and unwanted cysteine residues. N-linked glycosylation occurs by attachment of an oligosaccharide chain to an asparagine residue in the tripeptide sequence Asn-X-Ser or Asn-X-Thr, where X can be any amino acid other than Pro. Removal of N-glycosylation sites can be achieved by mutating the Asn or Ser / Thr residue to a different residue, specifically by way of conservative substitution. Deamidation of asparagine and glutamine residues can occur depending on factors such as pH and surface exposure. Asparagine residues are generally susceptible to deamidation, primarily when present in the sequence Asn-Gly, and to a lesser extent in other dipeptide sequences such as Asn-Ala. When such a deamidation site, particularly Asn-Gly, is present in the CDR sequence, it may thus be desirable to generally remove the site by conservative substitution to remove one of the residues involved. Substitutions in the CDR sequence to remove one of the residues involved are also intended to be encompassed by the present invention.

[0272] (Conventional) " fragment " of an antibody encompasses a part of a complete antibody, specifically the antigen-binding region or variable region of a complete antibody. Examples of antibody fragments include Fv, Fab, F(ab’)2, Fab’, dsFv, (dsFv)2, scFv, sC(Fv)2, diabodies, bispecific, and multispecific antibodies formed from antibody fragments. Fragments of conventional antibodies can also be single domain antibodies, such as heavy chain antibodies or VHHs.

[0273] The term " Fab " refers to an antibody fragment having a molecular weight of approximately 50,000 and antigen-binding activity, wherein in the fragment obtained by treating IgG with the protease papain, approximately half of the N-terminal side of the H chain and the entire L chain are bound together by a disulfide bond.

[0274] The term " F(ab’)2 " refers to an antibody fragment having a molecular weight of approximately 100,000 and antigen-binding activity, which in the fragment obtained by treating IgG with one protease, i.e., pepsin, is slightly larger than Fab bound by a disulfide bond via the hinge region.

[0275] The term "Fab′" refers to an antibody fragment having a molecular weight of about 50,000 and antigen-binding activity, which is obtained by cleaving the disulfide bonds in the hinge region of F(ab′)2.

[0276] A single-chain Fv ("scFv") polypeptide is a covalently linked VH::VL heterodimer, which is typically expressed by gene fusion of genes encoding VH and VL including a peptide-encoding linker. The human scFv fragments of the present invention include CDRs which are specifically maintained in the proper conformation by using genetic recombination techniques. Bivalent and multivalent antibody fragments can be formed by spontaneous association of monovalent scFvs, or can be generated by coupling monovalent scFvs through a peptide linker, such as bivalent sC(Fv)2. "dsFv" is a VH::VL heterodimer stabilized by a disulfide bond. "(dsFv)2" refers to two dsFvs coupled through a peptide linker.

[0277] The term " bispecific antibody " or " BsAb " generally refers to an antibody that binds the antigen-binding sites of two antibodies within a single molecule. Thus, a BsAb is capable of binding two different antigens simultaneously. Antibodies or antibody derivatives having a set of desired binding properties and effector functions have been designed, modified and produced with increasing frequency using genetic engineering, such as those described in EP 2050 764 A1.

[0278] The term "multispecific antibody" refers to an antibody that binds the antigen-binding sites of two or more antibodies within a single molecule.

[0279] The term " diabody " refers to a small antibody fragment having two antigen-binding sites, which fragment comprises a heavy-chain variable domain (VH) linked to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains on the other chain and form two antigen-binding sites.

[0280] The term " hybridoma " refers to a cell that is obtained by cell fusion of B cells prepared by immunizing a non-human mammal with an antigen and myeloma cells derived from a mouse or the like that produce a desired monoclonal antibody having antigen specificity.

[0281] When referring to a polypeptide (i.e., an antibody of the present invention) or a nucleotide sequence, " purify " and " isolate " are meant to indicate that the indicated molecule is present in the substantial absence of other biomacromolecules of the same type. Specifically, as used herein, the term " purify" means there is at least 75%, 85%, 95% or 98% by weight of the same type of biological macromolecule. A "separated" nucleic acid molecule encoding a specific polypeptide refers to a nucleic acid molecule substantially free of nucleic acid molecules that do not encode the subject polypeptide; however, the molecule may contain some other bases or moieties that do not adversely affect the basic properties of the composition.

[0282] 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 capable of binding to an epitope of the antigen. A target antigen may have one or more epitopes. For each target antigen recognized by an antibody or by an antibody-like binding protein, the antibody-like binding protein is capable of competing with the intact antibody that recognizes the target antigen.

[0283] " affinity " is theoretically defined by the equilibrium association between the intact antibody and the antigen. Affinity can be expressed, for example, by the half maximal effective concentration (EC 50 ).

[0284] The "half maximal effective concentration" is also referred to as " EC 50 ", and refers to the concentration of a drug, antibody or toxin that induces half of the response between the baseline and the maximum after a specific exposure time. EC 50 is negatively correlated with affinity. The lower the EC 50 value, the higher the affinity of the antibody.

[0285] " K D " is the equilibrium dissociation constant, which is the ratio of k off / k on between an antibody and its antigen. K D is negatively correlated with affinity. The K D value is related to the antibody concentration and the lower the K D value, the higher the affinity of the antibody. Affinity can be experimentally evaluated by a variety of known methods, such as measuring the association and dissociation rates by surface plasmon resonance or measuring EC 50. 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 from the sample attaches to the surface. Subsequently, another specific antibody is applied to the surface and it can then 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, most commonly a color change in the substrate. Fluorescence-activated cell sorting (FACS) provides a method for sorting a heterogeneous mixture of biological cells into two or more containers, one cell at a time, based on the specific light-scattering and fluorescence characteristics of each cell. In these assays, EC 50 is the antibody concentration that induces a response halfway between the baseline and the maximum after exposure to a defined antigen concentration by ELISA (enzyme-linked immunosorbent assay) or on cells expressing the antigen by FACS (fluorescence-activated cell sorting) for some specific time. Surface plasmon resonance is a label-free method in which the binding of molecules in the soluble phase (“analyte”) is directly measured as the binding of “ligand” molecules immobilized on the sensor surface. In the sensing device, the binding of the ligand is monitored by an optical phenomenon called surface plasmon. Specifically, when “analyte” molecules dissociate from “ligand” molecules, a decrease in the SPR signal (expressed in resonance units, RU) is observed. The association (‘on rate’, k a ) and dissociation rate (‘off rate’, k d ) are obtained from the signals of the association and dissociation processes, and the equilibrium dissociation constant (‘binding constant’, K D ) is calculated therefrom and especially calculated. The signal given in resonance units (RU) depends on the size of the ligand present in the analyte, however, in cases where the experimental conditions are the same, i.e., the ligand is the same molecule under the same conditions, the RU obtained can represent the affinity, where the higher the signal obtained in RU, the higher the binding.

[0286] When the EC 50 for two antigens is in a similar range, the monoclonal antibody that binds to antigen 1 (Ag1) “ cross-react ”. In the present application, the affinity is measured for two antigens by the same method, and when the ratio of the affinity of Ag2 to the affinity of Ag1 is equal to or less than 10 (specifically 5, 2, 1, or 0.5), the monoclonal antibody that binds to Ag1 cross-reacts with Ag2.

[0287] When the affinities for two antigens are very different, the monoclonal antibody that binds to Ag1 “ not significantly cross react”. If the binding response is too low, the affinity for Ag2 may be unmeasurable. In the present invention, in the same experimental setup and at the same antibody concentration, if the binding response of a monoclonal antibody to Ag2 is less than 5% of the binding response of the same monoclonal antibody to Ag1, then the monoclonal antibody that binds to Ag1 does not significantly cross-react with Ag2. In practice, the antibody concentration used can be the EC 50 or the concentration required to reach a saturation plateau with Ag1.

[0288] As used herein, “ specificity ” refers to the ability of an antibody to discriminate its binding (the “epitope”) target peptide sequence from highly related, highly homologous peptide sequences.

[0289] When it does not significantly cross-react with Ag2, the monoclonal antibody “ specific binding ” with Ag1.

[0290] “ domain ” can be any region of a protein, generally defined based on sequence homology and usually involving a specific structural or functional entity.

[0291] ″ recombinant ″ molecules are those molecules prepared, expressed, generated, or isolated recombinantly.

[0292] As used herein, “ subject ” refers to mammals such as rodents, felines, canines, and primates. Specifically, the subject according to the present invention is a human.

[0293] Anti-CD3 antibody

[0294] “ CD3 ” refers to an antigen that is expressed on T-cells as part of the multi-molecular T-cell receptor complex and consists of at least three different chains, CD3ε, CD3δ, and CD3γ. CD3δ and CD3γ have low sequence identity and / or similarity to human CD3ε (similarity and identity less than 20%). CD3ε and CDR3δ can together form a complex, which is referred to herein as the “CD3ε / δ-complex”. CD3ε also forms a complex with CDR3γ, which is called the “CD3ε / γ-complex”. Clustering of CD3 on T-cells (e.g., by immobilized anti-CD3-antibody) leads to T-cell receptor engagement and similar T-cell activation but is independent of its clonotypic specificity. “ CD3ε ” contains three domains, an intracellular domain, a transmembrane domain, and an extracellular domain.

[0295] Most of the prior art anti-CD3 antibodies recognize the CD3ε-chain. One of these prior art anti-CD3 antibodies is OKT3. T cell activation by antibody molecules has been described in the prior art, for example by using the antibody molecule OKT3. Anti-CD3 antibodies and their variants have been described in the prior art (US 4,361,549; US 4,361,549; US 5,885,573; US 5,929,212 and WO 98 / 52975 or US 5,955,358). OKT3 has further been used as a potent immunosuppressant in clinical transplantation to treat allograft rejection (Thistlethwaite 1984, Transplantation 38, 695-701; Woodle 1991, Transplantation 51, 1207-1212; Choi 2001, Eur. J. Immunol. 31(1), 94-106).

[0296] The major drawback of this therapy is that T cell activation results in cytokine release due to cross-linking between T cells and cells bearing FcγR and the human anti-mouse antibody (HAMA) response. Several publications have described that humanization of antibodies such as OKT3 reduces these side effects: US 5,929,212; US 5,885,573 and others. On the other hand, OKT3 or other anti-CD3 antibodies can be used as immunostimulants to stimulate T cell activation and proliferation (US 6,406,696 Bluestone; US 6,143,297 Bluestone; US 6,113,901 Bluestone; Yannelly 1990, J. Immunol. Meth. 1, 91-100). Anti-CD3 antibodies have also been described for use in combination with anti-CD28 antibodies to induce T cell proliferation (US 6,352,694). OKT3 has further been used by itself or as a component of a bispecific antibody to target cytotoxic T cells to tumor cells or virus-infected cells (Nitta 1990, Lancet 335, 368-376; Sanna 1995, Bio / Technology 13, 1221-1224; WO99 / 54440).

[0297] The ways of using antibodies as agents for recruiting T-cells that have been available so far have been limited by several findings. First, natural or engineered antibodies with high affinity for T-cells often do not activate the T-cells they bind to. Second, natural or engineered antibodies with low affinity for T-cells often fail to effectively trigger the T-cell-mediated cytotoxic ability.

[0298] A reference for the full-length human CD3ε protein including the signal peptide is available under Uniprot database accession number P07766 and is included herein under SEQ ID NO: 1 (available on December 12, 2014).

[0299] A reference sequence for the full-length cynomolgus monkey CD3ε protein including the signal peptide is available under Uniprot database accession number Q95LI5 and is included herein under SEQ ID NO: 2 (available on December 12, 2014).

[0300] The sequence of the mature human CD3ε His-tag Fc-fusion protein cloned by the inventors from genomic DNA is disclosed in SEQ ID NO: 3. The mature human CD3ε His-tag Fc-fusion protein comprises amino acids 23 to 126 of the full-length human CD3ε protein and thus comprises the extracellular domain of human CD3ε.

[0301] The sequence of the mature cynomolgus monkey CD3ε Fc-fusion protein cloned by the invention from genomic DNA is disclosed under SEQ ID NO: 4. The mature cynomolgus monkey CD3ε Fc-fusion protein comprises amino acids 23 - 117 of the full-length cynomolgus monkey CD3ε protein and thus comprises the extracellular domain of human or cynomolgus monkey CD3ε, containing an alanine to valine exchange at amino acid position 35 compared to amino acid position 57 of the wild-type sequence.

[0302] The domain organization of human and cynomolgus monkey CD3ε is as follows (based on the Uniprot P07766 sequence (human) and the Uniprot Q95LI5 sequence (cynomolgus monkey)):

[0303] CD3ε domain Position on SEQ ID NO: 1 (human) Position on SEQ ID NO: 2 (cynomolgus monkey) extracellular 23-126 22-117 transmembrane domain 127-152 118-138 cytoplasm 153-207 139-198

[0304] Accordingly, the extracellular domain of human CD3ε consists of the amino acids at positions 23 - 126 of SEQ ID NO: 1 and the extracellular domain of cynomolgus monkey CD3ε consists of the amino acids at positions 22 - 117 of SEQ ID NO: 2.

[0305] The inventors successfully generated, screened, and selected specific mouse and rat anti-CD3 antibodies. These anti-CD3 antibodies exhibit high affinity for human and cynomolgus monkey CD3 proteins, but have low T-cell activation in the absence of target cells.

[0306] The present invention has determined the sequences of the variable heavy and light chains of such monoclonal antibodies, which are designated as 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".

[0307] The anti-CD3 antibody designated "20G6-F3" comprises:

[0308] - a variable heavy domain, which consists of the following sequence

[0309] (SEQ ID NO: 5, where the CDRs are shown in bold and underlined), containing CDR1-H of sequence SEQ ID NO: 6, CDR2-H of sequence SEQ ID NO: 7, and CDR3-H of sequence SEQ ID NO: 8, and

[0310] - a variable light domain, which consists of the following sequence

[0311] (SEQ ID NO: 9, where the CDRs are shown in bold and underlined), containing CDR1-L of sequence SEQ ID NO: 10, CDR2-L consisting of the sequence 'KVS', and CDR3-L of sequence SEQ ID NO: 11.

[0312] The anti-CD3 antibody designated "4B4-D7" comprises:

[0313] - a variable heavy domain, which consists of the following sequence (SEQ ID NO: 12, where the CDRs are shown in bold and underlined), containing CDR1-H of sequence SEQ ID NO: 13, CDR2-H of sequence SEQ ID NO: 14, and CDR3-H of sequence SEQ ID NO: 15, and

[0314] - A light chain variable domain, which consists of the following sequences (SEQ ID NO: 16, where the CDRs are shown in bold and underlined), containing CDR1-L of sequence SEQ ID NO: 17, CDR2-L of the 'KVS' group, and CDR3-L of sequence SEQ ID NO: 11.

[0315] The anti-CD3 antibody designated "4E7-C9" comprises:

[0316] - A heavy chain variable domain, which consists of the following sequences (SEQ ID NO: 18, where the CDRs are shown in bold and underlined), containing CDR1-H of sequence SEQ ID NO: 13, CDR2-H of sequence SEQ ID NO: 19, and CDR3-H of sequence SEQ ID NO: 20, and

[0317] - A light chain variable domain, which consists of the following sequences (SEQ ID NO: 21, where the CDRs are shown in bold and underlined), containing CDR1-L of sequence SEQ ID NO: 22, CDR2-L composed of the 'KVS' sequence, and CDR3-L of sequence SEQ ID NO: 11.

[0318] The anti-CD3 antibody designated "18F5-H10" comprises:

[0319] - A heavy chain variable domain, which consists of the following sequences (SEQ ID NO: 23, where the CDRs are shown in bold and underlined), containing CDR1-H of sequence SEQ ID NO: 24, CDR2-H of sequence SEQ ID NO: 19, and CDR3-H of sequence SEQ ID NO: 25, and

[0320] - A light chain variable domain, which consists of the following sequences (SEQ ID NO: 26, where the CDRs are shown in bold and underlined), containing CDR1-L of sequence SEQ ID NO: 27, CDR2-L composed of the 'KVS' sequence, and CDR3-L of sequence SEQ ID NO: 28.

[0321] The anti-CD3 antibody designated "12D2-E5" comprises:

[0322] - A heavy chain variable domain, which consists of the following sequences (SEQ ID NO: 29, where the CDRs are shown in bold and underlined), containing CDR1-H of SEQ ID NO: 30, CDR2-H of SEQ ID NO: 31, and CDR3-H of SEQ ID NO: 32, and

[0323] - A light chain variable domain, which consists of the following sequences (SEQ ID NO: 33, where the CDRs are shown in bold and underlined), containing CDR1-L of SEQ ID NO: 34, CDR2-L consisting of the sequence 'RDD', and CDR3-L of SEQ ID NO: 35.

[0324] The anti-CD3 antibody designated "11D7-C3" comprises:

[0325] - A heavy chain variable domain, which consists of the following sequences (SEQ ID NO: 36, where the CDRs are shown in bold and underlined), containing CDR1-H of SEQ ID NO: 13, CDR2-H of SEQ ID NO: 37, and CDR3-H of SEQ ID NO: 38.

[0326] - A light chain variable domain, which consists of the following sequences (SEQ ID NO: 39, where the CDRs are shown in bold and underlined), containing CDR1-L of SEQ ID NO: 10, CDR2-L consisting of the sequence 'KVS', and CDR3-L of SEQ ID NO: 28.

[0327] The anti-CD3 antibody designated "11H3-E5" comprises:

[0328] - A heavy chain variable domain, which consists of the following sequences (SEQ ID NO: 40, where the CDRs are shown in bold and underlined), containing CDR1-H of SEQ ID NO: 13, CDR2-H of SEQ ID NO: 37, and CDR3-H of SEQ ID NO: 41, and

[0329] - A light chain variable domain, which consists of the following sequences (SEQ ID NO: 42, where the CDRs are shown in bold and underlined), comprising CDR1-L of SEQ ID NO: 17, CDR2-L consisting of the sequence 'KVS', and CDR3-L of SEQ ID NO: 11.

[0330] The anti-CD3 antibody designated "13H2-C2" comprises:

[0331] - A heavy chain variable domain consisting of the following sequence (SEQ ID NO: 43, where the CDRs are shown in bold and underlined), comprising CDR1-H of SEQ ID NO: 13, CDR2-H of SEQ ID NO: 37, and CDR3-H of SEQ ID NO: 44, and

[0332] - A light chain variable domain consisting of the following sequence (SEQ ID NO: 45, where the CDRs are shown in bold and underlined), comprising CDR1-L of SEQ ID NO: 10, CDR2-L consisting of the sequence 'KVS', and CDR3-L of SEQ ID NO: 11.

[0333] The anti-CD3 antibody designated "13C1-F6" comprises:

[0334] - A heavy chain variable domain consisting of the following sequence (SEQ ID NO: 46, where the CDRs are shown in bold and underlined), comprising CDR1-H of SEQ ID NO: 13, CDR2-H of SEQ ID NO: 37, and CDR3-H of SEQ ID NO: 47, and

[0335] - A light chain variable domain consisting of the following sequence (SEQ ID NO: 48, where the CDRs are shown in bold and underlined), comprising CDR1-L of SEQ ID NO: 10, CDR2-L consisting of the sequence 'KVS', and CDR3-L of SEQ ID NO: 11.

[0336] The anti-CD3 antibody designated "18H11-F10" comprises:

[0337] - A heavy chain variable domain consisting of the following sequence (SEQ ID NO: 49, where the CDRs are shown in bold and underlined), containing CDR1-H of SEQ ID NO: 50, CDR2-H of SEQ ID NO: 51, and CDR3-H of SEQ ID NO: 52, and

[0338] - a light chain variable domain, which consists of the following sequences (SEQ ID NO: 53, where the CDRs are shown in bold and underlined), containing CDR1-L of SEQ ID NO: 54, CDR2-L consisting of the sequence 'NAN', and CDR3-L of SEQ ID NO: 55.

[0339] The anti-CD3 antibody called "1E6-C9" comprises:

[0340] - a heavy chain variable domain, which consists of the following sequences (SEQ ID NO: 56, where the CDRs are shown in bold and underlined), containing CDR1-H of SEQ ID NO: 57, CDR2-H of SEQ ID NO: 37, and CDR3-H of SEQ ID NO: 58, and

[0341] - a light chain variable domain, which consists of the following sequences (SEQ ID NO: 59, where the CDRs are shown in bold and underlined), containing CDR1-L of SEQ ID NO: 10, CDR2-L consisting of the sequence 'KVS', and CDR3-L of SEQ ID NO: 28.

[0342] The anti-CD3 antibody called "10F4-C10" comprises:

[0343] - a heavy chain variable domain, which consists of the following sequences (SEQ ID NO: 60, where the CDRs are shown in bold and underlined), containing CDR1-H of SEQ ID NO: 13, CDR2-H of SEQ ID NO: 37, and CDR3-H of SEQ ID NO: 61, and

[0344] - a light chain variable domain, which consists of the following sequences (SEQ ID NO: 62, where the CDRs are shown in bold and underlined), comprising CDR1-L of SEQ ID NO: 10, CDR2-L consisting of the sequence 'KVS', and CDR3-L of SEQ ID NO: 11.

[0345] The anti-CD3 antibody designated "10E6-G6" comprises:

[0346] - A heavy chain variable domain consisting of the following sequence (SEQ ID NO: 63, where the CDRs are shown in bold and underlined), comprising CDR1-H of SEQ ID NO: 64, CDR2-H of SEQ ID NO: 65, and CDR3-L of SEQ ID NO: 47, and

[0347] - A light chain variable domain consisting of the following sequence (SEQ ID NO: 66, where the CDRs are shown in bold and underlined), comprising CDR1-L of SEQ ID NO: 67, CDR2-L consisting of the sequence 'KVS', and CDR3-L of SEQ ID NO: 28.

[0348] The anti-CD3 antibody designated "18G9-H11" comprises:

[0349] - A heavy chain variable domain consisting of the following sequence (SEQ ID NO: 68, where the CDRs are shown in bold and underlined), comprising CDR1-H of SEQ ID NO: 13, CDR2-H of SEQ ID NO: 37, and CDR3-H of SEQ ID NO: 69, and

[0350] - A light chain variable domain consisting of the following sequence (SEQ ID NO: 70, where the CDRs are shown in bold and underlined), comprising CDR1-L of SEQ ID NO: 10, CDR2-L consisting of the sequence 'KVS', and CDR3-L of SEQ ID NO: 71.

[0351] The anti-CD3 antibody designated "11F3-B9" comprises:

[0352] - A heavy chain variable domain consisting of the following sequence (SEQ ID NO: 72, where the CDRs are shown in bold and underlined), containing CDR1-H of SEQ ID NO: 13, CDR2-H of SEQ ID NO: 37, and CDR3-H of SEQ ID NO: 84, and

[0353] - A light chain variable domain, which consists of the following sequences (SEQ ID NO: 73, where the CDRs are shown in bold and underlined), containing CDR1-L of SEQ ID NO: 17, CDR2-L consisting of the sequence 'KVS', and CDR3-L of SEQ ID NO: 11.

[0354] The anti-CD3 antibody designated "12G3-E8" comprises:

[0355] - A heavy chain variable domain, which consists of the following sequences (SEQ ID NO: 74, where the CDRs are shown in bold and underlined), containing CDR1-H of SEQ ID NO: 75, CDR2-H of SEQ ID NO: 76, and CDR3-H of SEQ ID NO: 77, and

[0356] - A light chain variable domain, which consists of the following sequences (SEQ ID NO: 78, where the CDRs are shown in bold and underlined), containing CDR1-L of SEQ ID NO: 10, CDR2-L consisting of the sequence 'KVS', and CDR3-L of SEQ ID NO: 11.

[0357] The anti-CD3 antibody designated "5B1-G2" comprises:

[0358] - A heavy chain variable domain, which consists of the following sequences (SEQ ID NO: 79, where the CDRs are shown in bold and underlined), containing CDR1-H of SEQ ID NO: 80, CDR2-H of SEQ ID NO: 76, and CDR3-H of SEQ ID NO: 81, and

[0359] - A light chain variable domain, which consists of the following sequences (SEQ ID NO: 82, where the CDRs are shown in bold and underlined), comprising CDR1-L of sequence SEQ ID NO: 10, CDR2-L consisting of the sequence 'KVS', and CDR3-L of sequence SEQ ID NO: 11.

[0360] The anti-CD3 antibody designated "16F8-A7" comprises:

[0361] - A heavy chain variable domain which contains the sequence (SEQ ID NO: 83, where the CDRs are shown in bold and underlined), comprising CDR1-H of sequence SEQ ID NO: 13, CDR2-H of sequence SEQ ID NO: 37, and CDR3-H of sequence SEQ ID NO: 84, and

[0362] - A light chain variable domain which consists of the following sequences (SEQ ID NO: 85, where the CDRs are shown in bold and underlined), comprising CDR1-L of sequence SEQ ID NO: 10, CDR2-L consisting of the sequence 'KVS', and CDR3-L of sequence SEQ ID NO: 11.

[0363] The anti-CD3 antibody designated "11F9-F8" comprises:

[0364] - A heavy chain variable domain which consists of the following sequences (SEQ ID NO: 46, where the CDRs are shown in bold and underlined), comprising CDR1-H of sequence SEQ ID NO: 13, CDR2-H of sequence SEQ ID NO: 37, and CDR3-H of sequence SEQ ID NO: 47, and

[0365] - A light chain variable domain which consists of the following sequences (SEQ ID NO: 87, where the CDRs are shown in bold and underlined), comprising CDR1-L of sequence SEQ ID NO: 10, CDR2-L consisting of the sequence 'KVS', and CDR3-L of sequence SEQ ID NO: 88.

[0366] The anti-CD3 antibody designated "3G5-E10" comprises:

[0367] - A heavy chain variable domain which consists of the following sequences (SEQ ID NO: 89, where the CDRs are shown in bold and underlined), containing CDR1-H of SEQ ID NO: 90, CDR2-H of SEQ ID NO: 91, and CDR3-H of SEQ ID NO: 32, and

[0368] - A light chain variable domain, which consists of the following sequences (SEQ ID NO: 92, where the CDRs are shown in bold and underlined), containing CDR1-L of SEQ ID NO: 93, CDR2-L consisting of the sequence 'GAS', and CDR3-L of SEQ ID NO: 94.

[0369] The anti-CD3 antibody designated "9D7-F3" comprises:

[0370] - A heavy chain variable domain, which consists of the following sequences (SEQ ID NO: 95, where the CDRs are shown in bold and underlined), containing CDR1-H of SEQ ID NO: 96, CDR2-H of SEQ ID NO: 97, and CDR3-H of SEQ ID NO: 98, and

[0371] - A light chain variable domain, which consists of the following sequences (SEQ ID NO: 99, where the CDRs are shown in bold and underlined), containing CDR1-L of SEQ ID NO: 100, CDR2-L consisting of the sequence 'NTN', and CDR3-L of SEQ ID NO: 101.

[0372] The anti-CD3 antibody designated "8C2-F7" comprises:

[0373] - A heavy chain variable domain, which consists of the following sequences (SEQ ID NO: 102, where the CDRs are shown in bold and underlined), containing CDR1-H of SEQ ID NO: 103, CDR2-H of SEQ ID NO: 104, and CDR3-H of SEQ ID NO: 105, and

[0374] - A light chain variable domain, which consists of the following sequences (SEQ ID NO: 106, where the CDRs are shown in bold and underlined), comprising CDR1-L of SEQ ID NO: 10, CDR2-L consisting of the sequence 'KVS', and CDR3-L of SEQ ID NO: 11.

[0375] The anti-CD3 antibody designated "20E5-F10" comprises:

[0376] - A heavy chain variable domain consisting of the following sequences (SEQ ID NO: 107, where the CDRs are shown in bold and underlined), comprising CDR1-H of SEQ ID NO: 80, CDR2-H of SEQ ID NO: 19, and CDR3-H of SEQ ID NO: 108, and

[0377] - A light chain variable domain consisting of the following sequences (SEQ ID NO: 109, where the CDRs are shown in bold and underlined), comprising CDR1-L of SEQ ID NO: 10, CDR2-L consisting of the sequence 'KVS', and CDR3-L of SEQ ID NO: 11.

[0378] The anti-CD3 antibody designated "20B5-F10" comprises:

[0379] - A heavy chain variable domain consisting of the following sequences (SEQ ID NO: 110, where the CDRs are shown in bold and underlined), comprising CDR1-H of SEQ ID NO: 13, CDR2-H of SEQ ID NO: 37, and CDR3-H of SEQ ID NO: 111, and

[0380] - A light chain variable domain consisting of the following sequences (SEQ ID NO: 112, where the CDRs are shown in bold and underlined), comprising CDR1-L of SEQ ID NO: 113, CDR2-L consisting of the sequence 'KVS', and CDR3-L of SEQ ID NO: 114.

[0381] The anti-CD3 antibody designated "6C9-C9" comprises:

[0382] - A heavy chain variable domain consisting of the following sequences (SEQ ID NO: 115, in which the CDRs are shown in bold and underlined), containing CDR1-H of SEQ ID NO: 116, CDR2-H of SEQ ID NO: 117, and CDR3-H of SEQ ID NO: 118, and

[0383] - a light chain variable domain, which consists of the following sequences (SEQ ID NO: 119, in which the CDRs are shown in bold and underlined), containing CDR1-L of SEQ ID NO: 100, CDR2-L consisting of the sequence 'VTN', and CDR3-L of SEQ ID NO: 120.

[0384] The anti-CD3 antibody designated "3E8-G1" comprises:

[0385] - a heavy chain variable domain, which consists of the following sequences (SEQ ID NO: 121, in which the CDRs are shown in bold and underlined), containing CDR1-H of SEQ ID NO: 122, CDR2-H of SEQ ID NO: 123, and CDR3-H of SEQ ID NO: 124, and

[0386] - a light chain variable domain, which consists of the following sequences (SEQ ID NO: 125, in which the CDRs are shown in bold and underlined), containing CDR1-L of SEQ ID NO: 126, CDR2-L consisting of the sequence 'RDD', and CDR3-L of SEQ ID NO: 127.

[0387] The anti-CD3 antibody designated "3H6-D2" comprises:

[0388] - a heavy chain variable domain, which consists of the following sequences (SEQ ID NO: 128, in which the CDRs are shown in bold and underlined), containing CDR1-H of SEQ ID NO: 13, CDR2-H of SEQ ID NO: 19, and CDR3-H of SEQ ID NO: 129, and

[0389] - a light chain variable domain, which consists of the following sequences (SEQ ID NO: 130, where the CDRs are shown in bold and underlined), comprising CDR1-L of sequence SEQ ID NO: 10, CDR2-L consisting of the sequence 'KVS', and CDR3-L of sequence SEQ ID NO: 11.

[0390] The anti-CD3 antibody designated "8H2" comprises:

[0391] - A heavy chain variable domain, which consists of the following sequences (SEQ ID NO: 131, where the CDRs are shown in bold and underlined), comprising CDR1-H of sequence SEQ ID NO: 103, CDR2-H of sequence SEQ ID NO: 104, and CDR3-H of sequence SEQ ID NO: 105, and

[0392] - A light chain variable domain, which consists of the following sequences (SEQ ID NO: 132, where the CDRs are shown in bold and underlined), comprising CDR1-L of sequence SEQ ID NO: 133, CDR2-L consisting of the sequence 'LVS', and CDR3-L of sequence SEQ ID NO: 134.

[0393] In one embodiment, the anti-CD3 antibody of the invention binds to human CD3. In another embodiment, the anti-CD3 antibody of the invention also binds to cynomolgus monkey CD3. Specifically, the anti-CD3 antibody of the invention binds to the extracellular domain of human CD3 or both human and cynomolgus monkey CD3. More specifically, the antibody binds to CD3ε. More specifically, the anti-CD3 antibody binds to the extracellular domain of human and cynomolgus monkey CD3ε. The anti-CD3 antibody binds to CD3ε when present in a complex form such as the CD3ε / δ complex or as a single protein, whether expressed in isolated form or present in the soluble extracellular domain or full-length membrane-anchored CD3ε, for example, present in T-cells.

[0394] The anti-CD3 antibody according to the invention is specific for the surface human CD3 protein or both human and cynomolgus monkey CD3 proteins, particularly CD3ε.

[0395] In one embodiment, the ratio of the affinity of the anti-CD3 antibody according to the invention for cynomolgus monkey CD3 to its affinity for human CD3 (KD(cynomolgus monkey) / KD(human)) ≤ 10, specifically ≤ 6, ≤ 5, ≤ 4, ≤ 3, for example ≤ 2, ≤ 1 or ≤ 0.5. Such a polypeptide according to the invention can be used in toxicity studies conducted in monkeys, and the toxicity profile observed in monkeys is related to the potential side effects expected in humans.

[0396] Specifically, the anti-CD3 antibody of the present invention does not bind to CD3γ and / or CD3δ protein or does not significantly cross-react with CD3γ and / or CD3δ protein.

[0397] Specifically, the antibody does not bind to human and cynomolgus monkey CD3γ and / or CD3δ protein or does not significantly cross-react with human and cynomolgus monkey CD3γ and / or CD3δ protein.

[0398] The full-length human CD3δ protein sequence is available under Uniprot database accession number P04234 (SEQ ID NO: 86, available on December 14, 2014). The extracellular domain of human CD3δ consists of amino acids at positions 22-105 of SEQ ID NO: 86.

[0399] The sequence of the full-length human CD3γ protein is available under Uniprot database accession number P09693 (SEQ ID NO: 185, available on December 14, 2014). The extracellular domain of human CD3γ consists of amino acids at positions 23-116 of SEQ ID NO: 185.

[0400] In addition, the anti-CD3 antibody according to the present invention has an affinity (KD) for human CD3 or cynomolgus monkey CD3 or both of ≤ 90 nM, ≤ 50 nM or ≤ 30 nM, such as ≤ 20 nM, ≤ 10 nM, ≤ 8 nM, ≤ 6 nM, ≤ 4 nM or ≤ 2 nM, for example, having an affinity of 0.1 nM - 10 nM, specifically 0.1 nM - 8 nM or 0.1 nM - 4 nM.

[0401] The KD value of the affinity for human CD3 or for cynomolgus monkey CD3 is determined by surface plasmon resonance using a soluble recombinant CD3ε / δ complex from human and cynomolgus monkey as the capture antigen.

[0402] 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). Antigens containing a signal peptide in the form of an Fc-fusion protein as described in the examples, such as human CD3ε and human CD3δ subunit extracellular domain constructs, can be used. Alternatively, cynomolgus monkey CD3ε and cynomolgus monkey CD3δ subunit extracellular domain constructs containing a signal peptide in the form of an Fc-fusion protein as described in the examples can be used as antigens. The capture of the human or cynomolgus monkey CD3ε / δ-Fc fusion protein is achieved using, for example, a human antibody capture kit (GE Healthcare). For example, the captured antibody can be coupled to a CM5 chip (BR-1001-88, GE Healthcare) using an amine coupling kit (BR-100-50, GE Healthcare) to approximately 12,000 RU. The CD3ε / δ-Fc fusion protein is captured at 10 μl / min to approximately 70 RU to obtain an Rmax value of 30 RU. The binding kinetics of the anti-CD3 antibody can be measured for the association and dissociation phases of 240 s and 600 s, respectively, at 30 μl / min, for example. For example, two-fold dilutions of the anti-CD3 antibody in the range of 3 - 400 nM in the assay buffer can be used. The generation of the capture surface can be carried out by injecting a 3 M MgCl2 solution at 30 μl / min for 1 min. For data analysis, BIA evaluation software v.4.1 (GE Healthcare) can be used, for example. The data can be globally fitted using a 1:1 Langmuir model with mass transport.

[0403] In one embodiment, the anti-CD3 antibody of the invention also has an apparent EC50 determined by FACS analysis of human T-cells, which is ≤ 60 nM, for example ≤ 50 nM, ≤ 40 nM, ≤ 30 nM, ≤ 20 nM or ≤ 15 nM. Generally, the apparent EC50 is in the range of 1 - 60 nM, particularly 1 - 30 nM, for example 1 - 20 nM.

[0404] In one embodiment, in the absence of target cells, the anti-CD3 antibody of the invention has less than 10%, less than 8%, less than 6%, less than 4%, less than 2%, less than 1%, for example less than 0.5% T-cell activation.

[0405] The term " activation of T-cells " herein refers to triggering CD3 signaling involving cytotoxic granule fusion, transient cytokine release, and proliferation. In the presence of target cells, the antibody-like binding protein and anti-CD3 antibody of the invention target CD3ε and activate T-cells; this activity is also referred to as " T-cell engagement effect”. T-cell engagement effect induces cytotoxicity in target cells.

[0406] As is known to those skilled in the art, activation of T-cells induces the expression of surface markers such as CD69 and CD25. Activation of T-cells can thus be measured by detecting and measuring 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.

[0407] Methods for measuring T-cell activation are also disclosed in the Examples (Example 3.3) section. Accordingly, in the context of the present invention, T-cell activation is measured as the percentage % of cells expressing CD69 in the total cell population, or as the percentage % of cells expressing CD4 and CD69 in the total cell population, or as the percentage % of cells expressing CD8 and CD69 in the total cell population.

[0408] The anti-CD3 antibody in the context of the present invention low T-cell activation ” refers to less than 10%, less than 8%, less than 6%, less than 4%, less than 2%, less than 1%, such as less than 0.5% T-cell activation in the absence of target cells.

[0409] Sequence alignment of the VH and VL regions of 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". Comparison of the CDR-H and CDR-L sequences tends to indicate that structurally "20G6-F3", "4B4-D7", "4E7-C9", "18F5-H10", "11D7-C3", "11H3-E5", "13H2-C2", "13C1-F6", "1E6-C9", "10F4-C10", "10E6-G6", "18G9-H11", "11F3-B9", "12G3-E8", "5B1-G2", "16F8-A7", "11F9-F8", "20E5-F10", "20B5-F10", and "3H6-D2" are closely related, and these antibodies may bind to the same epitope. Comparison of the CDR-H and CDR-L sequences of the related antibodies is given in Figures 1 and 2, respectively. CDR positions that are strictly conserved among the antibodies and recognized as crucial for specificity have been identified, while other positions may tolerate substitutions.

[0410] Accordingly, the antibodies according to the invention comprise:

[0411] - a heavy chain variable domain comprising CDR1-H consisting of the sequence GFX1X2X3X4AW (SEQ ID NO: 331), where X1 is T or S, X2 is F or V, X3 is S or T and X4 is N, K, L or Y, or any combination thereof, and

[0412] CDR2-H consisting of the sequence IKX1X2X3NX4YX5T (SEQ ID NO: 332), where X1 is A or D, X2 is K or R, X3 is S or A, X4 is N or S and X5 is A or E, or any combination thereof; and

[0413] the sequence TWRHYYSSHTMDA (SEQ ID NO: 69) or RALTYYGYKRDAMDG (SEQ ID NO: 129) or RX1X2X3YX4X5X6X7X8X9X10 X 11 DX 12 (SEQ ID NO: 333)-composed CDR3-H, where X1 is Y, G or A, X2 is V, T or L, X3 is H, N, Y or Q, X4 is G, R or A, X5 is F or V or no amino acid, X6 is R or no amino acid, X7 is F, S or I or no amino acid, X8 is F, L, N, M, Y, S, A or G, X9 is Y, A, K, S, N, T, F or L, X 10 is A, P, G or T, X 11 is M, L, F or S and X 12 is A, V or Y, or any combination thereof, and

[0414] -light chain variable domain, which contains CDR1-L composed of the sequence QX1LX2HX3NGX4TY (SEQ ID NO: 334), where X1 is R or S, X2 is V or E, X3 is N, D or T and X4 is N or Y, or any combination thereof; and

[0415] CDR2-L composed of the sequence 'KVS'; and

[0416] CDR3-L composed of the sequence GQGX1X2YPFT (SEQ ID NO: 335), where X1 is T, A or S and X2 is H, E or Q, or any combination thereof.

[0417] According to the embodiment, the anti-CD3 antibody according to the present invention comprises the CDR sequences of the heavy chain and / or light chain of one of the 28 anti-CD3 antibodies listed above, namely "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".

[0418] Therefore, the present invention relates to an anti-CD3 antibody, which comprises:

[0419] a) A heavy chain variable domain that contains CDR1-H of sequence SEQ ID NO: 6 or a sequence that differs from SEQ ID NO: 6 by one amino acid substitution; CDR2-H of sequence SEQ ID NO: 7 or a sequence that differs from SEQ ID NO: 7 by one or more amino acid substitutions; CDR3-H of sequence SEQ ID NO: 8 or a sequence that differs from SEQ ID NO: 8 by one amino acid substitution; and

[0420] a light chain variable domain that contains CDR1-L of sequence SEQ ID NO: 10 or SEQ ID NO: 142 or a sequence that differs from SEQ ID NO: 10 or SEQ ID NO: 142 by one amino acid substitution; CDR2-L of the sequence 'KVS' or a sequence that differs from 'KVS' by one amino acid substitution and CDR3-L of sequence SEQ ID NO: 11 or a sequence that differs from SEQ ID NO: 11 by one amino acid substitution; or

[0421] b) A heavy chain variable domain that contains CDR1-H of sequence SEQ ID NO: 13 or a sequence that differs from SEQ ID NO: 13 by one amino acid substitution; CDR2-H of sequence SEQ ID NO: 14 or a sequence that differs from SEQ ID NO: 14 by one or more amino acid substitutions; CDR3-H of sequence SEQ ID NO: 15 or a sequence that differs from SEQ ID NO: 15 by one amino acid substitution; and

[0422] a light chain variable domain that contains CDR1-L of sequence SEQ ID NO: 17 or SEQ ID NO: 184 or a sequence that differs from SEQ ID NO: 17 or SEQ ID NO: 184 by one amino acid substitution; CDR2-L of the sequence 'KVS' or a sequence that differs from 'KVS' by one amino acid substitution and CDR3-L of sequence SEQ ID NO: 11 or a sequence that differs from SEQ ID NO: 11 by one amino acid substitution; or

[0423] c) A heavy chain variable domain that contains CDR1-H of sequence SEQ ID NO: 13 or a sequence that differs from SEQ ID NO: 13 by one amino acid substitution; CDR2-H of sequence SEQ ID NO: 19 or a sequence that differs from SEQ ID NO: 19 by one or more amino acid substitutions; CDR3-H of sequence SEQ ID NO: 20 or a sequence that differs from SEQ ID NO: 20 by one amino acid substitution; and

[0424] A light chain variable domain comprising CDR1-L having the sequence SEQ ID NO: 22 or a sequence differing from SEQ ID NO: 22 by one amino acid substitution; CDR2-L having the sequence 'KVS' or a sequence differing from 'KVS' by one amino acid substitution; and CDR3-L having the sequence SEQ ID NO: 11 or a sequence differing from SEQ ID NO: 11 by one amino acid substitution; or

[0425] d) A heavy chain variable domain comprising CDR1-H having the sequence SEQ ID NO: 24 or a sequence differing from SEQ ID NO: 24 by one amino acid substitution; CDR2-H having the sequence SEQ ID NO: 19 or a sequence differing from SEQ ID NO: 19 by one or more amino acid substitutions; CDR3-H having the sequence SEQ ID NO: 25 or a sequence differing from SEQ ID NO: 25 by one amino acid substitution; and

[0426] A light chain variable domain comprising CDR1-L having the sequence SEQ ID NO: 27 or a sequence differing from SEQ ID NO: 27 by one amino acid substitution; CDR2-L having the sequence 'KVS' or a sequence differing from 'KVS' by one amino acid substitution; and CDR3-L having the sequence SEQ ID NO: 28 or a sequence differing from SEQ ID NO: 28 by one amino acid substitution; or

[0427] e) A heavy chain variable domain comprising CDR1-H having the sequence SEQ ID NO: 30 or a sequence differing from SEQ ID NO: 30 by one amino acid substitution; CDR2-H having the sequence SEQ ID NO: 31 or a sequence differing from SEQ ID NO: 31 by one or more amino acid substitutions; CDR3-H having the sequence SEQ ID NO: 32 or a sequence differing from SEQ ID NO: 32 by one amino acid substitution; and

[0428] A light chain variable domain comprising CDR1-L having the sequence SEQ ID NO: 34 or a sequence differing from SEQ ID NO: 34 by one amino acid substitution; CDR2-L having the sequence 'RDD' or a sequence differing from 'RDD' by one amino acid substitution; and CDR3-L having the sequence SEQ ID NO: 35 or a sequence differing from SEQ ID NO: 35 by one amino acid substitution; or

[0429] f) A heavy chain variable domain comprising CDR1-H having the sequence SEQ ID NO: 13 or a sequence differing from SEQ ID NO: 13 by one amino acid substitution; CDR2-H having the sequence SEQ ID NO: 37 or a sequence differing from SEQ ID NO: 37 by one or more amino acid substitutions; CDR3-H having the sequence SEQ ID NO: 38 or a sequence differing from SEQ ID NO: 38 by one amino acid substitution; and

[0430] A light chain variable domain comprising CDR1-L having the sequence SEQ ID NO: 10 or a sequence differing from SEQ ID NO: 10 by one amino acid substitution; CDR2-L having the sequence 'KVS' or a sequence differing from 'KVS' by one amino acid substitution; and CDR3-L having the sequence SEQ ID NO: 28 or a sequence differing from SEQ ID NO: 28 by one amino acid substitution; or

[0431] g) A heavy chain variable domain comprising CDR1-H having the sequence SEQ ID NO: 13 or a sequence differing from SEQ ID NO: 13 by one amino acid substitution; CDR2-H having the sequence SEQ ID NO: 37 or a sequence differing from SEQ ID NO: 37 by one or more amino acid substitutions; CDR3-H having the sequence SEQ ID NO: 41 or a sequence differing from SEQ ID NO: 41 by one amino acid substitution; and

[0432] A light chain variable domain comprising CDR1-L having the sequence SEQ ID NO: 17 or a sequence differing from SEQ ID NO: 17 by one amino acid substitution; CDR2-L having the sequence 'KVS' or a sequence differing from 'KVS' by one amino acid substitution; and CDR3-L having the sequence SEQ ID NO: 11 or a sequence differing from SEQ ID NO: 11 by one amino acid substitution; or

[0433] h) A heavy chain variable domain comprising CDR1-H having the sequence SEQ ID NO: 13 or a sequence differing from SEQ ID NO: 13 by one amino acid substitution; CDR2-H having the sequence SEQ ID NO: 37 or a sequence differing from SEQ ID NO: 37 by one or more amino acid substitutions; CDR3-H having the sequence SEQ ID NO: 44 or a sequence differing from SEQ ID NO: 44 by one amino acid substitution; and

[0434] A light chain variable domain comprising a CDR1-L having the sequence SEQ ID NO:10 or a sequence differing from SEQ ID NO:10 by one amino acid substitution; a CDR2-L having the sequence 'KVS' or a sequence differing from 'KVS' by one amino acid substitution; and a CDR3-L having the sequence SEQ ID NO:11 or a sequence differing from SEQ ID NO:11 by one amino acid substitution; or

[0435] i) A heavy chain variable domain comprising a CDR1-H having the sequence SEQ ID NO:13 or a sequence differing from SEQ ID NO:13 by one amino acid substitution; a CDR2-H having the sequence SEQ ID NO:37 or a sequence differing from SEQ ID NO:37 by one or more amino acid substitutions; a CDR3-H having the sequence SEQ ID NO:47 or a sequence differing from SEQ ID NO:47 by one amino acid substitution; and

[0436] A light chain variable domain comprising a CDR1-L having the sequence SEQ ID NO:10 or a sequence differing from SEQ ID NO:10 by one amino acid substitution; a CDR2-L having the sequence 'KVS' or a sequence differing from 'KVS' by one amino acid substitution; and a CDR3-L having the sequence SEQ ID NO:11 or a sequence differing from SEQ ID NO:11 by one amino acid substitution; or

[0437] j) A heavy chain variable domain comprising a CDR1-H having the sequence SEQ ID NO:50 or a sequence differing from SEQ ID NO:50 by one amino acid substitution; a CDR2-H having the sequence SEQ ID NO:51 or a sequence differing from SEQ ID NO:51 by one or more amino acid substitutions; a CDR3-H having the sequence SEQ ID NO:52 or a sequence differing from SEQ ID NO:52 by one amino acid substitution; and

[0438] A light chain variable domain comprising a CDR1-L having the sequence SEQ ID NO:54 or a sequence differing from SEQ ID NO:54 by one amino acid substitution; a CDR2-L having the sequence 'NAN' or a sequence differing from 'NAN' by one amino acid substitution; and a CDR3-L having the sequence SEQ ID NO:55 or a sequence differing from SEQ ID NO:55 by one amino acid substitution; or

[0439] k) A heavy chain variable domain comprising CDR1-H having the sequence SEQ ID NO: 57 or a sequence differing from SEQ ID NO: 57 by one amino acid substitution; CDR2-H having the sequence SEQ ID NO: 37 or a sequence differing from SEQ ID NO: 37 by one or more amino acid substitutions; CDR3-H having the sequence SEQ ID NO: 58 or a sequence differing from SEQ ID NO: 58 by one amino acid substitution; and

[0440] a light chain variable domain comprising CDR1-L having the sequence SEQ ID NO: 10 or a sequence differing from SEQ ID NO: 10 by one amino acid substitution; CDR2-L having the sequence 'KVS' or a sequence differing from 'KVS' by one amino acid substitution; and CDR3-L having the sequence SEQ ID NO: 28 or a sequence differing from SEQ ID NO: 28 by one amino acid substitution; or

[0441] l) A heavy chain variable domain comprising CDR1-H having the sequence SEQ ID NO: 13 or a sequence differing from SEQ ID NO: 13 by one amino acid substitution; CDR2-H having the sequence SEQ ID NO: 37 or a sequence differing from SEQ ID NO: 37 by one or more amino acid substitutions; CDR3-H having the sequence SEQ ID NO: 61 or a sequence differing from SEQ ID NO: 61 by one amino acid substitution; and

[0442] a light chain variable domain comprising CDR1-L having the sequence SEQ ID NO: 10 or a sequence differing from SEQ ID NO: 10 by one amino acid substitution; CDR2-L having the sequence 'KVS' or a sequence differing from 'KVS' by one amino acid substitution; and CDR3-L having the sequence SEQ ID NO: 11 or a sequence differing from SEQ ID NO: 11 by one amino acid substitution; or

[0443] m) A heavy chain variable domain comprising CDR1-H having the sequence SEQ ID NO: 64 or a sequence differing from SEQ ID NO: 64 by one amino acid substitution; CDR2-H having the sequence SEQ ID NO: 65 or a sequence differing from SEQ ID NO: 65 by one or more amino acid substitutions; CDR3-H having the sequence SEQ ID NO: 47 or a sequence differing from SEQ ID NO: 47 by one amino acid substitution; and

[0444] A light chain variable domain comprising a CDR1-L having the sequence SEQ ID NO: 67 or a sequence differing from SEQ ID NO: 67 by one amino acid substitution; a CDR2-L having the sequence 'KVS' or a sequence differing from 'KVS' by one amino acid substitution; and a CDR3-L having the sequence SEQ ID NO: 28 or a sequence differing from SEQ ID NO: 28 by one amino acid substitution; or

[0445] n) A heavy chain variable domain comprising a CDR1-H having the sequence SEQ ID NO: 13 or a sequence differing from SEQ ID NO: 13 by one amino acid substitution; a CDR2-H having the sequence SEQ ID NO: 37 or a sequence differing from SEQ ID NO: 37 by one or more amino acid substitutions; a CDR3-H having the sequence SEQ ID NO: 69 or a sequence differing from SEQ ID NO: 69 by one amino acid substitution; and

[0446] A light chain variable domain comprising a CDR1-L having the sequence SEQ ID NO: 10 or a sequence differing from SEQ ID NO: 10 by one amino acid substitution; a CDR2-L having the sequence 'KVS' or a sequence differing from 'KVS' by one amino acid substitution; and a CDR3-L having the sequence SEQ ID NO: 71 or a sequence differing from SEQ ID NO: 71 by one amino acid substitution; or

[0447] o) A heavy chain variable domain comprising a CDR1-H having the sequence SEQ ID NO: 13 or a sequence differing from SEQ ID NO: 13 by one amino acid substitution; a CDR2-H having the sequence SEQ ID NO: 37 or a sequence differing from SEQ ID NO: 37 by one or more amino acid substitutions; a CDR3-H having the sequence SEQ ID NO: 84 or a sequence differing from SEQ ID NO: 84 by one amino acid substitution; and

[0448] A light chain variable domain comprising a CDR1-L having the sequence SEQ ID NO: 17 or a sequence differing from SEQ ID NO: 17 by one amino acid substitution; a CDR2-L having the sequence 'KVS' or a sequence differing from 'KVS' by one amino acid substitution; and a CDR3-L having the sequence SEQ ID NO: 11 or a sequence differing from SEQ ID NO: 11 by one amino acid substitution; or

[0449] p) A heavy chain variable domain comprising CDR1-H having the sequence SEQ ID NO: 75 or a sequence differing from SEQ ID NO: 75 by one amino acid substitution; CDR2-H having the sequence SEQ ID NO: 76 or a sequence differing from SEQ ID NO: 76 by one or more amino acid substitutions; CDR3-H having the sequence SEQ ID NO: 77 or a sequence differing from SEQ ID NO: 77 by one amino acid substitution; and

[0450] a light chain variable domain comprising CDR1-L having the sequence SEQ ID NO: 10 or a sequence differing from SEQ ID NO: 10 by one amino acid substitution; CDR2-L having the sequence 'KVS' or a sequence differing from 'KVS' by one amino acid substitution; and CDR3-L having the sequence SEQ ID NO: 11 or a sequence differing from SEQ ID NO: 11 by one amino acid substitution; or

[0451] q) A heavy chain variable domain comprising CDR1-H having the sequence SEQ ID NO: 80 or a sequence differing from SEQ ID NO: 80 by one amino acid substitution; CDR2-H having the sequence SEQ ID NO: 76 or a sequence differing from SEQ ID NO: 76 by one or more amino acid substitutions; CDR3-H having the sequence SEQ ID NO: 81 or a sequence differing from SEQ ID NO: 81 by one amino acid substitution; and

[0452] a light chain variable domain comprising CDR1-L having the sequence SEQ ID NO: 10 or a sequence differing from SEQ ID NO: 10 by one amino acid substitution; CDR2-L having the sequence 'KVS' or a sequence differing from 'KVS' by one amino acid substitution; and CDR3-L having the sequence SEQ ID NO: 11 or a sequence differing from SEQ ID NO: 11 by one amino acid substitution; or

[0453] r) A heavy chain variable domain comprising CDR1-H having the sequence SEQ ID NO: 13 or a sequence differing from SEQ ID NO: 13 by one amino acid substitution; CDR2-H having the sequence SEQ ID NO: 37 or a sequence differing from SEQ ID NO: 37 by one or more amino acid substitutions; CDR3-H having the sequence SEQ ID NO: 84 or a sequence differing from SEQ ID NO: 84 by one amino acid substitution; and

[0454] A light chain variable domain comprising a CDR1-L having the sequence SEQ ID NO: 10 or a sequence differing from SEQ ID NO: 10 by one amino acid substitution; a CDR2-L having the sequence 'KVS' or a sequence differing from 'KVS' by one amino acid substitution; and a CDR3-L having the sequence SEQ ID NO: 11 or a sequence differing from SEQ ID NO: 11 by one amino acid substitution; or

[0455] s) A heavy chain variable domain comprising a CDR1-H having the sequence SEQ ID NO: 13 or a sequence differing from SEQ ID NO: 13 by one amino acid substitution; a CDR2-H having the sequence SEQ ID NO: 37 or a sequence differing from SEQ ID NO: 37 by one or more amino acid substitutions; a CDR3-H having the sequence SEQ ID NO: 47 or a sequence differing from SEQ ID NO: 47 by one amino acid substitution; and

[0456] A light chain variable domain comprising a CDR1-L having the sequence SEQ ID NO: 10 or a sequence differing from SEQ ID NO: 10 by one amino acid substitution; a CDR2-L having the sequence 'KVS' or a sequence differing from 'KVS' by one amino acid substitution; and a CDR3-L having the sequence SEQ ID NO: 88 or a sequence differing from SEQ ID NO: 88 by one amino acid substitution; or

[0457] t) A heavy chain variable domain comprising a CDR1-H having the sequence SEQ ID NO: 90 or a sequence differing from SEQ ID NO: 90 by one amino acid substitution; a CDR2-H having the sequence SEQ ID NO: 91 or a sequence differing from SEQ ID NO: 91 by one or more amino acid substitutions; a CDR3-H having the sequence SEQ ID NO: 32 or a sequence differing from SEQ ID NO: 32 by one amino acid substitution, and

[0458] A light chain variable domain comprising a CDR1-L having the sequence SEQ ID NO: 93 or a sequence differing from SEQ ID NO: 93 by one amino acid substitution; a CDR2-L having the sequence 'GAS' or a sequence differing from 'GAS' by one amino acid substitution; and a CDR3-L having the sequence SEQ ID NO: 94 or a sequence differing from SEQ ID NO: 94 by one amino acid substitution; or

[0459] u) A heavy chain variable domain comprising a CDR1-H having the sequence SEQ ID NO: 96 or a sequence differing from SEQ ID NO: 96 by one amino acid substitution; a CDR2-H having the sequence SEQ ID NO: 97 or a sequence differing from SEQ ID NO: 97 by one or more amino acid substitutions; a CDR3-H having the sequence SEQ ID NO: 98 or a sequence differing from SEQ ID NO: 98 by one amino acid substitution; and

[0460] a light chain variable domain comprising a CDR1-L having the sequence SEQ ID NO: 100 or a sequence differing from SEQ ID NO: 100 by one amino acid substitution; a CDR2-L having the sequence 'NTN' or a sequence differing from 'NTN' by one amino acid substitution; and a CDR3-L having the sequence SEQ ID NO: 101 or a sequence differing from SEQ ID NO: 101 by one amino acid substitution; or

[0461] v) A heavy chain variable domain comprising a CDR1-H having the sequence SEQ ID NO: 103 or a sequence differing from SEQ ID NO: 103 by one amino acid substitution; a CDR2-H having the sequence SEQ ID NO: 104 or a sequence differing from SEQ ID NO: 104 by one or more amino acid substitutions; a CDR3-H having the sequence SEQ ID NO: 105 or a sequence differing from SEQ ID NO: 105 by one amino acid substitution; and

[0462] a light chain variable domain comprising a CDR1-L having the sequence SEQ ID NO: 10 or a sequence differing from SEQ ID NO: 10 by one amino acid substitution; a CDR2-L having the sequence 'KVS' or a sequence differing from 'KVS' by one amino acid substitution; and a CDR3-L having the sequence SEQ ID NO: 11 or a sequence differing from SEQ ID NO: 11 by one amino acid substitution; or

[0463] w) A heavy chain variable domain comprising a CDR1-H having the sequence SEQ ID NO: 80 or a sequence differing from SEQ ID NO: 80 by one amino acid substitution; a CDR2-H having the sequence SEQ ID NO: 19 or a sequence differing from SEQ ID NO: 19 by one or more amino acid substitutions; a CDR3-H having the sequence SEQ ID NO: 108 or a sequence differing from SEQ ID NO: 108 by one amino acid substitution; and

[0464] A light chain variable domain comprising a CDR1-L having the sequence SEQ ID NO: 10 or a sequence differing from SEQ ID NO: 10 by one amino acid substitution; a CDR2-L having the sequence 'KVS' or a sequence differing from 'KVS' by one amino acid substitution; and a CDR3-L having the sequence SEQ ID NO: 11 or a sequence differing from SEQ ID NO: 11 by one amino acid substitution; or

[0465] x) A heavy chain variable domain comprising a CDR1-H having the sequence SEQ ID NO: 13 or a sequence differing from SEQ ID NO: 13 by one amino acid substitution; a CDR2-H having the sequence SEQ ID NO: 37 or a sequence differing from SEQ ID NO: 37 by one or more amino acid substitutions; a CDR3-H having the sequence SEQ ID NO: 111 or a sequence differing from SEQ ID NO: 111 by one amino acid substitution; and

[0466] A light chain variable domain comprising a CDR1-L having the sequence SEQ ID NO: 113 or a sequence differing from SEQ ID NO: 113 by one amino acid substitution; a CDR2-L having the sequence 'KVS' or a sequence differing from 'KVS' by one amino acid substitution; and a CDR3-L having the sequence SEQ ID NO: 114 or a sequence differing from SEQ ID NO: 114 by one amino acid substitution; or

[0467] y) A heavy chain variable domain comprising a CDR1-H having the sequence SEQ ID NO: 116 or a sequence differing from SEQ ID NO: 116 by one amino acid substitution; a CDR2-H having the sequence SEQ ID NO: 117 or a sequence differing from SEQ ID NO: 117 by one or more amino acid substitutions; a CDR3-H having the sequence SEQ ID NO: 118 or a sequence differing from SEQ ID NO: 118 by one amino acid substitution; and

[0468] A light chain variable domain comprising a CDR1-L having the sequence SEQ ID NO: 100 or a sequence differing from SEQ ID NO: 100 by one amino acid substitution; a CDR2-L having the sequence 'VTN' or a sequence differing from 'VTN' by one amino acid substitution; and a CDR3-L having the sequence SEQ ID NO: 120 or a sequence differing from SEQ ID NO: 120 by one amino acid substitution; or

[0469] z) A heavy chain variable domain comprising a CDR1-H having the sequence SEQ ID NO: 122 or a sequence differing from SEQ ID NO: 122 by one amino acid substitution; a CDR2-H having the sequence SEQ ID NO: 123 or a sequence differing from SEQ ID NO: 123 by one or more amino acid substitutions; a CDR3-H having the sequence SEQ ID NO: 124 or a sequence differing from SEQ ID NO: 124 by one amino acid substitution; and

[0470] A light chain variable domain comprising a CDR1-L having the sequence SEQ ID NO: 126 or a sequence differing from SEQ ID NO: 126 by one amino acid substitution; a CDR2-L having the sequence 'RDD' or a sequence differing from 'RDD' by one amino acid substitution; and a CDR3-L having the sequence SEQ ID NO: 127 or a sequence differing from SEQ ID NO: 127 by one amino acid substitution; or

[0471] aa) A heavy chain variable domain comprising a CDR1-H having the sequence SEQ ID NO: 13 or a sequence differing from SEQ ID NO: 13 by one amino acid substitution; a CDR2-H having the sequence SEQ ID NO: 19 or a sequence differing from SEQ ID NO: 19 by one or more amino acid substitutions; a CDR3-H having the sequence SEQ ID NO: 129 or a sequence differing from SEQ ID NO: 129 by one amino acid substitution; and

[0472] A light chain variable domain comprising a CDR1-L having the sequence SEQ ID NO: 10 or a sequence differing from SEQ ID NO: 10 by one amino acid substitution; a CDR2-L having the sequence 'KVS' or a sequence differing from 'KVS' by one amino acid substitution; and a CDR3-L having the sequence SEQ ID NO: 11 or a sequence differing from SEQ ID NO: 11 by one amino acid substitution; or

[0473] bb) A heavy chain variable domain comprising a CDR1-H having the sequence SEQ ID NO: 103 or a sequence differing from SEQ ID NO: 103 by one amino acid substitution; a CDR2-H having the sequence SEQ ID NO: 104 or a sequence differing from SEQ ID NO: 104 by one or more amino acid substitutions; a CDR3-H having the sequence SEQ ID NO: 105 or a sequence differing from SEQ ID NO: 105 by one amino acid substitution; and

[0474] A light chain variable domain comprising CDR1-L having the sequence SEQ ID NO: 133 or a sequence differing from SEQ ID NO: 133 by one amino acid substitution; CDR2-L having the sequence 'LVS' or a sequence differing from 'LVS' by one amino acid substitution; and CDR3-L having the sequence SEQ ID NO: 134 or a sequence differing from SEQ ID NO: 134 by one amino acid substitution.

[0475] One or more individual amino acids in one or more of the above CDR sequences may be altered by substitution, particularly by conservative substitution. Such alterations are intended, for example, in the humanization of antibodies, to remove glycosylation sites or deamidation sites.

[0476] Based on sequence alignments 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", amino acid substitutions were identified. Thus, in one embodiment, amino acid substitutions are made at the following positions:

[0477] - at one or more of positions 3-6 in CDR1-H, such as at position 3, 5, or 6 of the sequence GFTFSNAW (SEQ ID NO: 13) CDR1-H or, for example, at positions 4 and 5 of the sequence GFTFSNAW (SEQ ID NO: 13) CDR1-H; and / or

[0478] - at one or more of positions 3-5, 7, and 9 in CDR2-H, such as at position 3, 4, 5, 7, or 9 of the sequence IKAKSNNYAT (SEQ ID NO: 37) CDR2-H or, for example, at positions 3 and 5 or 3 and 7 of the sequence IKAKSNNYAT (SEQ ID NO: 37) CDR2-H; and / or

[0479] - at one or more of positions 2-4, 7-10, and 12 in CDR3-H, such as positions 2, 3, 4, 6, 7, 8, 9, and 10 or positions 2, 4, 6, 8, 10, and 12 of the CDR3-H of the sequence RGVYYALSPFDY (SEQ ID NO: 8) or positions 2, 3, 7, and 8 of the CDR3-H of the sequence RGVYYALSPFDY (SEQ ID NO: 8) or positions 7 and 8 of the CDR3-H of the sequence RGLYYGLSPSDY (SEQ ID NO: 38) or positions 2, 3, 4, 6, 7, 8, 9, and 10 of the CDR3-H of the sequence RGLYYGLSPSDY (SEQ ID NO: 38); and / or

[0480] - at one or more of positions 2, 4, 6, and 9 in CDR1-L, such as position 6 of the CDR1-L of the sequence QSLVHDNGNTY (SEQ ID NO: 17) or QSLVHTNGNTY (SEQ ID NO: 27) or position 2 of the CDR1-L of the sequence QSLVHNNGNTY (SEQ ID NO: 10) or QRLVHNNGNTY (SEQ ID NO: 113) or positions 4, 6, and 9 of the CDR1-L of the sequence QSLVHNNGNTY (SEQ ID NO: 10); and / or

[0481] - at one or more of positions 4 and 5 in CDR3-L, such as position 4 of the CDR3-L of the sequence GQGSQYPFT (SEQ ID NO: 71) or GQGTQYPFT (SEQ ID NO: 11), or positions 4 and 5 of the CDR3-L of the sequence GQGAHYPFT (SEQ ID NO: 88) or position 5 of the CDR3-L of the sequence GQGTHYPFT (SEQ ID NO: 28) or GQGTEYPFT (SEQ ID NO: 114).

[0482] The anti-CD3 antibody according to the present invention is specifically a conventional antibody, particularly a conventional monoclonal antibody or antibody fragment, bispecific or multispecific antibody.

[0483] The anti-CD3 antibody according to the present invention specifically comprises IgG or a fragment thereof or consists of IgG or a fragment thereof.

[0484] According to another embodiment, the present invention relates to an anti-CD3 antibody, which comprises:

[0485] a) a heavy chain variable domain, which contains CDR1-H of sequence SEQ ID NO: 6, CDR2-H of sequence SEQ ID NO: 7, CDR3-H of sequence SEQ ID NO: 8, and

[0486] A light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 10 or SEQ ID NO: 142, CDR2-L of the sequence 'KVS', and CDR3-L of sequence SEQ ID NO: 11; or

[0487] b) A heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 13, CDR2-H of sequence SEQ ID NO: 14, CDR3-H of sequence SEQ ID NO: 15, and

[0488] A light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 17 or SEQ ID NO: 184, CDR2-L of the sequence 'KVS', and CDR3-L of sequence SEQ ID NO: 11; or

[0489] c) A heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 13, CDR2-H of sequence SEQ ID NO: 19, CDR3-H of sequence SEQ ID NO: 20, and

[0490] A light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 22, CDR2-L of the sequence 'KVS', and CDR3-L of sequence SEQ ID NO: 11; or

[0491] d) A heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 24, CDR2-H of sequence SEQ ID NO: 19, CDR3-H of sequence SEQ ID NO: 25, and

[0492] A light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 27, CDR2-L of the sequence 'KVS', and CDR3-L of sequence SEQ ID NO: 28; or

[0493] e) A heavy chain variable domain comprising CDR1-H of sequence SEQ ID NO: 30, CDR2-H of sequence SEQ ID NO: 31, CDR3-H of sequence SEQ ID NO: 32, and

[0494] A light chain variable domain comprising CDR1-L of sequence SEQ ID NO: 34, CDR2-L of the sequence 'RDD', and CDR3-L of sequence SEQ ID NO: 35; or

[0495] f) A heavy chain variable domain containing CDR1-H of SEQ ID NO: 13, CDR2-H of SEQ ID NO: 37, CDR3-H of SEQ ID NO: 38, and

[0496] A light chain variable domain containing CDR1-L of SEQ ID NO: 10, CDR2-L of the sequence 'KVS', and CDR3-L of SEQ ID NO: 28; or

[0497] g) A heavy chain variable domain containing CDR1-H of SEQ ID NO: 13, CDR2-H of SEQ ID NO: 37, CDR3-H of SEQ ID NO: 41, and

[0498] A light chain variable domain containing CDR1-L of SEQ ID NO: 17, CDR2-L of the sequence 'KVS', and CDR3-L of SEQ ID NO: 11; or

[0499] h) A heavy chain variable domain containing CDR1-H of SEQ ID NO: 13, CDR2-H of SEQ ID NO: 37, CDR3-H of SEQ ID NO: 44, and

[0500] A light chain variable domain containing CDR1-L of SEQ ID NO: 10, CDR2-L of the sequence 'KVS', and CDR3-L of SEQ ID NO: 11; or

[0501] i) A heavy chain variable domain containing CDR1-H of SEQ ID NO: 13, CDR2-H of SEQ ID NO: 37, CDR3-H of SEQ ID NO: 47, and

[0502] A light chain variable domain containing CDR1-L of SEQ ID NO: 10, CDR2-L of the sequence 'KVS', and CDR3-L of SEQ ID NO: 11; or

[0503] j) A heavy chain variable domain containing CDR1-H of SEQ ID NO: 50, CDR2-H of SEQ ID NO: 51, CDR3-H of SEQ ID NO: 52, and

[0504] A light chain variable domain containing CDR1-L of SEQ ID NO: 54, CDR2-L of the sequence 'NAN', and CDR3-L of SEQ ID NO: 55; or

[0505] k) A heavy chain variable domain comprising CDR1-H of SEQ ID NO: 57, CDR2-H of SEQ ID NO: 37, CDR3-H of SEQ ID NO: 58, and

[0506] a light chain variable domain comprising CDR1-L of SEQ ID NO: 10, CDR2-L of the sequence 'KVS', and CDR3-L of SEQ ID NO: 28; or

[0507] l) A heavy chain variable domain comprising CDR1-H of SEQ ID NO: 13, CDR2-H of SEQ ID NO: 37, CDR3-H of SEQ ID NO: 61, and

[0508] a light chain variable domain comprising CDR1-L of SEQ ID NO: 10, CDR2-L of the sequence 'KVS', and CDR3-L of SEQ ID NO: 11; or

[0509] m) A heavy chain variable domain comprising CDR1-H of SEQ ID NO: 64, CDR2-H of SEQ ID NO: 65, CDR3-H of SEQ ID NO: 47, and

[0510] a light chain variable domain comprising CDR1-L of SEQ ID NO: 67, CDR2-L of the sequence 'KVS', and CDR3-L of SEQ ID NO: 28; or

[0511] n) A heavy chain variable domain comprising CDR1-H of SEQ ID NO: 13, CDR2-H of SEQ ID NO: 37, CDR3-H of SEQ ID NO: 69, and

[0512] a light chain variable domain comprising CDR1-L of SEQ ID NO: 10, CDR2-L of the sequence 'KVS', and CDR3-L of SEQ ID NO: 71; or

[0513] o) A heavy chain variable domain comprising CDR1-H of SEQ ID NO: 13, CDR2-H of SEQ ID NO: 37, CDR3-H of SEQ ID NO: 84, and

[0514] A light chain variable domain containing CDR1-L of SEQ ID NO: 17, CDR2-L of the sequence 'KVS', and CDR3-L of SEQ ID NO: 11; or

[0515] p) A heavy chain variable domain containing CDR1-H of SEQ ID NO: 75, CDR2-H of SEQ ID NO: 76, CDR3-H of SEQ ID NO: 77, and

[0516] A light chain variable domain containing CDR1-L of SEQ ID NO: 10, CDR2-L of the sequence 'KVS', and CDR3-L of SEQ ID NO: 11; or

[0517] q) A heavy chain variable domain containing CDR1-H of SEQ ID NO: 80, CDR2-H of SEQ ID NO: 76, CDR3-H of SEQ ID NO: 81, and

[0518] A light chain variable domain containing CDR1-L of SEQ ID NO: 10, CDR2-L of the sequence 'KVS', and CDR3-L of SEQ ID NO: 11; or

[0519] r) A heavy chain variable domain containing CDR1-H of SEQ ID NO: 13, CDR2-H of SEQ ID NO: 37, CDR3-H of SEQ ID NO: 84, and

[0520] A light chain variable domain containing CDR1-L of SEQ ID NO: 10, CDR2-L of the sequence 'KVS', and CDR3-L of SEQ ID NO: 11; or

[0521] s) A heavy chain variable domain containing CDR1-H of SEQ ID NO: 13, CDR2-H of SEQ ID NO: 37, CDR3-H of SEQ ID NO: 47, and

[0522] A light chain variable domain containing CDR1-L of SEQ ID NO: 10, CDR2-L of the sequence 'KVS', and CDR3-L of SEQ ID NO: 88; or

[0523] t) A heavy chain variable domain containing CDR1-H of SEQ ID NO: 90, CDR2-H of SEQ ID NO: 91, CDR3-H of SEQ ID NO: 32, and

[0524] A light chain variable domain comprising CDR1-L of SEQ ID NO: 93, CDR2-L of the sequence 'GAS', and CDR3-L of SEQ ID NO: 94; or

[0525] u) A heavy chain variable domain comprising CDR1-H of SEQ ID NO: 96, CDR2-H of SEQ ID NO: 97, CDR3-H of SEQ ID NO: 98, and

[0526] A light chain variable domain comprising CDR1-L of SEQ ID NO: 100, CDR2-L of the sequence 'NTN', and CDR3-L of SEQ ID NO: 101; or

[0527] v) A heavy chain variable domain comprising CDR1-H of SEQ ID NO: 103, CDR2-H of SEQ ID NO: 104, CDR3-H of SEQ ID NO: 105, and

[0528] A light chain variable domain comprising CDR1-L of SEQ ID NO: 10, CDR2-L of the sequence 'KVS', and CDR3-L of SEQ ID NO: 11; or

[0529] w) A heavy chain variable domain comprising CDR1-H of SEQ ID NO: 80, CDR2-H of SEQ ID NO: 19, CDR3-H of SEQ ID NO: 108, and

[0530] A light chain variable domain comprising CDR1-L of SEQ ID NO: 10, CDR2-L of the sequence 'KVS', and CDR3-L of SEQ ID NO: 11; or

[0531] x) A heavy chain variable domain comprising CDR1-H of SEQ ID NO: 13, CDR2-H of SEQ ID NO: 37, CDR3-H of SEQ ID NO: 111, and

[0532] A light chain variable domain comprising CDR1-L of SEQ ID NO: 113, CDR2-L of the sequence 'KVS', and CDR3-L of SEQ ID NO: 114; or

[0533] y) A heavy chain variable domain that contains CDR1-H of SEQ ID NO: 116, CDR2-H of SEQ ID NO: 117, CDR3-H of SEQ ID NO: 118, and

[0534] A light chain variable domain that contains CDR1-L of SEQ ID NO: 100, CDR2-L of the sequence 'VTN', and CDR3-L of SEQ ID NO: 120; or

[0535] z) A heavy chain variable domain that contains CDR1-H of SEQ ID NO: 122, CDR2-H of SEQ ID NO: 123, CDR3-H of SEQ ID NO: 124, and

[0536] A light chain variable domain that contains CDR1-L of SEQ ID NO: 126, CDR2-L of the sequence 'RDD', and CDR3-L of SEQ ID NO: 127; or

[0537] aa) A heavy chain variable domain that contains CDR1-H of SEQ ID NO: 13, CDR2-H of SEQ ID NO: 19, CDR3-H of SEQ ID NO: 129, and

[0538] A light chain variable domain that contains CDR1-L of SEQ ID NO: 10, CDR2-L of the sequence 'KVS', and CDR3-L of SEQ ID NO: 11; or

[0539] bb) A heavy chain variable domain that contains CDR1-H of SEQ ID NO: 103, CDR2-H of SEQ ID NO: 104, CDR3-H of SEQ ID NO: 105; and

[0540] A light chain variable domain that contains CDR1-L of SEQ ID NO: 133, CDR2-L of the sequence 'LVS', and CDR3-L of SEQ ID NO: 134.

[0541] The present invention also provides anti-CD3 antibodies that comprise at least one of the heavy chain variable domains and / or light chain variable domains called anti-CD3 antibodies listed above.

[0542] Therefore, the present invention specifically relates to anti-CD3 antibodies that comprise:

[0543] a) The heavy chain variable domain of the sequence SEQ ID NO: 5 or a sequence that is at least 85% identical thereto, and / or the light chain variable domain of the sequence SEQ ID NO: 9 or a sequence that is at least 85% identical thereto; or

[0544] b) The heavy chain variable domain of the sequence SEQ ID NO: 12 or a sequence that is at least 85% identical thereto, and / or the light chain variable domain of the sequence SEQ ID NO: 16 or a sequence that is at least 85% identical thereto; or

[0545] c) The heavy chain variable domain of the sequence SEQ ID NO: 18 or a sequence that is at least 85% identical thereto, and / or the light chain variable domain of the sequence SEQ ID NO: 21 or a sequence that is at least 85% identical thereto; or

[0546] d) The heavy chain variable domain of the sequence SEQ ID NO: 23 or a sequence that is at least 85% identical thereto, and / or the light chain variable domain of the sequence SEQ ID NO: 26 or a sequence that is at least 85% identical thereto; or

[0547] e) The heavy chain variable domain of the sequence SEQ ID NO: 29 or a sequence that is at least 85% identical thereto, and / or the light chain variable domain of the sequence SEQ ID NO: 33 or a sequence that is at least 85% identical thereto; or

[0548] f) The heavy chain variable domain of the sequence SEQ ID NO: 36 or a sequence that is at least 85% identical thereto, and / or the light chain variable domain of the sequence SEQ ID NO: 39 or a sequence that is at least 85% identical thereto; or

[0549] g) The heavy chain variable domain of the sequence SEQ ID NO: 40 or a sequence that is at least 85% identical thereto, and / or the light chain variable domain of the sequence SEQ ID NO: 42 or a sequence that is at least 85% identical thereto; or

[0550] h) The heavy chain variable domain of the sequence SEQ ID NO: 43 or a sequence that is at least 85% identical thereto, and / or the light chain variable domain of the sequence SEQ ID NO: 45 or a sequence that is at least 85% identical thereto; or

[0551] i) The heavy chain variable domain of the sequence SEQ ID NO: 46 or a sequence that is at least 85% identical thereto, and / or the light chain variable domain of the sequence SEQ ID NO: 48 or a sequence that is at least 85% identical thereto; or

[0552] j) The heavy chain variable domain of the sequence SEQ ID NO: 49 or a sequence that is at least 85% identical thereto, and / or the light chain variable domain of the sequence SEQ ID NO: 53 or a sequence that is at least 85% identical thereto; or

[0553] k) The heavy chain variable domain of the sequence SEQ ID NO: 56 or a sequence that is at least 85% identical thereto, and / or the light chain variable domain of the sequence SEQ ID NO: 59 or a sequence that is at least 85% identical thereto; or

[0554] l) The heavy chain variable domain of the sequence SEQ ID NO: 60 or a sequence that is at least 85% identical thereto, and / or the light chain variable domain of the sequence SEQ ID NO: 62 or a sequence that is at least 85% identical thereto; or

[0555] m) The heavy chain variable domain of the sequence SEQ ID NO: 63 or a sequence that is at least 85% identical thereto, and / or the light chain variable domain of the sequence SEQ ID NO: 66 or a sequence that is at least 85% identical thereto; or

[0556] n) The heavy chain variable domain of the sequence SEQ ID NO: 68 or a sequence that is at least 85% identical thereto, and / or the light chain variable domain of the sequence SEQ ID NO: 70 or a sequence that is at least 85% identical thereto; or

[0557] o) The heavy chain variable domain of the sequence SEQ ID NO: 72 or a sequence that is at least 85% identical thereto, and / or the light chain variable domain of the sequence SEQ ID NO: 73 or a sequence that is at least 85% identical thereto; or

[0558] p) The heavy chain variable domain of the sequence SEQ ID NO: 74 or a sequence that is at least 85% identical thereto, and / or the light chain variable domain of the sequence SEQ ID NO: 78 or a sequence that is at least 85% identical thereto; or

[0559] q) The heavy chain variable domain of the sequence SEQ ID NO: 79 or a sequence that is at least 85% identical thereto, and / or the light chain variable domain of the sequence SEQ ID NO: 82 or a sequence that is at least 85% identical thereto; or

[0560] r) Containing the heavy chain variable domain of the sequence SEQ ID NO: 83 or a sequence that is at least 85% identical thereto, and / or the light chain variable domain of the sequence SEQ ID NO: 85 or a sequence that is at least 85% identical thereto; or

[0561] s) the heavy chain variable domain of the sequence SEQ ID NO: 46 or a sequence that is at least 85% identical thereto, and / or the light chain variable domain of the sequence SEQ ID NO: 87 or a sequence that is at least 85% identical thereto; or

[0562] t) the heavy chain variable domain of the sequence SEQ ID NO: 89 or a sequence that is at least 85% identical thereto, and / or the light chain variable domain of the sequence SEQ ID NO: 92 or a sequence that is at least 85% identical thereto; or

[0563] u) the heavy chain variable domain of the sequence SEQ ID NO: 95 or a sequence that is at least 85% identical thereto, and / or the light chain variable domain of the sequence SEQ ID NO: 99 or a sequence that is at least 85% identical thereto; or

[0564] v) the heavy chain variable domain of the sequence SEQ ID NO: 102 or a sequence that is at least 85% identical thereto, and / or the light chain variable domain of the sequence SEQ ID NO: 106 or a sequence that is at least 85% identical thereto; or

[0565] w) the heavy chain variable domain of the sequence SEQ ID NO: 107 or a sequence that is at least 85% identical thereto, and / or the light chain variable domain of the sequence SEQ ID NO: 109 or a sequence that is at least 85% identical thereto; or

[0566] x) the heavy chain variable domain of the sequence SEQ ID NO: 110 or a sequence that is at least 85% identical thereto, and / or the light chain variable domain of the sequence SEQ ID NO: 112 or a sequence that is at least 85% identical thereto; or

[0567] y) the heavy chain variable domain of the sequence SEQ ID NO: 115 or a sequence that is at least 85% identical thereto, and / or the light chain variable domain of the sequence SEQ ID NO: 119 or a sequence that is at least 85% identical thereto; or

[0568] z) the heavy chain variable domain of the sequence SEQ ID NO: 121 or a sequence that is at least 85% identical thereto, and / or the light chain variable domain of the sequence SEQ ID NO: 125 or a sequence that is at least 85% identical thereto; or

[0569] aa) the heavy chain variable domain of the sequence SEQ ID NO: 128 or a sequence that is at least 85% identical thereto, and / or the light chain variable domain of the sequence SEQ ID NO: 130 or a sequence that is at least 85% identical thereto; or

[0570] bb) The heavy chain variable domain of the sequence SEQ ID NO: 131 or a sequence that is at least 85% identical thereto, and / or the light chain variable domain of the sequence SEQ ID NO: 132 or a sequence that is at least 85% identical thereto.

[0571] For example, the sequence of the heavy or light chain variable domain may optionally differ from the reference sequences 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, particularly one or more conservative amino acid substitutions and / or substitutions with canonical residues. Specifically, the sequence of the heavy or light chain variable domain differs from the reference sequences 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.

[0572] The sequence changes compared to the sequences SEQ ID NO: 5, 9, 12, 16, 18, 21, 23, 26, 29, 33, 36, 39, 40, 42, 43, 45, 46, 48, 49, 53, 56, 59, 60, 62, 63, 66, 68, 70, 72, 73, 74, 78, 79, 82, 83, 85, 87, 89, 92, 95, 99, 102, 106, 107, 109, 110, 112, 115, 119, 121, 125, 128, 130, 131, 132 will specifically be substantially in one or more framework regions FR1-L, FR2-L, FR3-L, FR4-L and / or FR1-H, FR2-H, FR3-H, FR4-H.

[0573] However, amino acid substitutions in one or more CDRs are also possible. Specifically, the sequence of the light chain variable domain may differ from the sequence SEQ ID NO: 9 only by substitution of S to R at position 28 in SEQ ID NO: 9 (in CDR1-L), and / or at least by substitution of V to E at position 30 in SEQ ID NO: 9 (in CDR1-L), and / or at least by substitution of N to D or T at position 33 in SEQ ID NO: 9 (in CDR1-L), and / or at least by substitution of N to Y at position 35 in SEQ ID NO: 9 (in CDR1-L), and / or the light chain variable domain sequence may differ from the sequence SEQ ID NO: 9 at least by substitution of T to S or A at position 97 in SEQ ID NO: 9 (in CDR3-L), and / or at least by substitution of Q to H or E at position 98 in SEQ ID NO: 9 (in CDR3-L), and / or the heavy chain variable domain may differ from the sequence SEQ ID NO: 9 at least by substitution of T to N or S at position 28 in SEQ ID NO: 5 (in CDR1-H), and / or at least by substitution of F to V at position 29 in SEQ ID NO: 5 (in CDR1-H), or at least by substitution of T to N or Y at position 30 in SEQ ID NO: 5 (in CDR1-H), or at least by substitution of K to L or Y at position 31 in SEQ ID NO: 5 (in CDR1-H), and / or the heavy chain variable domain may differ from the sequence SEQ ID NO: 5 at least by substitution of D to A at position 53 in SEQ ID NO: 5 (in CDR2-H), or at least by substitution of K to R at position 54 in SEQ ID NO: 5 (in CDR2-H), or at least by substitution of S to A at position 55 in SEQ ID NO: 5 (in CDR2-H), or at least by substitution of S to N at position 57 in SEQ ID NO: 5 (in CDR2-H), or at least by substitution of A to E at position 59 in SEQ ID NO: 5 (in CDR2-H), and / or the heavy chain variable domain may differ from the sequence SEQ ID NO: 5 at least by substitution of G to A at position 100 in SEQ ID NO: 5 (in CDR3-H), and / or at least by substitution of T to V at position 101 in SEQ ID NO: 5 (in CDR3-H), at least by substitution of Q to Y at position 102 in SEQ ID NO: 5 (in CDR3-H).

[0574] In one embodiment, the anti-CD3 antibodies and fragments thereof of the present invention are rat antibodies and rat antibody fragments, respectively.

[0575] In one aspect of the present invention, the anti-CD3 antibody of the present invention may also be a chimeric antibody and specifically a rat / human antibody, such as an antibody comprising a rat heavy chain and light chain variable domains and human CH and CL domains.

[0576] In another aspect of the present invention, the anti-CD3 antibody may also be a humanized antibody or a humanized antibody fragment obtained, for example, by CDR-grafting or by the 4D method (US20110027266).

[0577] Accordingly, in one embodiment, the anti-CD3 antibody of the present invention is a humanized antibody comprising:

[0578] a) a heavy chain variable domain containing 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

[0579] a light chain variable domain containing 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

[0580] b) a heavy chain variable domain containing 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

[0581] 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.

[0582] According to another embodiment, the anti-CD3 antibody of the invention is a humanized antibody comprising

[0583] a) a heavy chain variable domain of sequence SEQ ID NO: 138 and / or a light chain variable domain of sequence SEQ ID NO: 143; or

[0584] b) a heavy chain variable domain of sequence SEQ ID NO: 171 and / or a light chain variable domain of sequence SEQ ID NO: 158; or

[0585] c) a heavy chain variable domain of sequence SEQ ID NO: 176 and / or a light chain variable domain of sequence SEQ ID NO: 164.

[0586] In one embodiment, the anti-CD3 antibody according to the invention comprises the three CDR sequences or variable domains referred to above as the anti-CD3 antibody heavy chain, or the six CDR sequences or variable domains of the heavy and light chains.

[0587] The invention also refers to the humanized anti-CD3 antibody fragments defined above. In one embodiment, the humanized anti-CD3 antibody described above is a chimeric antibody.

[0588] The anti-CD3 antibody according to the invention may also be a single domain antibody or a fragment thereof. Specifically, 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 described above. 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.

[0589] The single domain antibody or a fragment thereof may also comprise a framework region of a camel single domain antibody and optionally a constant domain of a camel single domain antibody.

[0590] The anti-CD3 antibody according to the present 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 diabody, especially a humanized antibody fragment.

[0591] Correspondingly, the anti-CD3 antibody of the present invention is a Fab, which comprises or consists of:

[0592] a) a heavy chain amino acid sequence SEQ ID NO: 186 and / or a light chain amino acid sequence SEQ ID NO: 187; or

[0593] b) a heavy chain amino acid sequence SEQ ID NO: 188 and / or a light chain amino acid sequence SEQ ID NO: 189; or

[0594] c) a heavy chain amino acid sequence SEQ ID NO: 190 and / or a light chain amino acid sequence SEQ ID NO: 191; or

[0595] d) a heavy chain amino acid sequence SEQ ID NO: 192 and / or a light chain amino acid sequence SEQ ID NO: 193.

[0596] In one embodiment, the CD3-antibody is a bispecific or multispecific antibody formed by at least one antibody fragment or at least one variable domain of the anti-CD3 antibody of the present invention. The multispecific antibody is a multivalent protein complex as described, for example, in EP 2 050 764 A1 or US 2005 / 0003403 A1.

[0597] The bispecific or multispecific CD3-antibody according to the present invention may be specific for (a) the human and human and cynomolgus monkey CD3 extracellular domains targeted by one of the above anti-CD3 antibodies and (b) at least one other antigen.

[0598] In one embodiment, the other antigen is CD123 and the correspondingly obtained bispecific antibody is a CD3 / CD123 bispecific antibody. Conventional bispecific antibodies can be produced by techniques known in the art.

[0599] The antibodies and anti-CD3 antibody fragments according to the present invention can be used in isolated (e.g., purified) form or incorporated into a carrier, such as a membrane or a lipid mediator (e.g., liposome).

[0600] In another embodiment, the anti-CD3 antibody of the present invention is used to prepare the antibody-like binding protein further defined in the "antibody-like binding protein" section of the present invention.

[0601] Any combination of the above embodiments constitutes a part of the present invention.

[0602] Anti-CD123 antibody

[0603] “ CD123 ” (cluster of differentiation 123), also known as “ interleukin 3 receptor, alpha (IL3RA) ” or “ IL3R ” or “ IL3RX ” or “ IL3RY ” or “ IL3RAY ” or “ hIL-3Ra ” and refers to the interleukin-3 specific subunit of the heterodimeric cytokine receptor. The functional interleukin-3 receptor is a heterodimer that includes a specific α chain (IL-3A; CD123) and an IL-3 receptor β chain (β0; CD131) shared with the receptors for granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin-5 (IL-5). CD123 is a type I integral transmembrane protein with a deduced molecular weight of approximately 43 kDa, which includes an extracellular domain involved in IL-3 binding, a transmembrane domain, and a short cytoplasmic tail of approximately 50 amino acids. The extracellular domain consists of two regions: an N-terminal region of approximately 100 amino acids, the sequence of which shows similarity to the equivalent regions of the GM-CSF and IL-5 receptor α-chains; and a proximal region of the transmembrane domain common to other members of this cytokine receptor family that contains four conserved cysteine residues and a WSXWS motif. The IL-3 binding domain contains a cytokine receptor motif (CRM) of approximately 200 amino acid residues consisting of two Ig-like fold domains. The extracellular domain of CD123 is highly glycosylated, with N-glycosylation required for both ligand binding and receptor signaling. The protein family consists of three members: IL3RA (CD123A), CSF2RA, and IL5RA. The overall structure is very conserved among the three members but the sequence homology is very low. A 300-amino acid-long isoform of CD123 has been discovered, but only at the RNA level, and it is available under the GenBank accession number ACM24116.1.

[0604] U.S. Patent No. 6,177,078 discloses the ability of the anti-IL-3 receptor α-chain (IL-3Rα, CD123) monoclonal antibodies 7G3 and 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 that by contacting cells with a composition of an antibody and a cytotoxic agent (selected from chemotherapeutic agents, toxins, or α-emitting radioisotopes), wherein the composition selectively binds to CD123 in an amount effective to cause cell death, to damage hematopoietic cancer precursor cells that express CD123 but do not significantly express CD131. However, it is unclear whether targeting CD123 functionally impairs AML-LSC.

[0605] The reference sequences of full-length human CD123 protein including the signal peptide are available from the NCBI database under accession number NP_002174.1 and under Uniprot accession number P26951, and are disclosed herein under SEQ ID NO: 194 (available on December 14, 2014). The reference sequences of full-length cynomolgus monkey CD123 protein including the signal peptide are available from the GenBank database under accession number EHH61867.1 and Uniprot accession number G8F3K3, and are disclosed herein under SEQ ID NO: 195 (available on December 14, 2014).

[0606] The mature human CD123 Strep-II tag Fc-fusion protein sequence cloned by the inventors from genomic DNA is disclosed under SEQ ID NO: 196. The mature human CD123 Fc-fusion protein comprises amino acids 19 - 305 of the full-length human CD123 protein and thus comprises the extracellular domain of human CD123.

[0607] The sequence of the mature cynomolgus monkey CD123 Strep-II tag Fc-fusion protein cloned by the inventors from genomic DNA is disclosed under SEQ ID NO: 197. The mature cynomolgus monkey CD123 Fc-fusion protein comprises amino acids 19 - 305 of the full-length cynomolgus monkey CD123 protein and thus comprises the extracellular domain of cynomolgus monkey CD123.

[0608] The domain organization of human and cynomolgus monkey CD123 is as follows (the human CD123 sequence can be obtained based on the human sequence available in the NCBI database under accession number NP_002174.1 (SEQ ID NO: 194), and the cynomolgus monkey CD123 sequence available under database accession number G8F3K3, SEQ ID NO: 195)):

[0609] human CD123 domain Position on SEQ ID NO: 194 (human) Position on SEQ ID NO: 195 (cynomolgus monkey) extracellular 19-305 19-305 transmembrane domain 306-325 306-325 cytoplasm 326-378 326-378

[0610] Accordingly, the extracellular domain of human CD123 consists of the amino acids at positions 19 - 305 of SEQ ID NO: 194.

[0611] CD123 (interleukin-3 receptor alpha chain IL-3Rα) is a tumor antigen overexpressed in a variety of hematological malignancies. A large number of AML blasts mainly express surface CD123 and this expression does not vary with AML subtypes. Higher expression of CD123 on AML at diagnosis has been reported to be associated with a poor prognosis. CD123 expression has been reported in other hematological malignancies including myelodysplasia, systemic mastocytosis, acute plasmacytoid dendritic cell neoplasm (BPDCN), ALL, and hairy cell leukemia.

[0612] CD123 is expressed on AML leukemia stem cells and increasing evidence suggests that AML arises from these LSCs, which show quiescence and relative resistance to DNA-damaging chemotherapy. It is hypothesized that the persistence of LSCs underlies relapse after initial remission, and thus eradication of LSCs is considered necessary for cure and is an important therapeutic goal.

[0613] “ leukemia stem cell (LSC) ” are cancer cells with properties associated with normal stem cells (i.e., the property of self-renewal and the ability to develop into multiple lineages). It has been proposed that such cells persist in blood cancers such as AML as a distinct population. The LCSs present in AML are also referred to as “AML-LCSs”.

[0614] “ acute myeloid leukemia (AML) ” is a clonal disorder that clinically presents due to an increase in the proliferation of heterogeneous and undifferentiated myeloid blasts. The leukemia hierarchy is maintained by a small population of LSCs (AML-LCSs) with special self-renewal capabilities and is capable of differentiating into leukemic blasts. These blasts generate large numbers of leukemic blasts that are readily detectable in patients at diagnosis and relapse, ultimately leading to death. AML-LSCs are generally reported as quiescent cells, distinct from rapidly dividing clonogenic progenitors. The properties of AML-LSCs render conventional chemotherapeutic agents that target proliferating cells less effective, potentially explaining the high proportion of AML patients who enter complete remission but almost always relapse, with <30% adult survival rates beyond 4 years. In addition, the occurrence of minimal residual disease at diagnosis and poor survival rates have been attributed to the high LSC frequency in AML patients. Therefore, there is an urgent need to develop new therapies for the long-term management of AML (and similar aforementioned blood cancer conditions) that specifically eliminate LSCs.

[0615] CD123 has been reported to be overexpressed on AML blasts and CD34 + / CD38 AML-LSCs relative to normal blood cells.

[0616] CD123 thus provides an important therapeutic target for cancer treatment, particularly for the treatment of cancers in patients with poor prognoses.

[0617] The inventors have successfully generated, screened, and selected specific murine and rat anti-CD123 antibodies that show high affinity for both human and cynomolgus monkey CD123 proteins and that do not significantly cross-react with human CSF2RA and IL5RA proteins and cynomolgus monkey CD3 protein.

[0618] The inventors have determined the sequences of the monoclonal antibody variable heavy and light chains of anti-CD123 antibodies designated "3E3-D3", "1E1-G5", "2B8-F3", "2F8-D6", "3B10-E6", "5A5-B4", "6B10-F4", "6C10-C4", "6D6-B8", "8B11-B7", "9B8-G6", "9D7-C8", and "9F6-G3".

[0619] The anti-CD123 antibody designated "3E3-D3" comprises:

[0620] - A variable heavy domain, which consists of the following sequence: (SEQ ID NO: 226, where the CDRs are shown in bold), containing CDR1-H of sequence SEQ ID NO: 227, CDR2-H of sequence SEQ ID NO: 228, and CDR3-H of sequence SEQ ID NO: 229, or

[0621] A variable heavy domain, which consists of the following sequence: (SEQ ID NO: 277, where the CDRs are shown in bold), containing CDR1-H of sequence SEQ ID NO: 227, CDR2-H of sequence SEQ ID NO: 353, and CDR3-H of sequence SEQ ID NO: 229, or

[0622] A variable heavy domain, which consists of the following sequence: (SEQ ID NO: 278, where the CDRs are shown in bold), containing CDR1-H of sequence SEQ ID NO: 227, CDR2-H of sequence SEQ ID NO: 279, and CDR3-H of sequence SEQ ID NO: 229, and a variable light domain which consists of the following sequence: (SEQ ID NO: 230, where the CDRs are shown in bold), containing CDR1-L of sequence SEQ ID NO: 231, CDR2-L consisting of the sequence 'RDD', and CDR3-L of sequence SEQ ID NO: 232.

[0623] The anti-CD123 antibody designated "1E1-G5" comprises:

[0624] - A variable heavy domain, which consists of the following sequence: (SEQ ID NO: 198, where the CDRs are in bold), containing CDR1-H of SEQ ID NO: 199, CDR2-H of SEQ ID NO: 200, and CDR3-H of SEQ ID NO: 201 and

[0625] - a light chain variable domain which consists of the following sequences: (SEQ ID NO: 202, where the CDRs are in bold), containing CDR1-L of SEQ ID NO: 203, CDR2-L consisting of the sequence 'DAN', and CDR3-L of SEQ ID NO: 204.

[0626] The anti-CD123 antibody designated "2B8-F3" comprises:

[0627] - a heavy chain variable domain which consists of the following sequences: (SEQ ID NO: 205, where the CDRs are in bold), containing CDR1-H of SEQ ID NO: 206, CDR2-H of SEQ ID NO: 207, and CDR3-H of SEQ ID NO: 208 and

[0628] - a light chain variable domain which consists of the following sequences: (SEQ ID NO: 209, where the CDRs are in bold), containing CDR1-L of SEQ ID NO: 210, CDR2-L consisting of the sequence 'ETS', and CDR3-L of SEQ ID NO: 211.

[0629] The anti-CD123 antibody designated "2F8-D6" comprises:

[0630] - a heavy chain variable domain which consists of the following sequences: (SEQ ID NO: 212, where the CDRs are in bold), containing CDR1-H of SEQ ID NO: 213, CDR2-H of SEQ ID NO: 214, and CDR3-H of SEQ ID NO: 215 and

[0631] - a light chain variable domain which consists of the following sequences: (SEQ ID NO: 216, where the CDRs are shown in bold), comprising CDR1-L of sequence SEQ ID NO: 217, CDR2-L consisting of the sequence 'NTN', and CDR3-L of sequence SEQ ID NO: 218.

[0632] The anti-CD123 antibody designated "3B10-E6" comprises:

[0633] - A heavy chain variable domain, which consists of the following sequences: (SEQ ID NO: 219, where the CDRs are shown in bold), comprising CDR1-H of sequence SEQ ID NO: 220, CDR2-H of sequence SEQ ID NO: 221, and CDR3-H of sequence SEQ ID NO: 222 and

[0634] - A light chain variable domain which consists of the following sequences: (SEQ ID NO: 223, where the CDRs are shown in bold), which comprises CDR1-L of sequence SEQ ID NO: 224, CDR2-L consisting of the sequence 'RVS', and CDR3-L of sequence SEQ ID NO: 225.

[0635] The anti-CD123 antibody designated "5A5-B4" comprises:

[0636] - A heavy chain variable domain, which consists of the following sequences: (SEQ ID NO: 233, where the CDRs are shown in bold), comprising CDR1-H of sequence SEQ ID NO: 234, CDR2-H of sequence SEQ ID NO: 235, and CDR3-H of sequence SEQ ID NO: 236 and

[0637] - A light chain variable domain which consists of the following sequences: (SEQ ID NO: 237, where the CDRs are shown in bold), comprising CDR1-L of sequence SEQ ID NO: 238, CDR2-L consisting of the sequence 'GAS', and CDR3-L of sequence SEQ ID NO: 239.

[0638] The anti-CD123 antibody designated "6B10-E4" comprises:

[0639] - A heavy chain variable domain, which consists of the following sequences: (SEQ ID NO: 240, where the CDRs are shown in bold), containing CDR1-H of SEQ ID NO: 241, CDR2-H of SEQ ID NO: 242, and CDR3-H of SEQ ID NO: 243 and

[0640] - A light chain variable domain consisting of the following sequences: (SEQ ID NO: 244, where the CDRs are shown in bold), containing CDR1-L of SEQ ID NO: 245, CDR2-L consisting of the sequence 'YAS', and CDR3-L of SEQ ID NO: 246.

[0641] The anti-CD123 antibody designated "6C10-C4" comprises:

[0642] - A heavy chain variable domain consisting of the following sequences: (SEQ ID NO: 247, where the CDRs are shown in bold), containing CDR1-H of SEQ ID NO: 248, CDR2-H of SEQ ID NO: 249, and CDR3-H of SEQ ID NO: 250 and

[0643] - A light chain variable domain consisting of the following sequences: (SEQ ID NO: 251, where the CDRs are shown in bold), containing CDR1-L of SEQ ID NO: 252, CDR2-L consisting of the sequence 'WAS', and CDR3-L of SEQ ID NO: 253.

[0644] The anti-CD123 antibody designated "6D6-B8" comprises:

[0645] - A heavy chain variable domain consisting of the following sequences: (SEQ ID NO: 254, where the CDRs are shown in bold), containing CDR1-H of SEQ ID NO: 255, CDR2-H of SEQ ID NO: 200, and CDR3-H of SEQ ID NO: 201 and

[0646] - A light chain variable domain consisting of the following sequences: (SEQ ID NO: 256, where the CDRs are in bold), containing CDR1-L of SEQ ID NO: 257, CDR2-L consisting of the sequence 'DAN', and CDR3-L of SEQ ID NO: 258.

[0647] The anti-CD123 antibody designated "8B11-B7" comprises:

[0648] - A heavy chain variable domain, which consists of the following sequences: (SEQ ID NO: 259, where the CDRs are in bold), containing CDR1-H of SEQ ID NO: 199, CDR2-H of SEQ ID NO: 260, and CDR3-H of SEQ ID NO: 201 and

[0649] - A light chain variable domain which consists of the following sequences: (SEQ ID NO: 261, where the CDRs are in bold), containing CDR1-L of SEQ ID NO: 262, CDR2-L consisting of the sequence 'DAS', and CDR3-L of SEQ ID NO: 263.

[0650] The anti-CD123 antibody designated "9B8-G6" comprises:

[0651] - A heavy chain variable domain, which consists of the following sequences: (SEQ ID NO: 264, where the CDRs are in bold), containing CDR1-H of SEQ ID NO: 265, CDR2-H of SEQ ID NO: 266, and CDR3-H of SEQ ID NO: 267 and

[0652] - A light chain variable domain which consists of the following sequences: (SEQ ID NO: 251, where the CDRs are in bold), containing CDR1-L of SEQ ID NO: 252, CDR2-L consisting of the sequence 'WAS', and CDR3-L of SEQ ID NO: 253.

[0653] The anti-CD123 antibody designated "9D7-C8" comprises:

[0654] - A heavy chain variable domain, which consists of the following sequences: (SEQ ID NO: 268, where the CDRs are in bold), containing CDR1-H of SEQ ID NO: 265, CDR2-H of SEQ ID NO: 266, and CDR3-H of SEQ ID NO: 269 and

[0655] - a light chain variable domain consisting of the following sequences: (SEQ ID NO: 270, where the CDRs are in bold), containing CDR1-L of SEQ ID NO: 271, CDR2-L consisting of the sequence 'WAS', and CDR3-L of SEQ ID NO: 253.

[0656] The anti-CD123 antibody designated "9F6-G3" comprises:

[0657] - a heavy chain variable domain consisting of the following sequences: (SEQ ID NO: 272, where the CDRs are in bold), containing CDR1-H of SEQ ID NO: 273, CDR2-H of SEQ ID NO: 274, and CDR3-H of SEQ ID NO: 201 and

[0658] - a light chain variable domain consisting of the following sequences: (SEQ ID NO: 275, where the CDRs are in bold), containing CDR1-L of SEQ ID NO: 276, CDR2-L consisting of the sequence 'DAS', and CDR3-L of SEQ ID NO: 258.

[0659] In one aspect of the invention, the anti-CD123 antibody binds to human CD123. In another embodiment, the anti-CD123 antibody further binds to cynomolgus monkey CD123. Specifically, the anti-CD123 antibody of the invention binds to the extracellular domain of human CD123 or both human and cynomolgus monkey CD123. More specifically, the anti-CD123 antibody binds to the distal portion of CD123, for example, binds to the amino acids at positions 19-49 of human CD123 of amino acid sequence SEQ ID NO: 194. The anti-CD123 antibody binds to CD123 whether expressed in isolated form or present in the soluble extracellular domain or full-length membrane-anchored CD123 in CD123-expressing cells such as AML cells or CD123-transfected cells. The anti-CD123 antibody according to the invention is specific for cells expressing human and cynomolgus monkey CD123 proteins on their surface, such as cancer cells expressing CD123.

[0660] The ratio of the affinity of the anti-CD123 antibody according to the present invention for cynomolgus monkey CD123 to the affinity for human CD123 (KD (cynomolgus monkey) / KD (human)) ≤ 10, specifically ≤ 6, ≤ 5, ≤ 4, ≤ 3, for example ≤ 2, ≤ 1 or ≤ 0.5. Thus, the polypeptide according to the present invention can be used in toxicity studies conducted in monkeys, and the toxicity profile observed in monkeys is related to the potential side effects expected in humans.

[0661] Specifically, the anti-CD123 antibody of the present invention does not bind to the CSF2RA and IL5RA proteins or does not significantly cross-react with the CSF2RA and IL5RA proteins.

[0662] Specifically, the antibody does not bind to the above-mentioned CSF2RA and IL5RA proteins of humans and cynomolgus monkeys, or does not significantly cross-react with the CSF2RA and IL5RA proteins of humans and cynomolgus monkeys.

[0663] In addition, the affinity (KD) of the anti-CD123 antibody according to the present invention for human CD123 or cynomolgus monkey CD123 or for both ≤ 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 example having an affinity of 0.1 nM - 20 nM, for example an affinity of 0.1 nM - 10 nM, specifically an affinity of 0.1 nM - 2 nM or 0.1 nM - 1 nM.

[0664] In one example, the KD value of the affinity for human CD3 or for cynomolgus monkey CD3 is determined by surface plasmon resonance using recombinant CD123 proteins from humans and cynomolgus monkeys (for example, human and cynomolgus monkey CD123-Fc fusion proteins) as capture antigens.

[0665] The antigen can be used, for example, for the extracellular domain of human or cynomolgus monkey CD123 containing a signal peptide corresponding to the amino acid sequence at positions M1 - R305 of the corresponding wild-type protein (SEQ ID NO: 194 (human), SEQ ID NO: 195 (cynomolgus monkey)). The amino acid sequences obtained for the mature proteins of human or cynomolgus monkey CD123 are listed in SEQ ID NO: 196 and SEQ ID NO: 197, respectively. Biacore measurements are known in the art. In the given example, the Biacore measurements can be carried out as described in the "anti-CD3 antibody" section above.

[0666] The anti-CD123 antibody of the present invention also has an apparent affinity constant (apparent KD) determined by FACS analysis (the analysis is performed, for example, on CD123-expressing cells such as CD123-transfected HEK293 cells with the anti-CD123 antibody purified from hybridoma supernatants), which is ≤20 nM, for example ≤15 nM, ≤10 nM, ≤6 nM, ≤5 nM, ≤4 nM, ≤3 nM, ≤2 nM or ≤1 nM. Generally, the apparent KD is in the range of 0.1 - 20 nM, specifically 0.1 - 10 nM, for example 0.1 - 5 nM.

[0667] The VH and VL region sequences of the anti-CD123 antibodies designated "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" were aligned. The CDR-H and CDR-L sequence comparisons tend to show 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 these antibodies bind appropriately to the same epitope. The alignments of the anti-CD123 antibodies designated "1E1-G5", "6D6-B8", "8B11-B7" and "9F6-G3" and "6C10-C4", "9B8-G6", "9D7-C8" are shown respectively in Figure 3 and Figure 4 . The CDR-H and CDR-L sequence comparisons further identified CDR positions that are strictly conserved between the two groups of antibodies and are therefore considered crucial for specificity, while other positions may support substitutions.

[0668] Accordingly, the antibody according to the present invention comprises:

[0669] a) a heavy chain variable domain that contains CDR1-H consisting of the sequence X1YTFTDX2I (SEQ ID NO: 336), where X1 is G or A and X2 is H, Y or N, or any combination thereof; and

[0670] CDR2-H consisting of the sequence INPYSX1GX2 (SEQ ID NO: 337), where X1 is G or D and X2 is T or A, or any combination thereof; and

[0671] CDR3-H consisting of the sequence ALNYGSYYAMDA (SEQ ID NO201), and

[0672] A light chain variable domain comprising CDR1-L consisting of the sequence X1DIX2X3N (SEQ ID NO: 338), where X1 is E or K, X2 is F, H or Y and X3 is N or S, or any combination thereof; and

[0673] CDR2-L consisting of the sequence 'DAN' or 'DAS'; and

[0674] CDR3-L consisting of the sequence X1QYNX2YPYT (SEQ ID NO: 339), where X1 is H or 0 and X2 is I, K or N, or any combination thereof; or

[0675] b) A heavy chain variable domain comprising CDR1-H consisting of the sequence GFSLTSYX1 (SEQ ID NO: 340), where X1 is H or S; and

[0676] CDR2-H consisting of the sequence MWX1DGDT (SEQ ID NO: 341), where X1 is S or N; and

[0677] CDR3-H consisting of the sequence ARGX1X2X3X4X5X6X7X8X9FX 10 Y (SEQ ID NO: 342), where X1 is D, Y or H, X2 is Y or R, X3 is S or T, X4 is S or P, X5 is Y or no amino acid, X6 is L, I or no amino acid, X7 is Y or no amino acid, X8 is L or no amino acid, X9 is W or no amino acid, and X 10 is A or D, or any combination thereof, and

[0678] A light chain variable domain comprising CDR1-L consisting of the sequence QSFLSSGDX1X2NY (SEQ ID NO: 343), where X1 is E or G and X2 is R or K, or any combination thereof; and

[0679] CDR2-L consisting of the sequence 'WAS'; and

[0680] CDR3-L consisting of the sequence QQYYDTPLT (SEQ ID NO: 253).

[0681] According to an embodiment, an anti-CD123 antibody according to the present invention comprises the heavy chain and / or light chain CDR sequences of one of the 13 anti-CD123 antibodies listed above, namely "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".

[0682] Accordingly, the present invention relates to anti-CD123 antibodies, which comprise:

[0683] a) a heavy chain variable domain comprising CDR1-H having the sequence SEQ ID NO: 227 or a sequence differing from SEQ ID NO: 227 by one amino acid substitution; CDR2-H having the sequence SEQ ID NO: 228 or SEQ ID NO: 353 or SEQ ID NO: 279 or a sequence differing from SEQ ID NO: 228 or SEQ ID NO: 353 or SEQ ID NO: 279 by one or more amino acid substitutions; CDR3-H having the sequence SEQ ID NO: 229 or a sequence differing from SEQ ID NO: 229 by one amino acid substitution; and

[0684] a light chain variable domain comprising CDR1-L having the sequence SEQ ID NO: 231 or a sequence differing from SEQ ID NO: 231 by one amino acid substitution; CDR2-L having the sequence 'RDD' or a sequence differing from 'RDD' by one amino acid substitution; and CDR3-L having the sequence SEQ ID NO: 232 or a sequence differing from SEQ ID NO: 232 by one amino acid substitution; or

[0685] b) a heavy chain variable domain comprising CDR1-H having the sequence SEQ ID NO: 199 or a sequence differing from SEQ ID NO: 199 by one amino acid substitution; CDR2-H having the sequence SEQ ID NO: 200 or a sequence differing from SEQ ID NO: 200 by one or more amino acid substitutions; CDR3-H having the sequence SEQ ID NO: 201 or a sequence differing from SEQ ID NO: 201 by one amino acid substitution; and

[0686] a light chain variable domain comprising CDR1-L having the sequence SEQ ID NO: 203 or a sequence differing from SEQ ID NO: 203 by one amino acid substitution; CDR2-L having the sequence 'DAN' or a sequence differing from 'DAN' by one amino acid substitution; and CDR3-L having the sequence SEQ ID NO: 204 or a sequence differing from SEQ ID NO: 204 by one amino acid substitution; or

[0687] c) A heavy chain variable domain comprising CDR1-H having the sequence SEQ ID NO: 206 or a sequence differing from SEQ ID NO: 206 by one amino acid substitution; CDR2-H having the sequence SEQ ID NO: 207 or a sequence differing from SEQ ID NO: 207 by one or more amino acid substitutions; CDR3-H having the sequence SEQ ID NO: 208 or a sequence differing from SEQ ID NO: 208 by one amino acid substitution; and

[0688] a light chain variable domain comprising CDR1-L having the sequence SEQ ID NO: 210 or a sequence differing from SEQ ID NO: 210 by one amino acid substitution; CDR2-L having the sequence 'ETS' or a sequence differing from 'ETS' by one amino acid substitution; and CDR3-L having the sequence SEQ ID NO: 211 or a sequence differing from SEQ ID NO: 211 by one amino acid substitution; or

[0689] d) A heavy chain variable domain comprising CDR1-H having the sequence SEQ ID NO: 213 or a sequence differing from SEQ ID NO: 213 by one amino acid substitution; CDR2-H having the sequence SEQ ID NO: 214 or a sequence differing from SEQ ID NO: 214 by one or more amino acid substitutions; CDR3-H having the sequence SEQ ID NO: 215 or a sequence differing from SEQ ID NO: 215 by one amino acid substitution; and

[0690] a light chain variable domain comprising CDR1-L having the sequence SEQ ID NO: 217 or a sequence differing from SEQ ID NO: 217 by one amino acid substitution; CDR2-L having the sequence 'NTN' or a sequence differing from 'NTN' by one amino acid substitution; and CDR3-L having the sequence SEQ ID NO: 218 or a sequence differing from SEQ ID NO: 218 by one amino acid substitution; or

[0691] e) A heavy chain variable domain comprising CDR1-H having the sequence SEQ ID NO: 220 or a sequence differing from SEQ ID NO: 220 by one amino acid substitution; CDR2-H having the sequence SEQ ID NO: 221 or a sequence differing from SEQ ID NO: 221 by one or more amino acid substitutions; CDR3-H having the sequence SEQ ID NO: 222 or a sequence differing from SEQ ID NO: 222 by one amino acid substitution of; and

[0692] A light chain variable domain comprising CDR1-L having the sequence SEQ ID NO: 224 or a sequence differing from SEQ ID NO: 224 by one amino acid substitution; CDR2-L having the sequence 'RVS' or a sequence differing from 'RVS' by one amino acid substitution; and CDR3-L having the sequence SEQ ID NO: 225 or a sequence differing from SEQ ID NO: 225 by one amino acid substitution; or

[0693] f) A heavy chain variable domain comprising CDR1-H having the sequence SEQ ID NO: 234 or a sequence differing from SEQ ID NO: 234 by one amino acid substitution; CDR2-H having the sequence SEQ ID NO: 235 or a sequence differing from SEQ ID NO: 235 by one or more amino acid substitutions; CDR3-H having the sequence SEQ ID NO: 236 or a sequence differing from SEQ ID NO: 236 by one amino acid substitution; and

[0694] A light chain variable domain comprising CDR1-L having the sequence SEQ ID NO: 238 or a sequence differing from SEQ ID NO: 238 by one amino acid substitution; CDR2-L having the sequence 'GAS' or a sequence differing from 'GAS' by one amino acid substitution; and CDR3-L having the sequence SEQ ID NO: 239 or a sequence differing from SEQ ID NO: 239 by one amino acid substitution; or

[0695] g) A heavy chain variable domain comprising CDR1-H having the sequence SEQ ID NO: 241 or a sequence differing from SEQ ID NO: 241 by one amino acid substitution; CDR2-H having the sequence SEQ ID NO: 242 or a sequence differing from SEQ ID NO: 242 by one or more amino acid substitutions; CDR3-H having the sequence SEQ ID NO: 243 or a sequence differing from SEQ ID NO: 243 by one amino acid substitution; and

[0696] A light chain variable domain comprising CDR1-L having the sequence SEQ ID NO: 245 or a sequence differing from SEQ ID NO: 245 by one amino acid substitution; CDR2-L having the sequence 'YAS' or a sequence differing from 'YAS' by one amino acid substitution; and CDR3-L having the sequence SEQ ID NO: 246 or a sequence differing from SEQ ID NO: 246 by one amino acid substitution; or

[0697] h) A heavy chain variable domain comprising CDR1-H having the sequence SEQ ID NO: 248 or a sequence differing from SEQ ID NO: 248 by one amino acid substitution; CDR2-H having the sequence SEQ ID NO: 249 or a sequence differing from SEQ ID NO: 249 by one or more amino acid substitutions; CDR3-H having the sequence SEQ ID NO: 250 or a sequence differing from SEQ ID NO: 250 by one amino acid substitution; and

[0698] A light chain variable domain comprising CDR1-L having the sequence SEQ ID NO: 252 or a sequence differing from SEQ ID NO: 252 by one amino acid substitution; CDR2-L having the sequence 'WAS' or a sequence differing from 'WAS' by one amino acid substitution; and CDR3-L having the sequence SEQ ID NO: 253 or a sequence differing from SEQ ID NO: 253 by one amino acid substitution; or

[0699] i) A heavy chain variable domain comprising CDR1-H having the sequence SEQ ID NO: 255 or a sequence differing from SEQ ID NO: 255 by one amino acid substitution; CDR2-H having the sequence SEQ ID NO: 200 or a sequence differing from SEQ ID NO: 200 by one or more amino acid substitutions; CDR3-H having the sequence SEQ ID NO: 201 or a sequence differing from SEQ ID NO: 201 by one amino acid substitution; and

[0700] A light chain variable domain comprising CDR1-L having the sequence SEQ ID NO: 257 or a sequence differing from SEQ ID NO: 257 by one amino acid substitution; CDR2-L having the sequence 'DAN' or a sequence differing from 'DAN' by one amino acid substitution; and CDR3-L having the sequence SEQ ID NO: 258 or a sequence differing from SEQ ID NO: 258 by one amino acid substitution; or

[0701] j) A heavy chain variable domain comprising CDR1-H having the sequence SEQ ID NO: 199 or a sequence differing from SEQ ID NO: 199 by one amino acid substitution; CDR2-H having the sequence SEQ ID NO: 260 or a sequence differing from SEQ ID NO: 260 by one or more amino acid substitutions; CDR3-H having the sequence SEQ ID NO: 201 or a sequence differing from SEQ ID NO: 201 by one amino acid substitution; and

[0702] A light chain variable domain comprising CDR1-L having the sequence SEQ ID NO: 262 or a sequence differing from SEQ ID NO: 262 by one amino acid substitution; CDR2-L having the sequence 'DAS' or a sequence differing from 'DAS' by one amino acid substitution; and CDR3-L having the sequence SEQ ID NO: 263 or a sequence differing from SEQ ID NO: 263 by one amino acid substitution; or

[0703] k) A heavy chain variable domain comprising CDR1-H having the sequence SEQ ID NO: 265 or a sequence differing from SEQ ID NO: 265 by one amino acid substitution; CDR2-H having the sequence SEQ ID NO: 266 or a sequence differing from SEQ ID NO: 266 by one or more amino acid substitutions; CDR3-H having the sequence SEQ ID NO: 267 or a sequence differing from SEQ ID NO: 267 by one amino acid substitution; and

[0704] A light chain variable domain comprising CDR1-L having the sequence SEQ ID NO: 252 or a sequence differing from SEQ ID NO: 252 by one amino acid substitution; CDR2-L having the sequence 'WAS' or a sequence differing from 'WAS' by one amino acid substitution; and CDR3-L having the sequence SEQ ID NO: 253 or a sequence differing from SEQ ID NO: 253 by one amino acid substitution; or

[0705] l) A heavy chain variable domain comprising CDR1-H having the sequence SEQ ID NO: 265 or a sequence differing from SEQ ID NO: 265 by one amino acid substitution; CDR2-H having the sequence SEQ ID NO: 266 or a sequence differing from SEQ ID NO: 266 by one or more amino acid substitutions; CDR3-H having the sequence SEQ ID NO: 269 or a sequence differing from SEQ ID NO: 269 by one amino acid substitution; and

[0706] A light chain variable domain comprising CDR1-L having the sequence SEQ ID NO: 271 or a sequence differing from SEQ ID NO: 271 by one amino acid substitution; CDR2-L having the sequence 'WAS' or a sequence differing from 'WAS' by one amino acid substitution; and CDR3-L having the sequence SEQ ID NO: 253 or a sequence differing from SEQ ID NO: 253 by one amino acid substitution; or

[0707] (m) A heavy chain variable domain comprising CDR1-H having the sequence SEQ ID NO: 273 or a sequence differing from SEQ ID NO: 273 by one amino acid substitution; CDR2-H having the sequence SEQ ID NO: 274 or a sequence differing from SEQ ID NO: 274 by one or more amino acid substitutions; CDR3-H having the sequence SEQ ID NO: 201 or a sequence differing from SEQ ID NO: 201 by one amino acid substitution; and

[0708] A light chain variable domain comprising CDR1-L having the sequence SEQ ID NO: 276 or a sequence differing from SEQ ID NO: 276 by one amino acid substitution; CDR2-L having the sequence 'DAS' or a sequence differing from 'DAS' by one amino acid substitution; and CDR3-L having the sequence SEQ ID NO: 258 or a sequence differing from SEQ ID NO: 258 by one amino acid substitution.

[0709] In one or more of the above CDR sequences, one or more individual amino acids may be altered by substitution, particularly by conservative substitution. Such alterations may be intended, for example, in antibody humanization, to remove glycosylation sites or deamidation sites.

[0710] Alignment of the VH and VL region sequences of the anti-CD123 antibodies "1E1-G5", "6D6-B8", "8B11-B7", "9F6-G3" identified different amino acid substitutions. Thus, in one embodiment, the following amino acid substitutions may be made:

[0711] - at one or more of positions 1 and 7 of CDR1-H, for example at position 7 of the CDR1-H sequence GYTFTDHI (SEQ ID NO: 199) or GYTFTDYI (SEQ ID NO: 273), for example at positions 1 and 5 of CDR1-H of the sequence AYTFTDNI (SEQ ID NO: 255); and / or

[0712] - at one or more of positions 6 and 8 of CDR2-H, for example at positions 6 and / or 8 of the CDR2-H sequence INPYSGGT (SEQ ID NO: 200) or INPYSDGT (SEQ ID NO: 274) or INPYSGGA (SEQ ID NO: 260); and / or

[0713] - at one or more of positions 1, 4 and 5 of CDR1-L, for example at position 1 or at positions 1, 4 and 5 of the CDR1-L sequence KDIYSN (SEQ ID NO: 262) or EDIFNN (SEQ ID NO: 257); and / or

[0714] - at position 3 of the sequence ‘DAN’ or ‘DAS’ of CDR2-L; and / or

[0715] - at one or more of positions 1 and 5 of CDR3, e.g., at position 1 and / or 5 of the CDR3-L sequences HQYNIYPYT (SEQ ID NO: 258) or QQYNKYPYT (SEQ ID NO: 204) or HQYNNYPYT (SEQ ID NO: 263).

[0716] In another embodiment, the following amino acid substitutions are made:

[0717] - at position 8 of CDR1-H, e.g., at position 8 of the CDR1-H sequences GFSLTSYH (SEQ ID NO: 265) or GFSLTSYS (SEQ ID NO: 248); and / or

[0718] - at position 3 of CDR2-H, e.g., at position 3 of the CDR2-H sequences MWSDGD (SEQ ID NO: 265) or MWNDGD (SEQ ID NO: 248); and / or

[0719] - at positions 4 and 9 of CDR3-H, e.g., at positions 4 and 9 of the CDR3-H sequences ARGDYSSYIYLWFAY (SEQ ID NO: 267) or ARGYYSSYLYLWFAY (SEQ ID NO: 269); and / or

[0720] - at one or more of positions 9 and 10 of CDR1-L, e.g., at positions 9 and 10 of the CDR1-L sequences QSFLSSGDERNY (SEQ ID NO: 252) or QSFLSSGDGKNY (SEQ ID NO: 271).

[0721] According to the embodiment, the antibody comprises:

[0722] a) a heavy chain variable domain that contains CDR1-H of sequence SEQ ID NO: 227, CDR2-H of sequences SEQ ID NO: 228, SEQ ID NO: 353, SEQ ID NO: 279, CDR3-H of sequence SEQ ID NO: 229, and

[0723] a light chain variable domain that contains CDR1-L of sequence SEQ ID NO: 231, CDR2-L of the sequence ‘RDD’, and CDR3-L of sequence SEQ ID NO: 232; or

[0724] n) A heavy chain variable domain containing CDR1-H of SEQ ID NO: 199, CDR2-H of SEQ ID NO: 200, and CDR3-H of SEQ ID NO: 201; and

[0725] A light chain variable domain containing CDR1-L of SEQ ID NO: 203, CDR2-L of the sequence 'DAN', and CDR3-L of SEQ ID NO: 204; or

[0726] o) A heavy chain variable domain containing CDR1-H of SEQ ID NO: 206, CDR2-H of SEQ ID NO: 207, and CDR3-H of SEQ ID NO: 208, and

[0727] A light chain variable domain containing CDR1-L of SEQ ID NO: 210, CDR2-L of the sequence 'ETS', and CDR3-L of SEQ ID NO: 211; or

[0728] p) A heavy chain variable domain containing CDR1-H of SEQ ID NO: 213, CDR2-H of SEQ ID NO: 214, and CDR3-H of SEQ ID NO: 215, and

[0729] A light chain variable domain containing CDR1-L of SEQ ID NO: 217, CDR2-L of the sequence 'NTN', and CDR3-L of SEQ ID NO: 218; or

[0730] q) A heavy chain variable domain containing CDR1-H of SEQ ID NO: 220, CDR2-H of SEQ ID NO: 221, and CDR3-H of SEQ ID NO: 222, and

[0731] A light chain variable domain containing CDR1-L of SEQ ID NO: 224, CDR2-L generated by the sequence 'RVS', and CDR3-L of SEQ ID NO: 225; or

[0732] r) A heavy chain variable domain containing CDR1-H of SEQ ID NO: 234, CDR2-H of SEQ ID NO: 235, and CDR3-H of SEQ ID NO: 236, and

[0733] A light chain variable domain comprising CDR1-L of SEQ ID NO: 238, CDR2-L of the sequence 'GAS', and CDR3-L of SEQ ID NO: 239; or

[0734] s) A heavy chain variable domain comprising CDR1-H of SEQ ID NO: 241, CDR2-H of SEQ ID NO: 242, CDR3-H of SEQ ID NO: 243, and

[0735] A light chain variable domain comprising CDR1-L of SEQ ID NO: 245, CDR2-L of the sequence 'YAS', and CDR3-L of SEQ ID NO: 246; or

[0736] t) A heavy chain variable domain comprising CDR1-H of SEQ ID NO: 248, CDR2-H of SEQ ID NO: 249, CDR3-H of SEQ ID NO: 250, and

[0737] A light chain variable domain comprising CDR1-L of SEQ ID NO: 252, CDR2-L of the sequence 'WAS', and CDR3-L of SEQ ID NO: 253; or

[0738] u) A heavy chain variable domain comprising CDR1-H of SEQ ID NO: 255, CDR2-H of SEQ ID NO: 200, CDR3-H of SEQ ID NO: 201, and

[0739] A light chain variable domain comprising CDR1-L of SEQ ID NO: 257, CDR2-L of the sequence 'DAN', and CDR3-L of SEQ ID NO: 258; or

[0740] v) A heavy chain variable domain comprising CDR1-H of SEQ ID NO: 199, CDR2-H of SEQ ID NO: 260, CDR3-H of SEQ ID NO: 201, and

[0741] A light chain variable domain comprising CDR1-L of SEQ ID NO: 262, CDR2-L of the sequence 'DAS', and CDR3-L of SEQ ID NO: 263; or

[0742] w) A heavy chain variable domain that contains CDR1-H of SEQ ID NO: 265, CDR2-H of SEQ ID NO: 266, CDR3-H of SEQ ID NO: 267, and

[0743] A light chain variable domain that contains CDR1-L of SEQ ID NO: 252, CDR2-L of the sequence 'WAS', and CDR3-L of SEQ ID NO: 253; or

[0744] x) A heavy chain variable domain that contains CDR1-H of SEQ ID NO: 265, CDR2-H of SEQ ID NO: 266, CDR3-H of SEQ ID NO: 269, and

[0745] A light chain variable domain that contains CDR1-L of SEQ ID NO: 271, CDR2-L of the sequence 'WAS', and CDR3-L of SEQ ID NO: 253; or

[0746] y) A heavy chain variable domain that contains CDR1-H of SEQ ID NO: 273, CDR2-H of SEQ ID NO: 274, CDR3-H of SEQ ID NO: 201, and

[0747] A light chain variable domain that contains CDR1-L of SEQ ID NO: 276, CDR2-L of the sequence 'DAS', and CDR3-L of SEQ ID NO: 258.

[0748] The anti-CD123 antibody according to the present invention is specifically a conventional antibody, especially a conventional monoclonal antibody or antibody fragment, bispecific or multispecific antibody.

[0749] The anti-CD123 antibody according to the present invention specifically comprises IgG or a fragment thereof or consists of IgG or a fragment thereof.

[0750] The present invention also provides an anti-CD123 antibody as defined above, which comprises at least a heavy chain variable domain and / or a light chain variable domain of one of the anti-CD123 antibodies listed above.

[0751] Therefore, the present invention specifically relates to an anti-CD123 antibody that comprises:

[0752] a) The heavy chain variable domain of the sequence SEQ ID NO: 226 or SEQ ID NO: 277 or SEQ ID NO: 278 or a sequence that is at least 85% identical thereto, and / or the light chain variable domain of the sequence SEQ ID NO: 230 or a sequence that is at least 85% identical thereto; or

[0753] b) The heavy chain variable domain of the sequence SEQ ID NO: 198 or a sequence that is at least 85% identical thereto, and / or the light chain variable domain of the sequence SEQ ID NO: 202 or a sequence that is at least 85% identical thereto; or

[0754] c) The heavy chain variable domain of the sequence SEQ ID NO: 205 or a sequence that is at least 85% identical thereto, and / or the light chain variable domain of the sequence SEQ ID NO: 209 or a sequence that is at least 85% identical thereto; or

[0755] d) The heavy chain variable domain of the sequence SEQ ID NO: 212 or a sequence that is at least 85% identical thereto, and / or the light chain variable domain of the sequence SEQ ID NO: 216 or a sequence that is at least 85% identical thereto; or

[0756] e) The heavy chain variable domain of the sequence SEQ ID NO: 219 or a sequence that is at least 85% identical thereto, and / or the light chain variable domain of the sequence SEQ ID NO: 223 or a sequence that is at least 85% identical thereto; or

[0757] f) The heavy chain variable domain of the sequence SEQ ID NO: 233 or a sequence that is at least 85% identical thereto, and / or the light chain variable domain of the sequence SEQ ID NO: 237 or a sequence that is at least 85% identical thereto; or

[0758] g) The heavy chain variable domain of the sequence SEQ ID NO: 240 or a sequence that is at least 85% identical thereto, and / or the light chain variable domain of the sequence SEQ ID NO: 244 or a sequence that is at least 85% identical thereto; or

[0759] h) The heavy chain variable domain of the sequence SEQ ID NO: 247 or a sequence that is at least 85% identical thereto, and / or the light chain variable domain of the sequence SEQ ID NO: 251 or a sequence that is at least 85% identical thereto; or

[0760] i) The heavy chain variable domain of the sequence SEQ ID NO: 254 or a sequence that is at least 85% identical thereto, and / or the light chain variable domain of the sequence SEQ ID NO: 256 or a sequence that is at least 85% identical thereto; or

[0761] j) The heavy chain variable domain of the sequence SEQ ID NO: 259 or a sequence that is at least 85% identical thereto, and / or the light chain variable domain of the sequence SEQ ID NO: 261 or a sequence that is at least 85% identical thereto; or

[0762] k) The heavy chain variable domain of the sequence SEQ ID NO: 264 or a sequence that is at least 85% identical thereto, and / or the light chain variable domain of the sequence SEQ ID NO: 251 or a sequence that is at least 85% identical thereto; or

[0763] l) The heavy chain variable domain of the sequence SEQ ID NO: 268 or a sequence that is at least 85% identical thereto, and / or the light chain variable domain of the sequence SEQ ID NO: 270 or a sequence that is at least 85% identical thereto; or

[0764] m) The heavy chain variable domain of the sequence SEQ ID NO: 272 or a sequence that is at least 85% identical thereto, and / or the light chain variable domain of the sequence SEQ ID NO: 275 or a sequence that is at least 85% identical thereto.

[0765] For example, the sequences of the heavy chain or light chain variable domains may differ from the reference sequences SEQ ID NO: 226, 277, 278, 230, 198, 202, 205, 209, 212, 216, 219, 223, 233, 237, 240, 244, 247, 251, 254, 256, 259, 261, 264, 251, 268, 270, 272 or 275 by one or more amino acid substitutions, specifically by one or more conservative amino acid substitutions and / or substitutions with canonical residues. Specifically, the sequences of the heavy chain or light chain variable domains may differ from the reference sequences 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 only by conservative amino acid substitutions.

[0766] The sequence changes compared to the sequences 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 are specifically present substantially in one or more of the following framework regions: FR1-L, FR2-L, FR3-L, FR4-L and / or FR1-H, FR2-H, FR3-H, FR4-H.

[0767] In one embodiment, the anti-CD123 antibodies and fragments thereof of the present invention are rat antibodies and fragments of rat antibodies, respectively.

[0768] The anti-CD123 antibody of the present invention may also be a chimeric antibody, specifically a rat / human antibody, such as an antibody comprising rat heavy and light chain variable domains and CH and CL domains from a human antibody. Fragments of such an antibody may be polypeptides. The anti-CD123 antibody may also be a humanized antibody or a fragment of a humanized antibody obtained by CDR-grafting or by the 4D method (US20110027266).

[0769] Accordingly, in one embodiment, the anti-CD123 antibody of the present invention is a humanized antibody comprising:

[0770] a) a heavy chain variable domain containing an amino acid sequence selected from the group consisting of SEQ ID NO: 280, SEQ ID NO: 281, SEQ ID NO: 282, SEQ ID NO: 283, SEQ ID NO: 284, SEQ ID NO: 285, SEQ ID NO: 286, SEQ ID NO: 287, SEQ ID NO: 288, SEQ ID NO: 289, SEQ ID NO: 290, SEQ ID NO: 291, SEQ ID NO: 301, and SEQ ID NO: 302; and

[0771] b) a light chain variable domain containing an amino acid sequence selected from the group consisting of SEQ ID NO: 292, SEQ ID NO: 293, SEQ ID NO: 294, SEQ ID NO: 295, SEQ ID NO: 296, SEQ ID NO: 297, SEQ ID NO: 298, SEQ ID NO: 299, SEQ ID NO: 300, SEQ ID NO: 303, SEQ ID NO: 304, and SEQ ID NO: 305.

[0772] In one embodiment, the anti-CD123 antibody according to the present invention comprises three CDR sequences or variable domains of the heavy chain of one of the anti-CD123 antibodies listed above, or six CDR sequences or variable domains of the heavy and light chains.

[0773] The present invention further relates to fragments of the humanized anti-CD123 antibody as listed above. In one embodiment, the humanized anti-CD123 antibody described above is a chimeric antibody.

[0774] The anti-CD123 antibody according to the invention may also be a single-domain antibody or a fragment thereof. Specifically, 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 described above. 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.

[0775] The single-domain antibody or a fragment thereof may also comprise a framework region of a camel single-domain antibody and optionally a constant domain of a camel single-domain antibody.

[0776] The anti-CD123 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 diabody, in particular a humanized antibody fragment.

[0777] The CD123-antibody may also be a bispecific or multispecific antibody formed by at least one antibody fragment or at least one variable domain of the anti-CD123 antibody according to the invention. Multispecific antibodies and multivalent protein complexes are as described, for example, in EP 2050 764 A1 or US 2005 / 0003403 A1.

[0778] The bispecific or multispecific CD123-antibody according to the invention may be specific for (a) the extracellular domain of human or human and cynomolgus monkey CD123 targeted by one of the above anti-CD123 antibodies and (b) at least one other antigen.

[0779] In a specific embodiment, the other antigen is CD3 and the bispecific antibody obtained accordingly is a CD3 / CD123 bispecific antibody. Conventional bispecific antibodies can be produced by techniques known in the art.

[0780] The antibodies and fragments thereof according to the invention may be used in isolated (e.g., purified) form or incorporated into a carrier, such as a membrane or a lipid mediator (e.g., liposome).

[0781] In another embodiment, the anti-CD123 antibody of the invention is used for the preparation of an antibody-like binding protein as further defined in the "antibody-like binding protein" section of the invention.

[0782] Any combination of the above embodiments constitutes part of the invention.

[0783] Antibody-like binding protein

[0784] The inventors have generated several antibody-like binding proteins, referred to as "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, specifically a CODV-Fab or CODV-Ig design.

[0785] " CODV form " in the context of the present invention refers to the cross-over dual variable (CODV) configuration of a bispecific or multispecific antibody. The CODV format allows for variable domain fold retention and interchangeability of ultimate affinity.

[0786] The CODV format has been previously described in international patent application WO2012 / 135345. Accordingly, in one embodiment, the antibody-like binding proteins of the present invention are in the CODV format as described in the previous international patent application WO2012 / 135345 (which is incorporated herein by reference).

[0787] In one embodiment, the present invention refers to antibody-like binding proteins in the CODV-Fab format. Accordingly, in one embodiment, the present invention refers to antibody-like binding proteins comprising two polypeptide chains forming two antigen-binding sites, wherein the first polypeptide has a structure represented by formula [I]:

[0788] V D1 -L1-V D2 -L2-C L [I]

[0789] and the second polypeptide has a structure represented by formula [II]:

[0790] V D3 -L3-V D4 -L4-C H1 [II]

[0791] wherein:

[0792] V D1 is the variable domain of the first immunoglobulin heavy or light chain;

[0793] V D2 is the variable domain of the second immunoglobulin heavy or light chain;

[0794] V D3 is the variable domain of the second immunoglobulin heavy or light chain;

[0795] VD4 is the variable domain of the first immunoglobulin heavy or light chain;

[0796] C L is the light chain constant domain of an immunoglobulin;

[0797] C H1 is the C of an immunoglobulin H1 heavy chain constant domain;

[0798] L1, L2, L3 and L4 are amino acid linkers;

[0799] and wherein said first and second polypeptides form a cross light - heavy chain pair, and

[0800] wherein V D1 and V D2 are both light chain variable domains or both heavy chain variable domains, and if V D1 and V D2 are light chain variable domains, then V D3 and V D4 are both heavy chain variable domains, or if V D1 and V D2 are heavy chain variable domains, then V D3 and V D4 are both light chain variable domains.

[0801] 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 the antibody - like binding protein of formula (II) in CODV - Fab increases the half - life of the antibody - like binding protein and thus improves the pharmacokinetic profile of the antibody - like binding protein. Adding an Fc region to CODV - Fab results in dimerization of the polypeptide containing the Fc domain and the obtained antibody - like binding protein is an antibody - like binding protein in the form of CODV - Ig. The present invention thus further refers to an antibody - like binding protein in the form of CODV - Ig.

[0802] 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 shown by formula [I]:

[0803] V D1 -L1 - V D2 -L2 - C L [I]

[0804] and two polypeptide chains have the structure shown by formula [III]:

[0805] V D3 -L3 - V D4 -L4 - C H1-F c [III]

[0806] Wherein:

[0807] V D1 is the variable domain of the first immunoglobulin heavy or light chain;

[0808] V D2 is the variable domain of the second immunoglobulin heavy or light chain;

[0809] V D3 is the variable domain of the second immunoglobulin heavy or light chain;

[0810] V D4 is the variable domain of the first immunoglobulin heavy or light chain;

[0811] C L is the constant domain of the immunoglobulin light chain;

[0812] C H1 is the C H1 constant domain of the immunoglobulin heavy chain;

[0813] F c is the immunoglobulin hinge region and the CH2, CH3 constant domains of the immunoglobulin heavy chain in the immunoglobulin;

[0814] L1, L2, L3, and L4 are amino acid linkers;

[0815] and wherein the polypeptide of formula I and the polypeptide of formula III form a cross light chain - heavy chain pair, and

[0816] where V D1 and V D2 are both light chain variable domains or both heavy chain variable domains, and if V D1 and V D2 are light chain variable domains, then V D3 and V D4 are both heavy chain variable domains, or if V D1 and V D2 are heavy chain variable domains, then V D3 and V D4 are both light chain variable domains.

[0817] In the CODV-Ig form, two polypeptide chains having the structure shown by formula [III] dimerize through their F c domain.

[0818] In another embodiment, the first F cA domain is added to an antibody-like binding protein CODV-Fab polypeptide of formula [II], and a second F c domain (designated F c2 ) is added to an antibody-like binding protein CODV-Fab polypeptide of formula [I]. In addition, in the same embodiment, a linker L5 is present in the polypeptide chain C of formula [I] between the L and F c2 domains to obtain a polypeptide chain of formula [IV].

[0819] Accordingly, the present invention further relates 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]:

[0820] V D1 -L1-V D2 -L2-C L -L5-F c2 [IV]

[0821] and one polypeptide chain has a structure represented by formula [III]:

[0822] V D3 -L3-V D4 -L4-C H1 -F c [III]

[0823] wherein:

[0824] V D1 is the variable domain of the first immunoglobulin heavy or light chain;

[0825] V D2 is the variable domain of the second immunoglobulin heavy or light chain;

[0826] V D3 is the variable domain of the second immunoglobulin heavy or light chain;

[0827] V D4 is the variable domain of the first immunoglobulin heavy or light chain;

[0828] C L is the light chain constant domain of the immunoglobulin;

[0829] C H1 is the C H1 heavy chain constant domain of the immunoglobulin;

[0830] F c is the immunoglobulin hinge region and the CH2 and CH3 immunoglobulin heavy chain constant domains of the immunoglobulin;

[0831] Fc2 is the immunoglobulin hinge region and the CH2 and CH3 immunoglobulin heavy chain constant domains;

[0832] L1, L2, L3, L4, and L5 are amino acid linkers; and

[0833] wherein the polypeptide of formula [IV] and the polypeptide of formula [III] form a cross light chain - heavy chain pair; and

[0834] wherein V D1 and V D2 are both light chain variable domains or both heavy chain variable domains, and if V D1 and V D2 are light chain variable domains, then V D3 and V D4 are both heavy chain variable domains, or if V D1 and V D2 are heavy chain variable domains, then V D3 and V D4 are both light chain variable domains.

[0835] In this CODV form, wherein the polypeptide chains shown by formulas [III] and [IV] dimerize through their respective F c2 and F c regions, and is referred to herein as CODV - Fab - TL.

[0836] In another embodiment of the CODV - Fab, a first F c domain is added to the polypeptide chain shown by formula [II] (to obtain formula [III]), and the antibody - like binding protein comprises a polypeptide chain containing or consisting of a second F c domain (referred to as F c3 ).

[0837] The present invention further relates to an antibody - like binding protein comprising three polypeptide chains that form two antigen - binding sites, wherein

[0838] the first polypeptide has a structure shown by formula [I]:

[0839] V D1 -L1 - V D2 -L2 - C L [I]

[0840] The second polypeptide has a structure shown by formula [III]:

[0841] V D3 -L3 - V D4 -L4 - C H1 -F c [III]

[0842] The third polypeptide F c3 is the immunoglobulin hinge region and the CH2 and CH3 immunoglobulin heavy chain constant domains (also referred to as F c stub);

[0843] wherein

[0844] V D1 is the variable domain of the first immunoglobulin heavy or light chain;

[0845] V D2 is the variable domain of the second immunoglobulin heavy or light chain;

[0846] V D3 is the variable domain of the second immunoglobulin heavy or light chain;

[0847] V D4 is the variable domain of the first immunoglobulin heavy or light chain;

[0848] C L is the constant domain of the immunoglobulin light chain;

[0849] C H1 is the C H1 constant domain of the immunoglobulin heavy chain;

[0850] F c is the immunoglobulin hinge region and the CH2 and CH3 immunoglobulin heavy chain constant domains;

[0851] L1, L2, L3, and L4 are amino acid linkers;

[0852] and wherein the polypeptide of formula [I] and the polypeptide of formula [III] form a cross light chain - heavy chain pair, and

[0853] wherein V D1 and V D2 are both light chain variable domains or both heavy chain variable domains, and if V D1 and V D2 are light chain variable domains, then V D3 and V D4 are both heavy chain variable domains, or if V D1 and V D2 are heavy chain variable domains, then V D3 and V D4 are both light chain variable domains;

[0854] and wherein the polypeptide of formula [III] heterodimerizes with the third polypeptide through its F c domain.

[0855] Accordingly, in the said embodiment, the so-called "Fc stump" (F c3 ) heterodimerizes with the polypeptide Fc region according to formula III. This CODV form is referred to herein as CODV-Fab-OL. This construct avoids the formation of aggregates of CODV-Fab.

[0856] In one embodiment of CODV-Fab-OL, F c and F c3 are both immunoglobulin variants in which the CH3 domain has been modified: according to the technique referred to as "Knob-into-Hole" described in particular in patents US5731168 and US8216805 (which are incorporated herein by reference), F c and F c3 have each been engineered at the CH3-CH3 interface to facilitate heteromultimer formation.

[0857] Accordingly, in one embodiment, the CH3 domain of one of F c and F c3 contains the mutations Y349C, T366S, L368A, and Y407V, while the CH3 domain of the other F c and F c3 contains the mutations S354C and T366W (the amino acid positions are represented by reference to the IgG1 sequence).

[0858] Examples of suitable F c and F c3 pairs include the pair of SEQ ID NO: 396 (F c ) and SEQ ID NO: 397 (F c3 ) and the pair of SEQ ID NO: 394 (F c ) and SEQ ID NO: 398 (F c3 ).

[0859] In one embodiment of the present invention, the first immunoglobulin or the second immunoglobulin is an anti-CD3 antibody as defined in the above "Anti-CD3 Antibody" section.

[0860] In another embodiment of the present invention, the first immunoglobulin or the second immunoglobulin is an anti-CD123 antibody as defined in the above "Anti-CD123 Antibody" section.

[0861] According to an embodiment of the present invention, the V D1 and V D2 of the polypeptide of formula I or formula [IV] are both light chain variable domains or both heavy chain variable domains, and the V D3 and V D4Both are heavy chain or both are light chain variable domains. This interchangeability is also referred to as "exchangeability" and thus determines the cross - dual variable (CODV) configuration of the antibody - like binding proteins of the present invention.

[0862] As defined above, V D1 and V D4 are the heavy chain or light chain variable domains of a first immunoglobulin, and V D2 and V D3 are the heavy chain or light chain variable domains of a second immunoglobulin, and V D1 and V D4 as well as V D2 and V D3 are thus considered as associated domains.

[0863] Accordingly, the term "cross" refers to the exchange ratio of the V D1 or V D2 of a polypeptide of formula [I] or formula [IV] corresponding to the associated variable domain V D4 or V D3 of its polypeptide of formula [II] or formula [III].

[0864] In a specific embodiment, V D1 and V D2 are light chain variable domains and V D3 and V D4 are heavy chain variable domains.

[0865] The antibody - like binding proteins of the present invention can use domains or sequences obtained from or derived from any human or non - human antibody, such as human, rat or humanized antibodies.

[0866] In one embodiment, the immunoglobulin is an IgG immunoglobulin.

[0867] Accordingly, in one embodiment, C L is the light chain constant domain of an IgG immunoglobulin. In another embodiment, C H1 is the C H1 heavy chain constant domain of an IgG immunoglobulin.

[0868] In one embodiment, the antibody - like binding proteins of the present invention can be prepared using the domains or sequences of the anti - CD3 antibody and anti - CD123 antibody described herein.

[0869] As used herein, the term "linker" refers to one or more amino acid residues inserted into the immunoglobulin domains that provide sufficient mobility for the light and heavy chain domains to fold into an exchanged dual variable domain immunoglobulin. In some embodiments, a linker consisting of 0 amino acids means that no linker is present. At the sequence level, the linker is inserted at the transition between variable domains or between variable and constant domains. Since the approximate size of immunoglobulin domains is well known, the transition between domains can be recognized. The precise location of the domain transition can be determined by locating peptide segments that do not form a secondary structural component (such as a β-sheet or an α-helix), as evidenced by experimental data or as hypothesized by modeling or secondary structure prediction. The linkers described in the context of the present invention are linkers L1, L2, L3, L4, and L5. L1 is located between the N-terminal V D1 domain and the V D2 domain; L2 is located between the V D2 and the C-terminal C L domain. Linkers L3 and L4 are located on the polypeptide as defined according to antibody-like protein format II or III. More precisely, L3 is located between the N-terminal V D3 and the V D4 domain and L4 is located between the V D4 and the C-terminal C H1 -Fc domain. L5 is located between the C L and the N-terminal F c2 intervals. Linkers L1, L2, L3, L4, and L5 are independent, but in some embodiments, they have the same sequence and / or length.

[0870] In some antibody-like binding proteins of the present invention, the length of L3 is at least twice the length of L1. In other antibody-like binding proteins of the present invention, the length of L4 is at least twice the length of L2. In some antibody-like binding proteins of the present invention, the length of L1 is at least twice the length of L3. In other antibody-like binding proteins of the present invention, the length of L2 is at least twice the length of L4.

[0871] In one embodiment, linkers L1, L2, L3, and L4 contain 0-20 amino acids. In one embodiment, L5 contains 0-10 amino acids.

[0872] In some antibody-like binding proteins of the present invention, L1 is 3-12 amino acid residues in length, L2 is 3-1 amino acid residues in length, L3 is 1-8 amino acid residues in length, and L4 is 1-3 amino acid residues in length. In other antibody-like binding proteins, L1 is 5-10 amino acid residues in length, L2 is 5-8 amino acid residues in length, L3 is 1-5 amino acid residues in length, and L4 is 1-2 amino acid residues in length. In preferred antibody-like binding proteins, L1 is 7 amino acid residues in length, L2 is 5 amino acid residues in length, L3 is 1 amino acid residue in length, and L4 is 2 amino acid residues in length.

[0873] In some antibody-like binding proteins of the present invention, L1 is 1-3 amino acid residues in length, L2 is 1-4 amino acid residues in length, L3 is 2-15 amino acid residues in length, and L4 is 2-15 amino acid residues in length. In other antibody-like binding proteins, L1 is 1-2 amino acid residues in length, L2 is 1-2 amino acid residues in length, L3 is 4-12 amino acid residues in length, and L4 is 2-12 amino acid residues in length. In preferred antibody-like binding proteins, L1 is 1 amino acid residue in length, L2 is 2 amino acid residues in length, L3 is 7 amino acid residues in length, and L4 is 5 amino acid residues in length.

[0874] In some antibody-like binding proteins of the present invention, L1, L3 or L4 may be equal to 0. However, in antibody-like binding proteins in which L3 or L4 is equal to 0, the corresponding linker between the variable region and the constant region or the dual variable domains on other chains cannot be 0. In some embodiments, L1 is equal to 0 and L3 is 2 or more amino acid residues, L3 is equal to 0 and L1 is 1 or more amino acid residues, or L4 is equal to 0 and L2 is 3 or more amino acid residues.

[0875] In some antibody-like binding proteins of the present invention, at least one of the linkers selected from the group consisting of L2, L3 and L4 contains at least one cysteine residue.

[0876] Examples of suitable linkers include single glycine, threonine or serine residues; dipeptides such as diglycine peptide, histidine-threonine peptide or glycine-serine dipeptide; tripeptides having three glycines, tripeptide Thr-His-Thr, tripeptide Gly-Gly-Ser; peptides having four glycine residues; peptides having five glycine residues; peptides having six glycine residues; peptides having seven glycine residues; peptides having eight glycine residues. Combinations of other amino acid residues can be used such as 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), and peptide Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 345). Other suitable linkers include single Ser and Val residues; 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: 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-G1y (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 IDNO: 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 IDNO: 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 containing randomly selected amino acids 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 for the antibody-like binding proteins of the invention.;

[0877] The identity and sequence of the amino acid residues in the linker can vary with the type of secondary structure elements to be achieved in the linker. For example, glycine, serine, and alanine are optimal for linkers with maximum flexibility. If a more rigid and extended linker is desired, some combinations of glycine, proline, threonine, and serine are effective. Any amino acid residue in combination with other amino acid residues can be recognized as a linker when constructing larger peptide linkers as needed according to the desired properties.

[0878] In one embodiment, linker L1 is the sequence Gly-Gln-Pro-Lys-Ala-Ala-Pro (SEQ ID NO: 307), linker L2 is the sequence Thr-Lys-Gly-Pro-Ser (SEQ ID NO: 309), linker L3 is the sequence 'S' and linker L4 is the sequence 'RT'.

[0879] In another embodiment, the L1, L2, L3, and L4 linker sequences are selected from the group consisting of: threonine; dipeptides such as histidine-threonine peptide; 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 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-Gl y(SEQ ID NO: 373) and Gly-Gly-Gly-Glu-Pro-Lys-Ser-Asp-Lys-Thr-His-Thr-Ser-Pro-Pro-Ser-Pro-Gly-Gly-Gly(SEQ ID NO: 374). In one embodiment, the sequence of linker L5 is selected from the group consisting of: a single serine residue; a dipeptide such as a glycine-serine dipeptide; and the tripeptides Gly-Gly-Ser, the peptide Gly-Gly-Gly-Ser (SEQ ID NO: 354), the peptide Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 344), the peptide Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 355), the peptide Gly-Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 356), the peptide Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 357), the peptide Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 358), the peptide Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 345) and the peptide Gly-Gly-Ser-Gly-Ser-Ser-Gly-Ser-Gly-Gly (SEQ ID NO: 389).;

[0880] The term " Fc domain " as used herein encompasses native Fc and Fc variants and sequences as defined above. When used in conjunction with Fc variants and native Fc molecules, the term "Fc domain" includes molecules in monomeric or multimeric form, whether produced by digestion of a complete antibody or by other means.

[0881] The term " native Fc″A molecule that includes sequences of non-antigen-binding fragments produced by antibody digestion or by other means, whether in monomeric or polymeric form, and may include a hinge region. The original immunoglobulin source of native Fc is specifically human and can be any immunoglobulin, although IgG1 and IgG2 are preferred. Native Fc molecules are composed of monomeric polypeptides that can be joined into dimeric or polymeric forms by covalent (i.e., disulfide bonds) and non-covalent associations. The number of intermolecular disulfide bonds between monomer subunits of native Fc molecules can be 1-4 depending on the class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, and IgGA2). An example of native Fc is the disulfide dimer obtained by papain digestion of IgG. The term ″native Fc″ as used herein is a collective term for monomeric, dimeric, and polymeric forms.

[0882] The term ″ Fc variant ″ refers to a molecule or sequence that is modified from native Fc but still contains a binding site for the salvage receptor FcRn (neonatal Fc receptor). Typical Fc variants and their interactions with the salvage receptor are known in the art. Thus, the term ″Fc variant″ can include molecules or sequences that are humanized from non-human native Fc. In addition, native Fc contains regions that can be removed because they provide structural features and biological activities that are not required for the antibody-like binding proteins of the present invention. Thus, the term ″Fc variant″ includes molecules or sequences that lack one or more native Fc sites or residues or in which one or more Fc sites or residues have been modified, where the modification affects or participates in: (1) disulfide bond formation, (2) incompatibility with the selected host cell, (3) N-terminal heterogeneity upon expression in the selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to an Fc receptor other than the salvage receptor, or (7) antibody-dependent cell cytotoxicity (ADCC).

[0883] In some embodiments, when the antibody-like binding protein includes two F c domains, i.e., in 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 the same immunoglobulin isotype or isotype subclass. Accordingly, in some embodiments, the F c and F c2 of CODV-Ig or the two F c domains of CODV-Fab-TL, or the F c and F c3All are of the IgG1 subclass, or the IgG2 subclass, or the IgG3 subclass, or the IgG4 subclass.

[0884] All antibody-like binding proteins as described herein do not have effector functions. This means that when the antibody-like binding protein contains one or more Fc domains of the IgG1 subclass (i.e., Fc in formula [III], Fc in formula [IV], and / or Fc), one or more Fc domains of the IgG1 backbone contain the double mutations L234A and L235A that abolish Fc effector functions (referred to as the "LALA mutation"). The Fc double mutations L234A and L235A do not bind 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). c Fc domain (i.e., Fc in formula [III]), c Fc in formula [IV], c2 and / or Fc c3 ), one or more Fc domains of the IgG1 backbone contain the double mutations L234A and L235A that abolish Fc effector functions (referred to as the "LALA mutation"). c Fc domain contains the double mutations L234A and L235A that abolish Fc effector functions (referred to as the "LALA mutation"). The Fc double mutations L234A and L235A do not bind 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).

[0885] In one instance, the Fc region contains the amino acid sequence SEQ ID NO: 330, SEQ ID NO: 394, or SEQ ID NO: 396. In one embodiment, the Fc region contains the two amino acid exchanges H435R and Y436F in the CH3 domain, as described in Jendeberg, L. et al. (1997, J. Immunological Meth. 201:25-34). Accordingly, in one embodiment, the Fc region contains the amino acid sequence SEQ ID NO: 327. In another embodiment, the Fc region contains the amino acid sequence SEQ ID NO: 392. c region contains the amino acid sequence SEQ ID NO: 330, SEQ ID NO: 394, or SEQ ID NO: 396. In one embodiment, the Fc C2 region contains the two amino acid exchanges H435R and Y436F in the CH3 domain, as described in Jendeberg, L. et al. (1997, J. Immunological Meth. 201:25-34). Accordingly, in one embodiment, the Fc C2 region contains the amino acid sequence SEQ ID NO: 327. In another embodiment, the Fc C2 region contains the amino acid sequence SEQ ID NO: 392.

[0886] The antibody-like binding protein designated CODV-Fab "7G3x20G6" contains:

[0887] - A polypeptide according to formula I, having the amino acid sequence: (SEQ ID NO: 306, with the linker in bold and underlined), which contains V of SEQ ID NO: 9, L1 of SEQ ID NO: 307, V of SEQ ID NO: 308, L2 of SEQ ID NO: 309, and C of SEQ ID NO: 310, and D1 , L1 of SEQ ID NO: 307, V of SEQ ID NO: 308, L2 of SEQ ID NO: 309, and C of SEQ ID NO: 310, and D2 , and L , and

[0888] - A polypeptide according to formula II, having the amino acid sequence: (SEQ ID NO: 311, the linker is shown in bold and underlined), which contains the V of sequence SEQ ID NO: 312 'RT' D3 of V of sequence SEQ ID NO: 5, L3 of amino acid sequence 'S' D4 of L4 of amino acid sequence and C of sequence SEQ ID NO: 313 H1 .

[0889] The antibody-like binding protein called CODV-Fab "7G3x4E7" contains:

[0890] - a polypeptide according to formula I, the amino acid sequence being: (SEQ ID NO: 314, the linker is shown in bold and underlined), which contains the V of sequence SEQ ID NO: 21 D1 of L1 of sequence SEQ ID NO: 307, V of sequence SEQ ID NO: 308 D2 of L2 of sequence SEQ ID NO: 309 and C of sequence SEQ ID NO: 310 L , and

[0891] - a polypeptide according to formula II, the amino acid sequence being: (SEQ ID NO: 315, the linker is shown in bold and underlined), which contains the V of sequence SEQ ID NO: 312 D3 of L3 of amino acid sequence 'S', V of sequence SEQ ID NO: 18 D4 of L4 of amino acid sequence 'RT' and C of sequence SEQ ID NO: 313 H1 .

[0892] The antibody-like binding protein called CODV-Fab "7G3x4B4" contains:

[0893] - a polypeptide according to formula I, the amino acid sequence being: (SEQ ID NO: 316, the linker is shown in bold and underlined), which contains the V of sequence SEQ ID NO: 16 D1 of L1 of sequence SEQ ID NO: 307, V of sequence SEQ ID NO: 308 D2 of L2 of sequence SEQ ID NO: 309 and C of sequence SEQ ID NO: 310 L , and

[0894] - A polypeptide according to Formula II, with the amino acid sequence: (SEQ ID NO: 317, with the linker in bold and underlined), which contains the V of SEQ ID NO: 312 D3 and L3 with the amino acid sequence 'S', the V of SEQ ID NO: 12 D4 , L4 with the amino acid sequence 'RT' and C of SEQ ID NO: 313 H1 .

[0895] The antibody-like binding protein called CODV-Fab "7G3x18F5" contains:

[0896] - A polypeptide according to Formula I, with the amino acid sequence: (SEQ ID NO: 318, with the linker in bold and underlined), which contains the V of SEQ ID NO: 26 D1 , L1 of SEQ ID NO: 307, the V of SEQ ID NO: 308 D2 , L2 of SEQ ID NO: 309 and C of SEQ ID NO: 310 L , and

[0897] - A polypeptide according to Formula II, with the amino acid sequence: (SEQ ID NO: 319, with the linker in bold and underlined), which contains the V of SEQ ID NO: 312 D3 , L3 with the amino acid sequence 'S', the V of SEQ ID NO: 23 D4 , L4 with the amino acid sequence 'RT' and C of SEQ ID NO: 313 H1 .

[0898] The antibody-like binding protein called CODV-Fab "hz20G6x7G3" contains:

[0899] - A polypeptide according to Formula I, with the amino acid sequence: (SEQ ID NO: 320, with the linker in bold and underlined), which contains the V of SEQ ID NO: 308 D1 , L1 of SEQ ID NO: 307, the V of SEQ ID NO: 143 D2 , L2 of SEQ ID NO: 309 and C of SEQ ID NO: 310 L , and

[0900] - A polypeptide according to Formula II, with the amino acid sequence: (SEQ ID NO: 321, linker in bold and underlined), which contains the V of SEQ ID NO: 138 D3 of L3 with the amino acid sequence 'S', the V of SEQ ID NO: 312 D4 of L4 with the amino acid sequence 'RT' and the C of SEQ ID NO: 313 H1 .

[0901] The antibody-like binding protein designated CODV-Fab "7G3xhz4B4" comprises:

[0902] - A polypeptide according to Formula I, with the amino acid sequence: (SEQ ID NO: 322, linker in bold and underlined), which contains the V of SEQ ID NO: 158 D1 of L1 of SEQ ID NO: 307, the V of SEQ ID NO: 308 D2 of L2 of SEQ ID NO: 309 and the C of SEQ ID NO: 310 L , and

[0903] - A polypeptide according to Formula II, with the amino acid sequence: (SEQ ID NO: 323, linker in bold and underlined), which contains the V of SEQ ID NO: 312 D3 of L3 with the amino acid sequence 'S', the V of SEQ ID NO: 171 D4 of L4 with the amino acid sequence 'RT' and the C of SEQ ID NO: 313 H1 .

[0904] The antibody-like binding protein designated CODV-Fab "hz4B4x3E3" comprises:

[0905] - A polypeptide according to Formula I, with the amino acid sequence: (SEQ ID NO: 324, linker in bold and underlined), which contains the VD1 of SEQ ID NO: 230, the L1 of SEQ ID NO: 307, the V of SEQ ID NO: 158 D2 of L2 of SEQ ID NO: 309 and the C of SEQ ID NO: 310 L, and

[0906] - A polypeptide according to formula II, with the amino acid sequence: (SEQ ID NO: 325, the linker is in bold and underlined), which contains V of SEQ ID NO: 171 D3 、L3 with the amino acid sequence 'S', V of SEQ ID NO: 226 D4 、L4 with the amino acid sequence 'RT' and C of SEQ ID NO: 313 H1 .

[0907] The antibody-like binding protein called CODV-Fab "hz20G6xhz7G3" contains:

[0908] - A polypeptide according to formula [I], which consists essentially of the following amino acid sequence:

[0909] (SEQ ID NO: 388, the linker is in bold and underlined), which contains V of SEQ ID NO: 385 D1 、L1 of SEQ ID NO: 389, V of SEQ ID NO: 141 D2 、L2 of SEQ ID NO: 389 and C of SEQ ID NO: 310 L , and

[0910] - A polypeptide according to formula [II], which consists essentially of the following amino acid sequence: (SEQ ID NO: 390), which contains V of SEQ ID NO: 138 D3 、L3 is 0 amino acids, V of SEQ ID NO: 383 D4 (italic and underlined), L4 is 0 amino acids and C of SEQ ID NO: 313 H1 .

[0911] In one instance, the polypeptide according to Formula II, referred to as the antibody-like binding proteins CODV-Fab "7G3x20G6", "7G3x4E7", "7G3x4B4", "7G3x18F5", "hz20G6x7G3", "7G3xhz4B4", "hz4B4x3E3", and "hz20G6xhz7G3", further comprises the hinge sequence and the sequence of His-tag EPKSCDKTHTHHHHHH (SEQ ID NO: 352) used correspondingly, for example, for purification.

[0912] The antibody-like binding protein referred to as CODV-Fab "hz20G6x7G3-TL4" (also referred to as CODV-Fab-TL4 "hz20G6x7G3") comprises:

[0913] - A polypeptide according to Formula [IV], which consists essentially of the following amino acid sequence: (SEQ ID NO: 326, the linker is in bold and underlined), which contains V of sequence SEQ ID NO: 308 D1 、sequence L1 of SEQ ID NO: 307, V of sequence SEQ ID NO: 143 D2 、sequence L2 of SEQ ID NO: 309, C of sequence SEQ ID NO: 310 L and F of sequence SEQ ID NO: 327 c2 (underlined), and

[0914] - A polypeptide according to Formula [III], which consists essentially of the following amino acid sequence: (SEQ ID NO: 328, the linker is in bold and underlined), which contains V of sequence SEQ ID NO: 138 D3 、L3 of amino acid sequence 'S', V of sequence SEQ ID NO: 312 D4 、L4 of amino acid sequence 'RT' and C of sequence SEQ ID NO: 329 H1 and F of sequence SEQ ID NO: 330 c .

[0915] In the above-mentioned CODV-Fab-TL4 "hz20G6x7G3" antibody-like binding protein, the Fc of sequence SEQ ID NO: 330 and the F of sequence SEQ ID NO: 327 c2 are from the IgG4 backbone. The antibody-like binding protein is in the form of CODV-Fab-TL and comprises or consists of a polypeptide of Formula III and a polypeptide of Formula IV.

[0916] The antibody-like binding protein designated CODV-Fab-TL1 “hz20G6xhz7G3” comprises:

[0917] - A polypeptide according to Formula IV consists essentially of the following amino acid sequence:

[0918] (SEQ ID NO: 391, the linker is in bold and underlined), which contains V of SEQ ID NO: 385 D1 、L1 of SEQ ID NO: 389, V of SEQ ID NO: 141 D2 、L2 of SEQ ID NO: 389, C of SEQ ID NO: 310 L 、L5 containing 0 amino acids and F of SEQ ID NO: 392 c2 (underlined); and

[0919] - A polypeptide according to Formula III, the amino acid sequence of which is:

[0920]

[0921]

[0922] (SEQ ID NO: 393), which contains V of SEQ ID NO: 138 D3 、L3 is 0 amino acids, V of SEQ ID NO: 383 D4 (italic), L4 is 0 amino acids, C of SEQ ID NO: 313 H1 and F of SEQ ID NO: 394 c (underlined).

[0923] In the antibody-like binding protein of CODV-Fab-TL1 “hz20G6x7G3”, the Fc of SEQ ID NO: 394 and the F of SEQ ID NO: 392 c2 are from the IgG1 backbone. The antibody-like binding protein is in the form of CODV-Fab-TL. It comprises or consists of a polypeptide of Formula IV and a polypeptide of Formula III.

[0924] The antibody-like binding protein designated CODV-Fab-OL1 “hz20G6xhz7G3” comprises:

[0925] - A polypeptide according to Formula I, the amino acid sequence of which is:

[0926]

[0927] (SEQ ID NO: 388, with the linker in bold and underlined), which contains V of SEQ ID NO: 385 D1 、L1 of SEQ ID NO: 389, V of SEQ ID NO: 141 D2 、L2 of SEQ ID NO: 389 and C of SEQ ID NO: 310 L ; and

[0928] - A polypeptide according to Formula III, whose amino acid sequence is:

[0929]

[0930]

[0931] (SEQ ID NO: 395), which contains V of SEQ ID NO: 138 D3 , L3 of 0 amino acids, V of SEQ ID NO: 383 D4 (italic and underlined), L4 of 0 amino acids, C of SEQ ID NO: 313 H1 and F of SEQ ID NO: 396 c (underlined);

[0932] and wherein the antibody - like binding protein designated as CODV - Fab - OL1 "hz20G6xhz7G3" further contains an Fc stump (F c3 ), whose amino acid sequence is: (SEQ ID NO: 397), and which heterodimerizes with the Fc region of the polypeptide according to Formula III.

[0933] The said antibody - like binding protein is in the form of CODV - Fab - OL, that is, it contains or consists of a polypeptide of Formula I, a polypeptide of Formula III and an Fc stump or is composed of the above. Its F c and F c3 sequences have been engineered according to the "Knob - into - Hole" technology and further contain the double mutations L234A and L235A.

[0934] F of SEQ ID NO: 396 cThe sequence has been designed to contain RF residues at positions 200 - 221 (bolded above), replacing the HY residues that are present at these positions in the Fc region. The HY>RF mutation (i.e., H435R and Y436F in the CH3 domain, described by Jendeberg, L. et al 1997, J. Immunological Meth., 201:25 - 34) is advantageous for purification purposes, as it abolishes binding to Protein A. In the case of CODV - Fab - OL1 "hz20G6xhz7G3", the F c stub contains HY residues (bolded above) at positions 217 - 218.

[0935] The antibody - like binding protein called CODV - Fab - OL1a "hz20G6xhz7G3" comprises:

[0936] - A polypeptide according to formula I, having the amino acid sequence:

[0937]

[0938] (SEQ ID NO: 388), which comprises V of SEQ ID NO: 385 D1 , L1 of SEQ ID NO: 389, V of SEQ ID NO: 141 D2 , L2 of SEQ ID NO: 389 and C of SEQ ID NO: 310 L ;

[0939] - A polypeptide according to formula III, having the amino acid sequence:

[0940]

[0941] (SEQ ID NO: 399), which comprises V of SEQ ID NO: 138 D3 , L3 of 0 amino acids, V of SEQ ID NO: 383 D4 (italicized and underlined), L4 of 0 amino acids, C of SEQ ID NO: 313 H1 and F of SEQ ID NO: 400 c (underlined);

[0942] - And wherein the antibody - like binding protein called CODV - Fab - OL1a "hz20G6xhz7G3" further comprises an Fc stub (F c3 ), having the amino acid sequence:

[0943] (SEQ ID NO: 398), which heterodimerizes with the Fc region of a polypeptide according to Formula III.

[0944] F of SEQ ID NO: 400 c contains the HY residues (bolded above) at positions 220 - 221 while the F of SEQ ID NO: 398 c stub contains the RF residues (bolded above) at positions 217 - 218.

[0945] The antibody - like binding protein is in the form of CODV - Fab - OL, i.e., it comprises or consists of a polypeptide of Formula I, a polypeptide of Formula III, and an Fc stub or is composed of the foregoing. Its F c and F c3 sequences have been engineered according to the "Knob - into - Hole" technology and further contain the double mutations L234A and L235A.

[0946] In one embodiment, the first immunoglobulin or the second immunoglobulin is selected from the group consisting of anti - CD123 antibodies: the anti - CD123 antibodies designated "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", or humanized forms thereof, or the anti - CD123 antibody "7G3" described below, e.g., the anti - CD123 antibody "3E3 - D3" or "7G3", or humanized forms thereof.

[0947] In one embodiment, the first immunoglobulin or the second immunoglobulin is an anti-CD3 antibody selected from the group consisting of the anti-CD3 antibodies designated "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", or a humanized form thereof, such as the anti-CD3 antibodies designated "20G6-F3", "4B4-D7", "4E7-C9", "18F5-H10", and "hz20G6", such as the anti-CD3 antibodies designated "20G6-F3", "4B4-D7".

[0948] Accordingly, V D1 and V D4 or V D2 and V D3 are variable domains of an anti-CD3 antibody heavy or light chain, wherein the anti-CD3 antibody comprises:

[0949] a) a heavy chain variable domain containing CDR1-H of SEQ ID NO: 6, CDR2-H of SEQ ID NO: 7, CDR3-H of SEQ ID NO: 8, and a light chain variable domain containing CDR1-L of SEQ ID NO: 10 or SEQ ID NO: 142, CDR2-L of the sequence 'KVS', and CDR3-L of SEQ ID NO: 11; or

[0950] b) a heavy chain variable domain containing CDR1-H of SEQ ID NO: 13, CDR2-H of SEQ ID NO: 14, CDR3-H of SEQ ID NO: 15, and a light chain variable domain containing CDR1-L of SEQ ID NO: 17 or SEQ ID NO: 184, CDR2-L of the sequence 'KVS', and CDR3-L of SEQ ID NO: 11; or

[0951] c) A heavy chain variable domain containing CDR1-H of SEQ ID NO: 13, CDR2-H of SEQ ID NO: 19, CDR3-H of SEQ ID NO: 20, and a light chain variable domain containing CDR1-L of SEQ ID NO: 22, CDR2-L of the sequence 'KVS', and CDR3-L of SEQ ID NO: 11; or

[0952] d) A heavy chain variable domain containing CDR1-H of SEQ ID NO: 24, CDR2-H of SEQ ID NO: 19, CDR3-H of SEQ ID NO: 25, and a light chain variable domain containing CDR1-L of SEQ ID NO: 27, CDR2-L of the sequence 'KVS', and CDR3-L of SEQ ID NO: 28, and

[0953] wherein if V D4 is the variable domain of the light chain, then 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, then V D1 is the variable domain of the light chain as defined above, or

[0954] if V D3 is the variable domain of the light chain, then 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, then V D3 is the variable domain of the light chain as defined above.

[0955] In another embodiment, V D1 and V D4 or V D2 and V D3 are variable domains of an anti-CD3 antibody heavy chain or light chain, wherein the anti-CD3 antibody is a humanized antibody and comprises:

[0956] a) A heavy chain variable domain of SEQ ID NO: 13 or a sequence at least 85% identical thereto and / or a light chain variable domain of SEQ ID NO: 143 or a sequence at least 85% identical thereto; or

[0957] b) A heavy chain variable domain of SEQ ID NO: 171 or a sequence at least 85% identical thereto and / or a light chain variable domain of SEQ ID NO: 158 or a sequence at least 85% identical thereto; or

[0958] c) the heavy chain variable domain of the sequence SEQ ID NO: 176 or a sequence that is at least 85% identical thereto and / or the light chain variable domain of the sequence SEQ ID NO: 164 or a sequence that is at least 85% identical thereto; or

[0959] wherein if V D4 is the variable domain of the light chain, then 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, then V D1 is the variable domain of the light chain as defined above, or

[0960] if V D3 is the variable domain of the light chain, then 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, then V D3 is the variable domain of the light chain as defined above.

[0961] In the sequences that are 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 6 CDR sequences are unchanged compared to the 6 CDRs present in the reference sequences 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.

[0962] 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 monkey CD3. Specifically, the antibody-like binding protein of the present invention binds to the extracellular domain of human CD3 or both human and cynomolgus monkey CD3. More specifically, the antibody binds to CD3ε. More specifically, the antibody-like binding protein binds to the extracellular domain of human or human and cynomolgus monkey CD3ε. Whether present in the form of a complex such as the CD3ε / δ complex or as a single protein, whether expressed in isolated form or present as a soluble extracellular domain or full-length membrane-anchored CD3ε, for example, in T cells, the antibody-like binding protein binds to CD3ε. The antibody-like binding protein according to the present invention specifically targets the surface human CD3 protein or both human and cynomolgus monkey CD3 proteins, particularly CD3ε.

[0963] The ratio of the affinity of the antibody-like binding according to the invention for cynomolgus CD3 to the affinity for human CD3 (KD (cynomolgus) / KD (human)) ≤ 10, specifically ≤ 6, ≤ 5, ≤ 4, ≤ 3, ≤ 2, ≤ 1 or ≤ 0.5. Thus, such polypeptides according to the invention can be used in toxicity studies carried out in monkeys, and the toxicity profiles observed in monkeys are related to the potential side effects expected in humans.

[0964] Furthermore, the antibody-like binding protein according to the invention has an affinity (KD) for human CD3 or cynomolgus CD3 or both ≤ 50 nM, ≤ 40 nM or ≤ 30 nM, for example ≤ 20 nM, for example an affinity of 0.1 nM - 30 nM, specifically an affinity of 0.4 nM - 20 nM or 0.4 nM - 15 nM.

[0965] In one embodiment, in the absence of target cells, the antibody-like binding protein of the invention has lower T-cell activation of less than 20%, less than 18%, less than 16%, less than 14%, less than 12%, less than 10%.

[0966] In one embodiment, in the presence of target cells, the antibody-like binding protein of the invention has higher T-cell activation of more than 55%, more than 60%, more than 62%, more than 64%, more than 66%, more than 68%, more than 70%.

[0967] “ low T-cell activation ” In the context of the antibody-like binding protein of the invention refers to T-cell activation of less than 20%, less than 18%, less than 16%, less than 14%, less than 12%, less than 10%.

[0968] As used herein, “ target cell ” refers to cells expressing a second antigen, and in one example of target cells herein refers to cells expressing CD123 such as THP-1 cells.

[0969] As used herein, “ high T-cell activation ” refers to T-cell activation of more than 50%, more than 55%, more than 60%, more than 62%, more than 64%, more than 66%, more than 68%, more than 70%.

[0970] In another embodiment, the invention relates to an antibody-like binding protein having biological and immunological specificity for at least one other target antigen.

[0971] Thus, 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 capable of binding two different antigen targets or epitopes.

[0972] Accordingly, in one embodiment, if the second immunoglobulin is a CD123 antibody as defined in the Anti-CD123 Antibody section above, the first immunoglobulin is an immunoglobulin against at least one other target, or if the first immunoglobulin is a CD123 antibody as defined in the Anti-CD123 Antibody section above, the second immunoglobulin is an immunoglobulin against at least one other target.

[0973] In another embodiment, if the second immunoglobulin is a CD3 antibody as defined in the Anti-CD3 Antibody section above, the first immunoglobulin is an immunoglobulin against at least one other target, or if the first immunoglobulin is a CD3 antibody as defined in the Anti-CD3 Antibody section above, the second immunoglobulin is an immunoglobulin against at least one other target.

[0974] The antibody-like binding proteins of the present invention have a T-cell engaging effect. This T-cell engaging effect induces cytotoxicity in target cells. In one embodiment, the target cells are cells expressing CD123, such as cancer cells expressing CD123, for example THP-1 or TF-1.

[0975] Accordingly, in one embodiment, the antibody-like binding proteins according to the present invention can bind and be able to engage primary T-cells and be able to lyse cells in vitro, wherein (EC 50 )≤40 pM, ≤35 pM, for example ≤30 pM.

[0976] “ cytotoxicity ” herein refers to the property of a compound such as the antibody-like binding proteins of the present invention or an anti-CD123 antibody to be toxic to cells. Cytotoxicity can be induced by different reaction mechanisms and can thus be classified as cell-mediated toxicity, apoptosis, antibody-dependent cell-mediated toxicity (ADCC) or complement-dependent cytotoxicity (CDC).

[0977] “ antibody-dependent cell-mediated cytotoxicity ” or “ ADCC ” refers to a cell-mediated immune defense mechanism whereby effector cells of the immune system actively lyse target cells whose membrane surface antigens have been bound by specific antibodies.

[0978] “ complement-dependent cytotoxicity ” or “ CDC ” in the context of the present invention refers to the lysis of target cells in the presence of complement system proteins.

[0979] “ cell-mediated cytotoxicity ” refers to the cell lysis of target cells by effector lymphocytes such as cytotoxic T lymphocytes or natural killer cells, and can thus be classified as T-cell-mediated cytotoxicity and NK cell cytotoxicity.

[0980] In one embodiment, the cytotoxicity herein refers to cell-mediated cytotoxicity such as T-cell-mediated cytotoxicity.

[0981] In addition, in one embodiment, cell-mediated cytotoxicity refers to cell-mediated cytotoxicity by T cells.

[0982] Accordingly, the antibody-like binding protein of the present invention induces cell-mediated cytotoxicity in target cells mediated by T cells.

[0983] Methods for measuring cytotoxicity are known to those skilled in the art and include using the 51 chromium (Cr) release assay, live / dead cell staining of target cells including propidium iodide, 7-AAD and other dyes known to those skilled in the art, detection of lytic molecules released by T cells including granzyme and perforin by flow cytometry or ELISA, detection of lactate dehydrogenase (LDH) released from damaged cells such as the culture medium as a detection of cytotoxicity and cell lysis of cells, detection of cell surface mobilization of CD107a, Annexin V (calcium-dependent phospholipid-binding protein) staining of apoptotic target cells, and detection of (CASP3) such as activation of Caspase-3. In addition, those skilled in the art can distinguish different cytotoxicity mechanisms based on the selected test and experimental settings.

[0984] In one example, cell-mediated cytotoxicity can be measured, for example, as described above using CFSE to label target cells and 7-AAD to label dead cells, such as in Example 3.2.

[0985] In another embodiment, the antibody-like binding protein is capable of binding to CD3 and at least one other antigen target, such as CD123.

[0986] In one embodiment, the antibody-like binding protein is capable of inhibiting the function of the other antigen target, such as CD123.

[0987] In one aspect of the invention, the antibody-like binding protein binds to human CD123. In another embodiment, the antibody-like binding protein also binds to cynomolgus monkey CD123. Specifically, the antibody-like binding protein of the invention binds to the extracellular domain of human CD123 or both human and cynomolgus monkey CD123. More specifically, the antibody-like binding protein binds to the distal portion of CD123, for example, binds to the amino acids at positions 19 - 49 of human CD123 starting from the amino acid sequence SEQ ID NO: 104. The antibody-like binding protein binds to CD123 whether in isolated form or in the soluble extracellular domain or full-length membrane-anchored CD123 present in CD123-expressing cells such as AML cells or CD123-transfected cells. The antibody-like binding protein according to the invention is specific for cells expressing human and cynomolgus monkey CD123 proteins on their surface, such as cancer cells expressing CD123.

[0988] Accordingly, the antibody-like binding protein according to the invention has an affinity (KD) for human CD123 or cynomolgus monkey CD123 or both of ≤ 20 nM, ≤ 15 nM or ≤ 10 nM, for example ≤ 5 nM, for example an affinity of 0.01 nM - 5 nM, more specifically 0.1 nM - 5 nM.

[0989] Accordingly, in one embodiment, the first immunoglobulin is an anti-CD3 antibody selected from the group consisting of: the anti-CD3 antibodies designated "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", or a humanized form thereof, e.g., the anti-CD3 antibodies designated "20G6-F3", "4B4-D7", "4E7-C9", "18F5-H10", "hz4B4", and "hz20G6", and the second immunoglobulin is an anti-CD123 antibody selected from the group consisting of: the anti-CD123 antibodies designated "3E3-D3", "1E1-G5", "2B8-F3", "2F8-D6", "3B10-E6", "5A5-B4", "6B10-E4", "6C10-C4", "6D6-B8", "8B 11-B7", "9B8-G6", "9D7-C8", and "9F6-G3".

[0990] Accordingly, in another embodiment, the second immunoglobulin is an anti-CD3 antibody selected from the group consisting of the anti-CD3 antibodies designated "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", or a humanized form thereof, such as the anti-CD3 antibodies designated "20G6-F3", "4B4-D7", "4E7-C9", "18F5-H10", "hz4B4", and "hz20G6", and the first immunoglobulin is an anti-CD123 antibody selected from the group consisting of the anti-CD123 antibodies designated "3E3-D3", "1E1-G5", "2B8-F3", "2F8-D6", "3B10-E6", "5A5-B4", "6B10-E4", "6C10-C4", "6D6-B8", "8B 11-B7", "9B8-G6", "9D7-C8", and "9F6-G3".

[0991] Accordingly, 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, which are defined respectively by three CDR sequences of one of the 13 anti-CD123 antibodies designated "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" as described above or by the heavy chain and light chain variable domain sequences,

[0992] wherein if both V D2 and V D3 comprise three CDR sequences of a heavy chain and a light chain variable domain sequence of an anti-CD3 antibody as defined above, then V D1 and V D4Each contains three CDR sequences of an anti-CD123 antibody heavy and light chain variable domain sequence as defined above, or

[0993] wherein if V D1 and V D4 each contain a CDR sequence of an anti-CD3 antibody heavy and light chain variable domain sequence as defined above, then V D2 and V D3 each contain three CDRs of an anti-CD123 antibody heavy and light chain variable domain sequence as defined above.

[0994] Accordingly, in one embodiment V D1 and V D4 are variable domains of an anti-CD3 antibody heavy or light chain, wherein the anti-CD3 antibody comprises a heavy chain variable domain containing CDR1-H of sequence SEQ ID NO: 6, CDR2-H of sequence SEQ ID NO: 7, CDR3-H of sequence SEQ ID NO: 8 and a light chain variable domain containing CDR1-L of sequence SEQ ID NO: 10 or SEQ ID NO: 142, CDR2-L of sequence 'KVS' and CDR3-L of sequence SEQ ID NO: 11 and V D2 and V D3 are variable domains of an anti-CD123 antibody heavy or light chain 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 "Anti-CD123 Antibodies" section,

[0995] wherein if V D3 and V D4 are both heavy chain variable domains, then V D1 and V D2 are both light chain variable domains, or if V D3 and V D4 are both light chain variable domains, then V D1 and V D2 are both heavy chain variable domains.

[0996] Accordingly, in another embodiment, V D2 and V D3is a variable domain of an anti-CD3 antibody heavy or light chain, wherein the anti-CD3 antibody comprises a heavy chain variable domain comprising CDR1-H having the sequence SEQ ID NO: 6, CDR2-H having the sequence SEQ ID NO: 7, CDR3-H having the sequence SEQ ID NO: 8, and a light chain variable domain comprising CDR1-L having the sequence SEQ ID NO: 10 or SEQ ID NO: 142, CDR2-L having the sequence 'KVS', and CDR3-L having the sequence SEQ ID NO: 11, and V D1 and V D4 is a variable domain of an anti-CD123 antibody heavy or light chain 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 "Anti-CD123 Antibodies" section,

[0997] wherein if V D3 and V D4 are both heavy chain variable domains, then V D1 and V D2 are both light chain variable domains, or if V D3 and V D4 are both light chain variable domains, then V D1 and V D2 are both heavy chain variable domains.

[0998] Accordingly, in another embodiment, V D1 and V D4 is a variable domain of an anti-CD3 antibody heavy or light chain, wherein the anti-CD3 antibody comprises a heavy chain variable domain comprising CDR1-H having the sequence SEQ ID NO: 13, CDR2-H having the sequence SEQ ID NO: 14, CDR3-H having the sequence SEQ ID NO: 15, and a light chain variable domain comprising CDR1-L having the sequence SEQ ID NO: 17 or SEQ ID NO: 184, CDR2-L having the sequence 'KVS', and CDR3-L having the sequence SEQ ID NO: 11, and V D2 and V D3is a variable domain of an anti-CD123 antibody heavy or light chain selected from the group: "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 "Anti-CD123 Antibodies" section,

[0999] where if V D3 and V D4 are both heavy chain variable domains, then V D1 and V D2 are both light chain variable domains, or if V D3 and V D4 are both light chain variable domains, then V D1 and V D2 are both heavy chain variable domains.

[1000] Accordingly, in another embodiment, V D1 and V D4 are variable domains of an anti-CD3 antibody heavy or light chain, where the anti-CD3 antibody comprises a heavy chain variable domain containing CDR1-H of sequence SEQ ID NO: 13, CDR2-H of sequence SEQ ID NO: 14, CDR3-H of sequence SEQ ID NO: 15 and a light chain variable domain containing CDR1-L of sequence SEQ ID NO: 17 or SEQ ID NO: 184, CDR2-L of sequence 'KVS' and CDR3-L of sequence SEQ ID NO: 11, and V D2 and V D3 are variable domains of an anti-CD123 antibody heavy or light chain selected from the group: "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 "Anti-CD123 Antibodies" section,

[1001] where if V D3 and V D4 are both heavy chain variable domains, then V D1 and V D2 are both light chain variable domains, or if V D3 and V D4 are both light chain variable domains, then V D1 and VD2 Both are variable domains of the heavy chain.

[1002] In another embodiment, V D1 and V D4 are variable domains of a humanized anti-CD3 antibody heavy or light chain, wherein the anti-CD3 antibody comprises a variable heavy chain variable domain of SEQ ID NO: 138 and / or a light chain variable domain of SEQ ID NO: 143, and V D2 and V D3 are variable domains of an anti-CD123 antibody heavy or light chain 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 "Anti-CD123 Antibodies" section,

[1003] wherein if V D3 and V D4 are both variable domains of the heavy chain, then V D1 and V D2 are both variable domains of the light chain, or if V D3 and V D4 are both variable domains of the light chain, then V D1 and V D2 are both variable domains of the heavy chain.

[1004] In another embodiment, V D2 and V D3 are variable domains of a humanized anti-CD3 antibody heavy or light chain, wherein the anti-CD3 antibody comprises a heavy chain variable domain of SEQ ID NO: 138 and / or a light chain variable domain of SEQ ID NO: 143, and V D1 and V D4 are variable domains of an anti-CD123 antibody heavy or light chain selected from the group consisting of: "3E3-D3", "1E1-G5", "2B8-F3", "2F8-D6", "3B10-E6", "5A5-B4", "6B 10-E4", "6C10-C4", "6D6-B8", "8B11-B7", "9B8-G6", "9D7-C8", "9F6-G3" anti-CD123 antibodies, as described above in the "Anti-CD 123 Antibodies" section,

[1005] wherein if V D3 and V D4 are both variable domains of the heavy chain, then V D1and V D2 are both light chain variable domains, or if V D3 and V D4 are both light chain variable domains, then V D1 and V D2 are both heavy chain variable domains.

[1006] In another embodiment, V D1 and V D4 are variable domains of a humanized anti-CD3 antibody heavy or light chain, wherein the anti-CD3 antibody comprises the heavy chain variable domain of SEQ ID NO: 171 and / or the light chain variable domain of SEQ ID NO: 158, and V D2 and V D3 are variable domains of an anti-CD123 antibody heavy or light chain 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 "Anti-CD123 Antibodies" section,

[1007] wherein if V D3 and V D4 are both heavy chain variable domains, then V D1 and V D2 are both light chain variable domains, or if V D3 and V D4 are both light chain variable domains, then V D1 and V D2 are both heavy chain variable domains.

[1008] In another embodiment, V D2 and V D3 are variable domains of a humanized anti-CD3 antibody heavy or light chain, wherein the anti-CD3 antibody comprises the heavy chain variable domain of SEQ ID NO: 171 and / or the light chain variable domain of SEQ ID NO: 158, and V D1 and V D4is a variable domain of an anti-CD123 antibody heavy or light chain selected from the group: "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 antibody, as described above in the "Anti-CD123 Antibody" section,

[1009] wherein if V D3 and V D4 are both heavy chain variable domains, then V D1 and V D2 are both light chain variable domains, or if V D3 and V D4 are both light chain variable domains, then V D1 and V D2 are both heavy chain variable domains.

[1010] In another embodiment, V D1 and V D4 are variable domains of a humanized anti-CD3 antibody heavy or light chain, wherein the anti-CD3 antibody comprises a heavy chain variable domain of SEQ ID NO: 176 and / or a light chain variable domain of SEQ ID NO: 164, and V D2 and V D3 are variable domains of an anti-CD123 antibody heavy or light chain selected from the group: "3E3-D3", "1E1-G5", "2B8-F3", "2F8-D6", "3B 10-E6", "5A5-B4", "6B10-E4", "6C10-C4", "6D6-B8", "8B11-B7", "9B8-G6", "9D7-C8", "9F6-G3" anti-CD123 antibody, as described above in the "Anti-CD123 Antibody" section,

[1011] wherein if V D3 and V D4 are both heavy chain variable domains, then V D1 and V D2 are both light chain variable domains, or if V D3 and V D4 are both light chain variable domains, then V D1 and V D2 are both heavy chain variable domains.

[1012] In another embodiment, V D2 and V D3is a variable domain of a humanized anti-CD3 antibody heavy or light chain, wherein said anti-CD3 antibody comprises a heavy chain variable domain of sequence SEQ ID NO: 176 and / or a light chain variable domain of sequence SEQ ID NO: 164, and V D1 and V D4 are variable domains of an anti-CD123 antibody heavy or light chain 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 "Anti-CD123 Antibodies" section,

[1013] wherein if V D3 and V D4 are both heavy chain variable domains, then V D1 and V D2 are both light chain variable domains, or if V D3 and V D4 are both light chain variable domains, then V D1 and V D2 are both heavy chain variable domains.

[1014] According to another 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 a heavy chain variable domain and a light chain variable domain as defined by the CDR sequences of the antibody 7G3 heavy chain and light chain variable domain sequences as defined hereinbelow. In one embodiment, V D1 and V D4 or V D2 and V D3 comprise the heavy chain variable domain and the light chain variable domain of the antibody 7G3 as described in patent application WO2013 / 173820 (which is incorporated herein by reference).

[1015] Accordingly, the anti-CD123 antibody referred to as "7G3" as used herein comprises:

[1016] - a heavy chain variable domain consisting of the following sequences: (SEQ ID NO: 312, where the CDRs are shown in bold), which contains CDR1-H of SEQ ID NO: 375, CDR2-H of SEQ ID NO: 376, and CDR3-H of SEQ ID NO: 377 and

[1017] - A light chain variable domain consisting of the following sequences: (SEQ ID NO: 308, where the CDRs are shown in bold), which contains CDR1-L of SEQ ID NO: 378, CDR2-L of the sequence 'WAS', and CDR3-L of SEQ ID NO: 379.

[1018] In another aspect of the present invention, antibody 7G3 can also be a humanized antibody or a fragment of a humanized antibody. Accordingly, in one embodiment, the antibody 7G3 of the present invention is a humanized antibody, which comprises:

[1019] - A heavy chain variable domain consisting of the following sequences: (SEQ ID NO: 380, where the CDRs are shown in bold), which contains CDR1-H of SEQ ID NO: 381, CDR2-H of SEQ ID NO: 377, and CDR3-H of SEQ ID NO: 382, or

[1020] A heavy chain variable domain consisting of the following sequences: (SEQ ID NO: 383, where the CDRs are shown in bold), which contains CDR1-H of SEQ ID NO: 381, CDR2-H of SEQ ID NO: 384, and CDR3-H of SEQ ID NO: 382, or

[1021] - A light chain variable domain consisting of the following sequences: (SEQ ID NO: 385, where the CDRs are shown in bold), which contains CDR1-L of SEQ ID NO: 378, CDR2-L of the sequence 'WAS', and CDR3-L of SEQ ID NO: 379.

[1022] In one embodiment, an antibody-like binding protein that specifically binds to human CD3ε and human CD123 comprises:

[1023] a) The light chain variable domain (V of the first immunoglobulin D1), which consists of the amino acid sequence SEQ ID NO: 9 or a sequence that is at least 85% identical thereto, the light chain variable domain (V of the second immunoglobulin D2 ), which consists of the amino acid sequence SEQ ID NO: 308 or a sequence that is at least 85% identical thereto, the heavy chain variable domain (V of the second immunoglobulin D3 ), which consists of the amino acid sequence SEQ ID NO: 312 or a sequence that is at least 85% identical thereto, and the heavy chain variable domain (V of the first immunoglobulin D4 ), which consists of the amino acid sequence SEQ ID NO: 5 or a sequence that is at least 85% identical thereto, or

[1024] b) The light chain variable domain (V of the first immunoglobulin D1 ), which consists of the amino acid sequence SEQ ID NO: 21 or a sequence that is at least 85% identical thereto, the light chain variable domain (V of the second immunoglobulin D2 ), which consists of the amino acid sequence SEQ ID NO: 308 or a sequence that is at least 85% identical thereto, the heavy chain variable domain (V of the second immunoglobulin D3 ), which consists of the amino acid sequence SEQ ID NO: 312 or a sequence that is at least 85% identical thereto, and the heavy chain variable domain (V of the first immunoglobulin D4 ), which consists of the amino acid sequence SEQ ID NO: 18 or a sequence that is at least 85% identical thereto, or

[1025] c) The light chain variable domain (V of the first immunoglobulin D1 ), which consists of the amino acid sequence SEQ ID NO: 16 or a sequence that is at least 85% identical thereto, the light chain variable domain (V of the second immunoglobulin D2 ), which consists of the amino acid sequence SEQ ID NO: 308 or a sequence that is at least 85% identical thereto, the heavy chain variable domain (V of the second immunoglobulin D3 ), which consists of the amino acid sequence SEQ ID NO: 312 or a sequence that is at least 85% identical thereto, and the heavy chain variable domain (V of the first immunoglobulin D4 ), which consists of the amino acid sequence SEQ ID NO: 12 or a sequence that is at least 85% identical thereto, or

[1026] d) The light chain variable domain (V of the first immunoglobulin D1 ), which consists of the amino acid sequence SEQ ID NO: 26 or a sequence that is at least 85% identical thereto, the light chain variable domain (V of the second immunoglobulin D2), which consists of the amino acid sequence SEQ ID NO: 308 or a sequence that is at least 85% identical thereto, the heavy chain variable domain (V of the second immunoglobulin D3 ), which consists of the amino acid sequence SEQ ID NO: 312 or a sequence that is at least 85% identical thereto, and the heavy chain variable domain (V of the first immunoglobulin D4 ), which consists of the amino acid sequence SEQ ID NO: 23 or a sequence that is at least 85% identical thereto, or

[1027] e) The light chain variable domain (V of the first immunoglobulin D1 ), which consists of the amino acid sequence SEQ ID NO: 308 or a sequence that is at least 85% identical thereto, the light chain variable domain (V of the second immunoglobulin D2 ), which consists of the amino acid sequence SEQ ID NO: 143 or a sequence that is at least 85% identical thereto, the heavy chain variable domain (V of the second immunoglobulin D3 ), which consists of the amino acid sequence SEQ ID NO: 138 or a sequence that is at least 85% identical thereto, and the heavy chain variable domain (V of the first immunoglobulin D4 ), which consists of the amino acid sequence SEQ ID NO: 312 or a sequence that is at least 85% identical thereto, or

[1028] f) The light chain variable domain (V of the first immunoglobulin D1 ), which consists of the amino acid sequence SEQ ID NO: 158 or a sequence that is at least 85% identical thereto, the light chain variable domain (V of the second immunoglobulin D2 ), which consists of the amino acid sequence SEQ ID NO: 308 or a sequence that is at least 85% identical thereto, the heavy chain variable domain (V of the second immunoglobulin D3 ), which consists of the amino acid sequence SEQ ID NO: 312 or a sequence that is at least 85% identical thereto, and the heavy chain variable domain (V of the first immunoglobulin D4 ), which consists of the amino acid sequence SEQ ID NO: 171 or a sequence that is at least 85% identical thereto,

[1029] g) The light chain variable domain (V of the first immunoglobulin D1 ), which consists of the amino acid sequence SEQ ID NO: 230 or a sequence that is at least 85% identical thereto, the light chain variable domain (V of the second immunoglobulin D2 ), which consists of the amino acid sequence SEQ ID NO: 158 or a sequence that is at least 85% identical thereto, the heavy chain variable domain (V of the second immunoglobulin D3), which consists of the amino acid sequence SEQ ID NO: 171 or a sequence at least 85% identical thereto, and the variable heavy domain (V D4 ), which consists of the amino acid sequence SEQ ID NO: 226 or a sequence at least 85% identical thereto,

[1030] h) The variable light domain (V D1 ) of the first immunoglobulin, which consists of the amino acid sequence SEQ ID NO: 385 or a sequence at least 85% identical thereto, the variable light domain (V D2 ) of the second immunoglobulin, which consists of the amino acid sequence SEQ ID NO: 141 or a sequence at least 85% identical thereto, the variable heavy domain (V D3 ) of the second immunoglobulin, which consists of the amino acid sequence SEQ ID NO: 138 or a sequence at least 85% identical thereto, and the variable heavy domain (V D4 ) of the first immunoglobulin, which consists of the amino acid sequence SEQ ID NO: 383 or a sequence at least 85% identical thereto.

[1031] Among the sequences at least 85% identical to the reference sequence described above (e.g., sequences at least 85% identical to SEQ ID NO: 383 or SEQ ID NO: 385), compared with the 6 CDRs in the reference sequence of the present invention, the sequences of the 6 CDRs have not changed.

[1032] In one embodiment, the antibody-binding protein defined according to any one of a)-g) further comprises the following linkers: L1 of sequence SEQ ID NO: 307, L2 of sequence SEQ ID NO: 309, L3 of the amino acid sequence 'S', L4 of the amino acid sequence 'RT', and C of sequence SEQ ID NO: 313 H1 .

[1033] In one embodiment, the antibody-binding protein defined according to any one of a)-g) further comprises F of sequence SEQ ID NO: 327 c2 .

[1034] In one embodiment, the antibody-binding protein defined according to h) further comprises the following linkers: L1 of sequence SEQ ID NO: 389, L2 of sequence SEQ ID NO: 389, L3 and L4 consisting of 0 amino acids, and C of sequence SEQ ID NO: 313 H1 .

[1035] In one embodiment, the antibody-binding protein defined according to h) further comprises F of sequence SEQ ID NO: 392 c2 .

[1036] In one embodiment, L5 of the antibody-binding protein as defined in any one of a)-h) comprises 0 amino acids.

[1037] In another embodiment, the antibody-binding protein as defined in any one of a)-g) further comprises the following linkers: L1 of SEQ ID NO: 307, L2 of SEQ ID NO: 309, L3 of the amino acid sequence 'S', L4 of the amino acid sequence 'RT', C of SEQ ID NO: 329 H1 and F of SEQ ID NO: 330 c .

[1038] In another embodiment, the antibody-binding protein as defined in h) further comprises the following linkers: L1 of SEQ ID NO: 389, L2 of SEQ ID NO: 389, L3 and L4 consisting of 0 amino acids, C of SEQ ID NO: 313 H1 and F of SEQ ID NO: 394 c .

[1039] In another embodiment, the antibody-binding protein as defined in h) further comprises an Fc stump 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.

[1040] In one embodiment, an antibody-binding protein that specifically binds to human CD3ε and human CD123 comprises or consists essentially of the following:

[1041] a) A polypeptide according to formula [I], which consists of an amino acid sequence SEQ ID NO: 388 containing V of SEQ ID NO: 385 D1 , L1 of SEQ ID NO: 389, V of SEQ ID NO: 141 D2 , L2 of SEQ ID NO: 389 and C of SEQ ID NO: 310 L , or

[1042] a sequence that is at least 85% identical to SEQ ID NO: 388, wherein the 3 CDRs of SEQ ID NO: 378, 'WAS' and SEQ ID NO: 379 in the hz7G3 light chain variable domain (the sequence of V of SEQ ID NO: 385 D1 ), and the hz20G6 light chain variable domain (V of SEQ ID NO: 141 D2In the sequences of SEQ ID NO: 142, 'KVS' and SEQ ID NO: 11, three CDRs remain unchanged, and

[1043] b) A polypeptide according to formula [II], which consists of a V containing the sequence SEQ ID NO: 138 D3 (where L3 is 0 amino acids), a V of sequence SEQ ID NO: 383 D4 (where L4 is 0 amino acids) and a C of sequence SEQ ID NO: 313 H1 forming the amino acid sequence SEQ ID NO: 390, or

[1044] a sequence that is at least 85% identical to SEQ ID NO: 390, wherein in the hz7G3 heavy chain variable domain (the V of SEQ ID NO: 383 D4 sequence), three CDRs of SEQ ID NO: 381, SEQ ID NO: 384 and SEQ ID NO: 382 and in the hz20G6 heavy chain variable domain (the V of SEQ ID NO: 138 D3 sequence), three CDRs of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 remain unchanged;

[1045] and wherein the polypeptide of formula [I] and the polypeptide of formula [II] form a cross light chain - heavy chain pair.

[1046] In one embodiment, an antibody - binding protein that specifically binds to human CD3ε and human CD123 comprises or consists essentially of:

[1047] a) A polypeptide according to formula [IV], which consists of a V containing the sequence SEQ ID NO: 385 D1 , an L1 of sequence SEQ ID NO: 389, a V of sequence SEQ ID NO: 141 D2 , an L2 of sequence SEQ ID NO: 389, a C of sequence SEQ ID NO: 310 L , an L5 containing 0 amino acids and an F of sequence SEQ ID NO: 392 c2 forming the amino acid sequence SEQ ID NO: 391, or

[1048] a sequence that is at least 85% identical to SEQ ID NO: 391, wherein in the hz7G3 light chain variable domain (the V of SEQ ID NO: 385 D1In the sequences of SEQ ID NO: 378, 'WAS' and the three CDRs of SEQ ID NO: 379 and the variable domain of the hz20G6 light chain (V of SEQ ID NO: 141) D2 In the sequences of SEQ ID NO: 142, 'KVS' and the three CDRs of SEQ ID NO: 11 remained unchanged; and

[1049] b) A polypeptide according to formula [III], which consists of a V containing the sequence SEQ ID NO: 138 D3 (where L3 is 0 amino acids), a V of SEQ ID NO: 383 D4 (where L4 is 0 amino acids) and a C of SEQ ID NO: 313 H1 and an F of SEQ ID NO: 394 c to form the amino acid sequence SEQ ID NO: 393, or

[1050] a sequence that is at least 85% identical to SEQ ID NO: 393, wherein in the sequence of the hz7G3 heavy chain variable domain (V of SEQ ID NO: 383) D4 the three CDRs of SEQ ID NO: 381, SEQ ID NO: 384 and SEQ ID NO: 382, and in the sequence of the hz20G6 heavy chain variable domain (V of SEQ ID NO: 138) D3 the three CDRs of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 remained unchanged;

[1051] and wherein the polypeptide of formula [IV] and the polypeptide of formula [III] form a cross light chain - heavy chain pair.

[1052] In the antibody - like binding protein, the polypeptide chains represented by formulas [III] and [IV] dimerize through their respective F c2 and F c regions.

[1053] In one embodiment, an antibody - binding protein that specifically binds to human CD3ε and human CD123 comprises or consists essentially of the following:

[1054] a) A polypeptide according to formula [I], which consists of a V containing the sequence SEQ ID NO: 385 D1 an L1 of SEQ ID NO: 389, a V of SEQ ID NO: 141 D2 an L2 of SEQ ID NO: 389, and a C of SEQ ID NO: 310 Lcomprises the amino acid sequence SEQ ID NO: 388, or

[1055] a sequence that is at least 85% identical to SEQ ID NO: 388, wherein the three CDRs of SEQ ID NO: 378, 'WAS', and SEQ ID NO: 379 in the hz7G3 light chain variable domain (the V of SEQ ID NO: 385 D1 sequence) and the three CDRs of SEQ ID NO: 142, 'KVS', and SEQ ID NO: 11 in the hz20G6 light chain variable domain (the V of SEQ ID NO: 141 D2 sequence) are unchanged; and

[1056] b) A polypeptide according to formula [III] that consists of or comprises the V of SEQ ID NO: 138 D3 (where L3 is 0 amino acids), the V of SEQ ID NO: 383 D4 (where L4 is 0 amino acids), and the C of SEQ ID NO: 313 H1 and the F of SEQ ID NO: 396 c and has the amino acid sequence SEQ ID NO: 395, or

[1057] a sequence that is at least 85% identical to SEQ ID NO: 395, wherein the three CDRs of SEQ ID NO: 381, SEQ ID NO: 384, and SEQ ID NO: 382 in the hz7G3 heavy chain variable domain (the V of SEQ ID NO: 383 D4 sequence) and the three CDRs of SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8 in the hz20G6 heavy chain variable domain (the V of SEQ ID NO: 138 D3 sequence) are unchanged; and

[1058] c) An Fc stump (polypeptide F c3 ) that is the sequence SEQ ID NO: 397 or a sequence that is at least 85% identical thereto, wherein said stump F c3 or a sequence that is at least 85% identical thereto heterodimerizes with the F c region of the polypeptide according to formula [III]; and

[1059] wherein the polypeptide of formula [I] and the polypeptide of formula [III] form a cross light chain - heavy chain pair.

[1060] In one embodiment, an antibody - binding protein that specifically binds to human CD3ε and human CD123 comprises or consists essentially of the following:

[1061] a) A polypeptide according to formula [I], which is composed of a V comprising the sequence SEQ ID NO: 385 D1 , an L1 of sequence SEQ ID NO: 389, a V of sequence SEQ ID NO: 141 D2 , an L2 of sequence SEQ ID NO: 389 and a C of sequence SEQ ID NO: 310 L of the amino acid sequence SEQ ID NO: 388, or

[1062] a sequence that is at least 85% identical to SEQ ID NO: 388, wherein the 3 CDRs of SEQ ID NO: 378, 'WAS' and SEQ ID NO: 379 in the hz7G3 light chain variable domain (sequence of V of SEQ ID NO: 385 D1 ), and the 3 CDRs of SEQ ID NO: 142, 'KVS' and SEQ ID NO: 11 in the hz20G6 light chain variable domain (sequence of V of SEQ ID NO: 141 D2 ) are unchanged; and

[1063] b) A polypeptide according to formula [III], which is composed of a V comprising the sequence SEQ ID NO: 138 D3 (L3 is 0 amino acids), a V of sequence SEQ ID NO: 383 D4 (L4 is 0 amino acids) and a C of sequence SEQ ID NO: 313 H1 and a sequence SEQ ID NO: 400 of F c of the amino acid sequence SEQ ID NO: 399, or

[1064] a sequence that is at least 85% identical to SEQ ID NO: 399, wherein the 3 CDRs of SEQ ID NO: 381, SEQ ID NO: 384 and SEQ ID NO: 382 in the hz7G3 heavy chain variable domain (sequence of V of SEQ ID NO: 383 D4 ), and the 3 CDRs of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 in the hz20G6 heavy chain variable domain (sequence of V of SEQ ID NO: 138 D3 ) are unchanged; and

[1065] c) An Fc stump (polypeptide F c3 ), which is the sequence SEQ ID NO: 398 or a sequence that is at least 85% identical thereto, wherein the stump F c3 or a sequence that is at least 85% identical thereto and the F of the polypeptide according to formula [III]c Zonal heterodimerization; and

[1066] wherein the polypeptide of formula [I] and the polypeptide of formula [III] form cross light chain - heavy chain pairs.

[1067] Immunoconjugate

[1068] In one embodiment, the anti - CD123 antibody of the present invention is conjugated or linked to a growth inhibitor, a cytotoxic agent, or a prodrug - activating enzyme. Specifically, the anti - CD123 antibody of the present invention is effective in targeting the growth inhibitor, the cytotoxic agent, or the prodrug to cancer cells that express or over - express CD123 on their surface.

[1069] Nucleic acids, vectors, and recombinant host cells

[1070] Another 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.

[1071] Generally, the nucleic acid is a DNA or RNA molecule, which can be included in any suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage, or viral vector.

[1072] The terms " vector ", " cloning vector ", and " expression vector " mean a mediator through which a DNA or RNA sequence (e.g., a foreign gene) can be transferred into a host cell, thereby transforming the host and promoting the expression of the introduced gene (e.g., transcription and translation).

[1073] Accordingly, another object of the present invention relates to a vector comprising the nucleic acid of the present invention.

[1074] Such a vector may contain regulatory components, such as promoters, enhancers, terminators, etc., to cause or direct the expression of the polypeptide when administered to a subject. Examples of promoters and enhancers used in expression vectors for animal cells include the SV40 early promoter and enhancer (Mizukami T. et al. 1987), the enhancer of the LTR promoter of Moloney mouse leukemia virus (Kuwana Y et al. 1987), the promoter (Mason JO et al. 1985), and the immunoglobulin H - chain enhancer (Gillies SD et al. 1983), etc.

[1075] 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), pSG1βd2-4- (Miyaji H et al. 1990), etc. Other examples of plasmids include plasmids containing an origin of replication, or integrating plasmids such as pUC, pcDNA, pBR, etc.

[1076] Other examples of viral vectors include adenovirus, retrovirus, herpesvirus, and AAV vectors. Such recombinant viruses can be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection using a replication plasmid or virus. Typical examples of viral packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc. Detailed protocols for producing such replication-defective 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.

[1077] Another object of the present invention relates to cells that have been transfected, infected, or transformed with the nucleic acid and / or vector according to the present invention.

[1078] The term " transform " means introducing an "exogenous" (i.e., foreign) gene, DNA, or RNA sequence into a host cell, and then the host cell will express the introduced gene or sequence to produce the desired substance, usually a protein or enzyme encoded by the introduced gene or sequence. The host cell that receives and expresses the introduced DNA or RNA has been " transform ".

[1079] 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 a host cell and a compatible vector under suitable conditions, for example, a protein encoded by exogenous DNA is carried by the vector and introduced into the host cell for expression.

[1080] General expression systems include Escherichia coli host cells and plasmid vectors, insect host cells and baculovirus vectors, and mammalian host cells and vectors. Examples of other host cells include, but are not limited to, prokaryotic cells (such as bacteria) and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include Escherichia coli (E. coli), Kluyveromyces, or Saccharomyces yeasts, mammalian cell lines (such as Vero cells, CHO cells, 3T3 cells, COS cells, etc.), and primary or established mammalian cell cultures (such as those derived from lymphoblasts, fibroblasts, embryonic cells, epithelial cells, nerve cells, adipocytes, etc.). Examples also include mouse SP2 / 0-Ag14 cells (ATCC CRL1581), mouse P3X63-Ag8.653 cells (ATCC CRL1580), CHO cells deficient in the dihydrofolate reductase gene (hereinafter referred to as the "DHFR gene"), rat YB2 / 3HL.P2.G11.16Ag.20 cells (ATCC CRL1662, hereinafter referred to as "YB2 / 0 cells"), etc. Preferably, they are YB2 / 0 cells, since when expressed in these cells, the ADCC activity of chimeric or humanized antibodies is enhanced.

[1081] Specifically, for the expression of humanized antibodies or antibody-like binding proteins, the expression vector can be of the 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 the type in which the two genes are present on the same vector (tandem type). Considering 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 (WO 97 / 10354), pEE18, etc.

[1082] The present invention also relates to a method for producing recombinant host cells expressing an anti-CD3 antibody, an anti-CD123 antibody, or an antibody-like binding protein according to the present invention, the method comprising or consisting of the following steps: (i) introducing the recombinant nucleic acid or vector as described above into competent host cells in vitro or ex vivo, (ii) culturing the obtained recombinant host cells in vitro or ex vivo, and (iii) optionally, selecting cells that express and / or secrete the antibody.

[1083] These recombinant host cells can be used to produce the anti-CD3 antibody, at least one anti-CD123 antibody, or at least one antibody-like binding protein of the present invention.

[1084] Methods for generating antibodies and antibody-like binding proteins of the present invention

[1085] One embodiment of the present invention provides a method for preparing an antibody-like binding protein comprising two polypeptide chains forming two antigen-binding sites, wherein the first polypeptide has a structure represented by formula [I]:

[1086] V D1 -L1-V D2 -L2-C L [I]

[1087] and the second polypeptide has a structure represented by formula [II]:

[1088] V D3 -L3-V D4 -L4-C H1 [II]

[1089] wherein:

[1090] V D1 is the variable domain of the first immunoglobulin heavy or light chain;

[1091] V D2 is the variable domain of the second immunoglobulin heavy or light chain;

[1092] V D3 is the variable domain of the second immunoglobulin heavy or light chain;

[1093] V D4 is the variable domain of the first immunoglobulin heavy or light chain;

[1094] C L is the light chain constant domain of the immunoglobulin;

[1095] C H1 is the C H1 heavy chain constant domain of the immunoglobulin;

[1096] L1, L2, L3 and L4 are amino acid linkers;

[1097] and wherein the first and second polypeptides form a cross light chain-heavy chain pair, and

[1098] where V D1 and V D2 are both light chain variable domains or both heavy chain variable domains, and if V D1 and V D2 are light chain variable domains, then V D3 and V D4 are both heavy chain variable domains, or if VD1 and V D2 is a heavy chain variable domain, then V D3 and V D4 are both light chain variable domains.

[1099] In another embodiment, the present invention provides a method for preparing an antibody-like binding protein comprising four polypeptide chains forming four antigen-binding sites, wherein two of the polypeptide chains have a structure represented by formula [I]:

[1100] V D1 -L1-V D2 -L2-C L [I]

[1101] and one of the polypeptide chains has a structure represented by formula [III]:

[1102] V D3 -L3-V D4 -L4-C H1 -F c [III]

[1103] Wherein:

[1104] V D1 is the variable domain of the first immunoglobulin heavy or light chain;

[1105] V D2 is the variable domain of the second immunoglobulin heavy or light chain;

[1106] V D3 is the variable domain of the second immunoglobulin heavy or light chain;

[1107] V D4 is the variable domain of the first immunoglobulin heavy or light chain;

[1108] C L is the light chain constant domain of the immunoglobulin;

[1109] C H1 is the C H1 heavy chain constant domain of the immunoglobulin;

[1110] F c is the immunoglobulin hinge region and the CH2, CH3 immunoglobulin heavy chain constant domains of the immunoglobulin;

[1111] L1, L2, L3 and L4 are amino acid linkers;

[1112] and wherein the polypeptide of formula I and the polypeptide of formula III form cross light chain-heavy chain pairs, and

[1113] wherein VD1 and V D2 are both light chain variable domains or both heavy chain variable domains, and if V D1 and V D2 is a light chain variable domain, then V D3 and V D4 are both heavy chain variable domains, or if V D1 and V D2 is a heavy chain variable domain, then V D3 and V D4 are both light chain variable domains.

[1114] In another embodiment, the present invention provides a method for preparing an antibody-like binding protein comprising four polypeptide chains forming four antigen-binding sites, wherein two polypeptide chains have the structure shown by formula [IV]:

[1115] V D1 -L1-V D2 -L2-C L -L5-F c2 [IV]

[1116] and two polypeptide chains have the structure shown by formula [III]:

[1117] V D3 -L3-V D4 -L4-C H1 -F c [III]

[1118] Wherein:

[1119] V D1 is the variable domain of the first immunoglobulin heavy or light chain;

[1120] V D2 is the variable domain of the second immunoglobulin heavy or light chain;

[1121] V D3 is the variable domain of the second immunoglobulin heavy or light chain;

[1122] V D4 is the variable domain of the first immunoglobulin heavy or light chain;

[1123] C L is the light chain constant domain of the immunoglobulin;

[1124] C H1 is the C H1 heavy chain constant domain of th...

Claims

1. An antibody-like binding protein that specifically binds to human CD3ε and human CD123, which comprises two polypeptide chains forming two antigen-binding sites, wherein the structure of the first polypeptide chain is as shown in formula [IV]: V D1 -L1-V D2 -L2-C L -L5-Fc2[IV] and the structure of the second polypeptide chain is as shown in formula [III]: V D3 -L3-V D4 -L4-C H1 -Fc[III] 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 first immunoglobulin; C L is the constant domain of the light chain of an immunoglobulin; C H1 is the C constant domain of an immunoglobulin H1 heavy chain; Fc is the immunoglobulin hinge region and the CH2 and CH3 immunoglobulin heavy chain constant domains of an immunoglobulin; Fc2 is the immunoglobulin hinge region and the CH2 and CH3 immunoglobulin heavy chain constant domains of an immunoglobulin; and L1, L2, L3, L4, and L5 are amino acid linkers; wherein the first polypeptide and the second polypeptide form a cross light chain-heavy chain pair; wherein V D1 and V D2 are both variable domains of the light chain, or both variable domains of the heavy chain, and if V D1 and V D2 are variable domains of the light chain then V D3 and V D4 are both variable domains of the heavy chain, and if V D1 and V D2 are variable domains of the heavy chain then V D3 and V D4 are both variable domains of the light chain; and wherein the first immunoglobulin or the second immunoglobulin is an anti-CD3 antibody, the anti-CD3 antibody comprises a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain comprises CDR1-H consisting of the amino acid sequence of SEQ ID NO:6, CDR2-H consisting of the amino acid sequence of SEQ ID NO:7, and CDR3-H consisting of the amino acid sequence of SEQ ID NO:8, and the light chain variable domain comprises CDR1-L consisting of the amino acid sequence of SEQ ID NO:142, CDR2-L consisting of the amino acid sequence KVS, and CDR3-L consisting of the amino acid sequence of SEQ ID NO:11; and wherein the first immunoglobulin or the second immunoglobulin is an antibody that specifically binds to the extracellular domain of human CD123, which comprises a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain comprises CDR1-H consisting of the amino acid sequence of SEQ ID NO:375, CDR2-H consisting of the amino acid sequence of SEQ ID NO:376, and CDR3-H consisting of the amino acid sequence of SEQ ID NO:377, and the light chain variable domain comprises CDR1-L consisting of the amino acid sequence of SEQ ID NO:378, CDR2-L consisting of the amino acid sequence WAS, and CDR3-L consisting of the amino acid sequence of SEQ ID NO:

379.

2. The antibody-like binding protein according to claim 1, wherein the first and second immunoglobulins are IgG immunoglobulins.

3. The antibody-like binding protein according to claim 1, wherein C L is the light chain constant domain of an IgG immunoglobulin and / or C H1 is the C H1 heavy chain constant domain of an IgG immunoglobulin.

4. The antibody-like binding protein according to claim 1, wherein: (a) The length of L3 is at least twice the length of L1; or (b) The length of L4 is at least twice the length of L2.

5. The antibody-like binding protein according to claim 1, wherein: (a) The length of L1 is at least twice the length of L3; or (b) The length of L2 is at least twice the length of L4.

6. The antibody-like binding protein according to claim 1, wherein: The length of L1 is 3 to 12 amino acid residues; The length of L2 is 3 to 14 amino acid residues; The length of L3 is 1 to 8 amino acid residues; and The length of L4 is 1 to 3 amino acid residues.

7. The antibody-like binding protein according to claim 6, wherein: The length of L1 is 5 to 10 amino acid residues; The length of L2 is 5 to 8 amino acid residues; The length of L3 is 1 to 5 amino acid residues; and The length of L4 is 1 to 2 amino acid residues.

8. The antibody-like binding protein according to claim 7, wherein: The length of L1 is 7 amino acid residues; The length of L2 is 5 amino acid residues; The length of L3 is 1 amino acid residue; and The length of L4 is 2 amino acid residues.

9. The antibody-like binding protein according to claim 1, wherein: The length of L1 is 1 to 3 amino acid residues; The length of L2 is 1 to 4 amino acid residues; The length of L3 is 2 to 15 amino acid residues; and The length of L4 is 2 to 15 amino acid residues.

10. The antibody-like binding protein according to claim 9, wherein: The length of L1 is 1 to 2 amino acid residues; The length of L2 is 1 to 2 amino acid residues; The length of L3 is 4 to 12 amino acid residues; and The length of L4 is 2 to 12 amino acid residues.

11. The antibody-like binding protein according to claim 10, wherein: The length of L1 is 1 amino acid residue; The length of L2 is 2 amino acid residues; The length of L3 is 7 amino acid residues; and The length of L4 is 5 amino acid residues.

12. The antibody-like binding protein according to claim 1, wherein at least one of the linkers selected from L2, L3, and L4 contains at least one cysteine residue.

13. The antibody-like binding protein according to claim 1, wherein: (a) The length of L1 is 0 amino acid residues and the length of L3 is 2 or more amino acid residues; (b) The length of L3 is 0 amino acid residues and the length of L1 is at least 1 amino acid residue; or (c) The length of L4 is 0 amino acid residues and the length of L2 is 3 or more amino acid residues.

14. The antibody-like binding protein according to claim 13, wherein the length of L1 is 0 amino acid residues and the length of L3 is 2 or more amino acid residues.

15. The antibody-like binding protein according to claim 13, wherein the length of L4 is 0 amino acid residues and the length of L2 is 3 or more amino acid residues.

16. The antibody-like binding protein according to claim 1, wherein the length of L5 is 0 to 10 amino acid residues.

17. The antibody-like binding protein according to claim 1, wherein the antibody-like binding protein is bispecific and capable of binding two different antigen targets.

18. A pharmaceutical composition comprising the antibody-like binding protein according to any one of claims 1 to 17 and a pharmaceutically acceptable carrier.

19. An isolated nucleic acid comprising a sequence encoding the antibody-like binding protein according to any one of claims 1 to 17.

20. An expression vector comprising the nucleic acid according to claim 19.

21. A host cell that has been transfected, infected, or transformed with the nucleic acid according to claim 19.

22. A host cell that has been transfected, infected, or transformed with the expression vector according to claim 20.

23. A kit comprising at least one antibody-like binding protein according to any one of claims 1 to 17.

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