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BISPECIFIC BINDING MOLECULES

20220306740 · 2022-09-29

    Inventors

    Cpc classification

    International classification

    Abstract

    The present disclosure provides novel bispecific molecules that binds to human Survivin and human CD3, and methods of making and using the same.

    Claims

    1. A bispecific molecule that binds to human Survivin and human CD3 comprising: a) a sTCR comprising: (1) a V.sub.β comprising SEQ ID NO: 20 (CDR1), SEQ ID NO: 24 (CDR2), and SEQ ID NO: 28 (CDR3), and (2) a V.sub.α comprising SEQ ID NO: 19 (CDR1), SEQ ID NO: 23 (CDR2), and SEQ ID NO: 27 (CDR3), wherein the V.sub.β and V.sub.α regions of the sTCR are connected via a first peptide linker (L1); b) a Fab comprising: (1) a heavy chain region comprising a V.sub.H comprising SEQ ID NO: 21 (CDR1), SEQ ID NO: 25 (CDR2), and SEQ ID NO: 29 (CDR3); and a CH1 comprising the amino acid sequence of SEQ ID NO: 18, and (2) a light chain region comprising a V.sub.L comprising SEQ ID NO: 22 (CDR1), SEQ ID NO: 26 (CDR2), and SEQ ID NO: 30 (CDR3); and a C.sub.K comprising the amino acid sequence of SEQ ID NO: 17, wherein the V.sub.α of the sTCR and the V.sub.H of the Fab are connected via a second peptide linker (L2); and c) a Fc region comprising: (1) a first constant region comprising a first CH2 and a first CH3, wherein the first constant region (CH2CH3) comprises the amino acid sequence of SEQ ID NO: 13, and (2) a second constant region comprising a second CH2′ and a second CH3′, wherein the second constant region (CH2′CH3′) comprises the amino acid sequence of SEQ ID NO: 16, wherein the bispecific molecule is in the form of
    V.sub.β-L1-V.sub.α-L2-V.sub.H-CH1CH2CH3V.sub.L-CκCH2′CH3′.

    Description

    BRIEF DESCRIPTION OF THE FIGURES

    [0076] FIGS. 1A-1E show the illustrations for the bispecific binding proteins of the present invention. FIG. 1A depicts V.sub.αV.sub.β-FTab; FIG. 1B depicts V.sub.βV.sub.α-FTab-hb-1, V.sub.βV.sub.α-FTab-hb-2, V.sub.βV.sub.α-FTab-hb-3, V.sub.βV.sub.α-FTab-hb-4, and V.sub.βV.sub.α-FTab-hb-5; FIG. 1C depicts V.sub.βV.sub.α-FTab-KiH and V.sub.βV.sub.α-FTab-KiH-2; FIG. 1D depicts V.sub.βV.sub.α-FTab-1, V.sub.βV.sub.α-FTab-2, and V.sub.βV.sub.α-FTab-3; and FIG. 1E depicts V.sub.αV.sub.β-FTab-hb-1.

    [0077] FIG. 2 shows Survivin TCR/CD3 bispecific molecule V.sub.βV.sub.α-FTab-KiH induced potent killing of OCI-AML2 across 4 healthy CD3+ T cell donors, as described in Example 4.

    [0078] FIG. 3 shows Survivin TCR/CD3 bispecific molecule V.sub.βV.sub.α-FTab-KiH induced potent killing of OCI-AML3 across 4 healthy CD3+ T cell donors, as described in Example 4.

    [0079] FIG. 4 shows Survivin TCR/CD3 bispecific molecule V.sub.βV.sub.α-FTab-KiH did not induce killing of OCI-Ly19, as described in Example 4.

    [0080] FIG. 5 shows Survivin TCR/CD3 bispecific molecule V.sub.βV.sub.α-FTab-KiH induced potent activation of CD3+ T cells across 4 healthy CD3+ T cell donors, against OCI-AML2, as described in Example 6.

    [0081] FIG. 6 shows Survivin TCR/CD3 bispecific molecule V.sub.βV.sub.α-FTab-KiH induced potent activation of CD3+ T cells across 4 healthy CD3+ T cell donors, against OCI-AML3, as described in Example 6.

    [0082] FIG. 7 shows Survivin TCR/CD3 bispecific molecule V.sub.βV.sub.α-FTab-KiH induced minimal activation of CD3+ T cells across 4 healthy CD3+ T cell donors, against OCI-Ly19, as described in Example 6.

    [0083] FIG. 8 shows Survivin TCR/CD3 bispecific half-life in monkey serum, as described in Example 7.

    [0084] FIG. 9 shows at molar equivalent doses, the bispecific molecule with KiH (i.e., V.sub.βV.sub.α-FTab-KiH′ which was also designated as V.sub.βV.sub.α-FTab-KiH-2) exhibited greater in vivo anti-tumor efficacy than the bispecific molecule that does not contain KiH (i.e., V.sub.βV.sub.α-FTab-hb-5), as described in Example 3.

    [0085] FIG. 10 shows the schematic representation of Survivin TCR/CD3 bispecific molecule V.sub.βV.sub.α-FTab-KiH.

    [0086] FIG. 11 shows V.sub.βV.sub.α-FTab-KiH TCR specificity screen.

    [0087] FIG. 12 shows T cell proliferation induced by V.sub.βV.sub.α-FTab-KiH at varying effector to target ratios.

    DETAILED DESCRIPTION OF THE INVENTION

    [0088] Described herein are novel bispecific molecules comprising an anti-CD3 binding domain and a soluble single chain T cell receptor targeting human Survivin. These bispecific molecules exhibit several unexpected properties, including, for example, unexpectedly long half-life, remarkable binding specificity directed towards a Survivin-derived peptide complexed to HLA-A2, and potent induction of T cell activation and proliferation.

    Abbreviations

    [0089] The bispecific binding molecules and polynucleotides described herein are, in many embodiments, described by way of their respective polypeptide or polynucleotide sequences. Unless indicated otherwise, polypeptide sequences are provided in N-terminus to C-terminus orientation, and polynucleotide sequences in 5′.fwdarw.3′ orientation. For polypeptide sequences, the conventional three or one-letter abbreviations for the genetically encoded amino acids are used.

    TABLE-US-00001 Abbreviation Term MACS magnetic-activated cell sorting PBS phosphate-buffered saline BSA bovine serum albumin EDTA ethylenediaminetetraacetic acid DMSO dimethyl sulfoxide FACS fluorescence-activated cell sorting Tris tris(hydroxymethyl)aminomethane SEC size-exclusion chromatography SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis PBMC peripheral blood mononuclear cells FBS fetal bovine serum MABEL minimum anticipated biological effect level PBMC peripheral blood mononuclear cell MeCN Acetonitrile TFA trifluoroacetic acid NMR nuclear magnetic resonance DMSO dimethyl sulfoxide LC/MS or LCMS liquid chromatography- mass spectrometry MeOH Methanol tBME tert-butyl methyl ether min Minute mL Milliliter μL Microliter g Gram mg Milligram mmol Millimoles HPLC high pressure liquid chromatography ppm parts per million pm Micrometer

    Embodiments

    [0090] Described herein are bispecific molecules that comprise a CD3 binding part that binds to human CD3 and a Survivin binding part that binds to human Survivin.

    [0091] The term “human CD3” as used herein relates to human cluster of differentiation 3 protein (CD3) described under UniProt P07766 (CD3E-HUMAN).

    [0092] The term “human Survivin” or “Survivin” as used herein relates to an inhibitor of apoptosis protein (IAP) described under UniProt O15392 (BIRC5_Human) which is a tumor-associated antigen that is expressed in human cancer cells.

    [0093] “Binding to CD3 or human Survivin” refers to a molecule that is capable of binding CD3 or human Survivin with sufficient affinity such that the molecule is useful as a therapeutic agent in targeting CD3 or human Survivin.

    [0094] Survivin Binding Part

    [0095] In one embodiment, Survivin binding part of the bispecific molecules of the present invention refers to a single-chain soluble T cell receptor (sTCR).

    [0096] The term “T cell receptors (TCRs)” as used herein are antigen-specific molecules that are responsible for recognizing antigenic peptides presented in the context of a product of the major histocompatibility complex (MHC) on the surface of antigen presenting cells (APCs) or any nucleated cell (e.g., all human cells in the body, except red blood cells).

    [0097] In one embodiment, the sTCR of the present invention is a modified TCR comprising a variable alpha region (V.sub.α) and a variable beta region (V.sub.β) derived from a wild type T cell receptor, wherein the V.sub.α, the V.sub.β, or both, comprise at least one mutation in one or more complementarity determining regions (CDRs) relative to the wild type T cell receptor, wherein the modified T cell receptor binds to a complex of the peptide (i.e., the Survivin peptide LTLGEFLKL (SEQ ID NO: 40)) and a MHC product known as HLA-A2 molecule.

    [0098] In one embodiment, the sTCR of the present invention comprises a V.sub.β and a V.sub.α, wherein the sTCR binds to a complex of the peptide comprising the amino acid sequence of SEQ ID NO: 40 and the HLA-A2 molecule.

    [0099] In one embodiment, the sTCR of the present invention comprises a V.sub.β and a V.sub.α, wherein the sTCR binds to a peptide (SEQ ID NO: 40) derived from human Survivin in complex with HLA-A2.

    [0100] In embodiments, the compounds of the present disclosure bind to survivin peptide/MHC with a K.sub.D of 1×10.sup.−7M or less, such as between about 1×10.sup.−7M and about 1×10.sup.−10 M, or between about 1×10.sup.−8M and about 1×10.sup.=10 M. In embodiments, the compounds of the present disclosure bind to survivin peptide/MHC complex with a K.sub.D of less than about 3×10.sup.−9M, or less than about 2.5×10.sup.−9M, or less than about 2.0×10.sup.−9M, or less than about 1.5×10.sup.−9M.

    [0101] In one embodiment, the V.sub.α of the sTCR of the present invention comprises SEQ ID NO: 19 (CDR1), SEQ ID NO: 23 (CDR2), and SEQ ID NO: 27 (CDR3).

    [0102] In one embodiment, the V.sub.α of the sTCR of the present invention comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8.

    [0103] In one preferred embodiment, the V.sub.α of the sTCR of the present invention comprises the amino acid sequence of SEQ ID NO: 6.

    [0104] In one embodiment, the V.sub.β of the sTCR of the present invention comprises SEQ ID NO: 20 (CDR1), SEQ ID NO: 24 (CDR2) and SEQ ID NO: 28 (CDR3) or SEQ ID NO: 31 (CDR3).

    [0105] In one preferred embodiment, the V.sub.β of the sTCR of the present invention comprises SEQ ID NO: 20 (CDR1), SEQ ID NO: 24 (CDR2) and SEQ ID NO: 28 (CDR3).

    [0106] In one embodiment, the V.sub.β of the sTCR of the present invention comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5.

    [0107] In one embodiment, the V.sub.β of the sTCR of the present invention comprises the amino acid sequence of SEQ ID NO: 2.

    [0108] In one embodiment, the sTCR variable beta region (V.sub.β) and the sTCR variable alpha region (V.sub.α) are connected via a first peptide linker (L1). The linker may be selected to increase expression, solubility, stability (for example, as measured by lower aggregation levels, lower rate of aggregation, higher melting temperature, and/or longer plasma half-life), and/or titer of a bispecific molecule of the present invention.

    [0109] In one embodiment, the sTCR variable beta region (V.sub.β) and the sTCR variable alpha region (V.sub.α) are connected via a first peptide linker (L1) comprising the amino acid sequence of SEQ ID NO: 1.

    [0110] In certain embodiments, the sTCR variable beta region (V.sub.β) is connected to the sTCR variable alpha region (V.sub.α) via a disulfide bridge. In embodiments, the disulfide bridge connecting the V.sub.α and V.sub.β regions is between cysteine 43 of the V.sub.α region and cysteine 235 of the V.sub.β region. In embodiments, the disulfide bridge connecting the V.sub.α and V.sub.β regions is between cysteine 43 and cysteine 235 of SEQ ID NO: 36 or SEQ ID NO: 88. In embodiments, the disulfide bridge connecting the V.sub.α and V.sub.β regions is between cysteine 43 of SEQ ID NO: 5 or SEQ ID NO: 2, and cysteine 100 of SEQ ID NO: 6.

    [0111] In one embodiment, the single-chain soluble T cell receptor (sTCR) of the present invention, which binds to a complex of the peptide Survivin, wherein the complex comprises the amino acid sequence of SEQ ID NO: 40 and the HLA-A2 molecule, wherein the sTCR comprises: [0112] (1) a sTCR variable beta region (V.sub.β) comprising SEQ ID NO: 20 (CDR1), SEQ ID NO: 24 (CDR2), and SEQ ID NO: 28 (CDR3) or SEQ ID NO: 31 (CDR3), and [0113] (2) a sTCR variable alpha region (V.sub.α) comprising SEQ ID NO: 19 (CDR1), SEQ ID NO: 23 (CDR2), and SEQ ID NO: 27 (CDR3), wherein the V.sub.β and V.sub.α regions of the sTCR are connected via a first peptide linker (L1).

    [0114] In one embodiment, the single-chain soluble T cell receptor (sTCR) of the present invention, which binds to a complex of the peptide Survivin, wherein the complex comprises the amino acid sequence of SEQ ID NO: 40 and the HLA-A2 molecule, wherein the sTCR comprises: [0115] (1) a sTCR variable beta region (V.sub.β) comprising SEQ ID NO: 20 (CDR1), SEQ ID NO: 24 (CDR2), and SEQ ID NO: 28 (CDR3), and [0116] (2) a sTCR variable alpha region (V.sub.α) comprising SEQ ID NO: 19 (CDR1), SEQ ID NO: 23 (CDR2), and SEQ ID NO: 27 (CDR3),

    [0117] wherein the V.sub.β and V.sub.α regions of the sTCR are connected via a first peptide linker (L1).

    [0118] In one embodiment, the single-chain soluble T cell receptor (sTCR) of the present invention, which binds to a complex of the peptide Survivin, wherein the complex comprises the amino acid sequence of SEQ ID NO: 40 and the HLA-A2 molecule, wherein the sTCR comprises: [0119] (1) a sTCR variable beta region (V.sub.β) comprising the amino acid sequence selected from a group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, and [0120] (2) a sTCR variable alpha region (V.sub.α) comprising the amino acid sequence selected from a group consisting of SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8,

    [0121] wherein the V.sub.β and V.sub.α regions of the sTCR are connected via a first peptide linker (L1).

    [0122] In one embodiment, the single-chain soluble T cell receptor (sTCR) of the present invention, which binds to a complex of the peptide Survivin, wherein the complex comprises the amino acid sequence of SEQ ID NO: 40 and the HLA-A2 molecule, wherein the sTCR comprises: [0123] (1) a sTCR variable beta region (V.sub.β) comprising the amino acid sequence selected from a group consisting of SEQ ID NO: 2, and [0124] (2) a sTCR variable alpha region (V.sub.α) comprising the amino acid sequence selected from a group consisting of SEQ ID NO: 6,

    [0125] wherein the V.sub.β and V.sub.α regions of the sTCR are connected via a first peptide linker (L1).

    [0126] In one embodiment, the first peptide linker (L1) of the present invention comprises the amino acid sequence of SEQ ID NO: 1.

    [0127] CD3 Binding Part

    [0128] In one embodiment, CD3 binding part of the bispecific molecules of the present invention is a combination of an antibody heavy chain comprising a heavy chain variable domain (V.sub.H) and a constant heavy chain domain 1 (CH1) and an antibody light chain comprising a light chain variable domain (V.sub.L) and a kappa (κ) light chain (constant domain Cκ), and preferably the V.sub.H, CH1, V.sub.L and Cκ as enclosed in an antigen binding fragment (Fab) that binds to human CD3 (anti-CD3-Fab), wherein the light chain (V.sub.L-Cκ) is covalently bound by a disulfide bridge to the heavy chain (V.sub.H—CH1). In some embodiments, the Cκ is replaced with a lambda light constant region.

    [0129] The “variable domain” (variable domain of a light chain (V.sub.L), variable region of a heavy chain (V.sub.H)) as used herein denotes each of the pair of light and heavy chains which are involved directly in binding the antibody to the target. The domains of variable human light and heavy chains have the same general structure and each domain comprises at least one complementary determining region (CDR), preferably three CDRs, which play a particularly important role in the binding specificity/affinity of the antibodies according to the invention and therefore provide a further object of the invention.

    [0130] In one embodiment, the V.sub.H of the anti-CD3-Fab of the present invention comprises SEQ ID NO: 21 (CDR1) or SEQ ID NO: 32 (CDR1), SEQ ID NO: 25 (CDR2) or SEQ ID NO: 33 (CDR2), and SEQ ID NO: 29 (CDR3) or SEQ ID NO: 34 (CDR3).

    [0131] In one preferred embodiment, the V.sub.H of the anti-CD3-Fab of the present invention comprises SEQ ID NO: 21 (CDR1), SEQ ID NO: 25 (CDR2), and SEQ ID NO: 29 (CDR3).

    [0132] In one embodiment, the V.sub.H of the anti-CD3-Fab of the present invention comprises the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10.

    [0133] In one preferred embodiment, the V.sub.H of the anti-CD3-Fab of the present invention comprises the amino acid sequence of SEQ ID NO: 9.

    [0134] In one embodiment, the V.sub.L of the anti-CD3-Fab of the present invention comprises SEQ ID NO: 22 (CDR1) or SEQ ID NO: 81 (CDR1), SEQ ID NO: 26 (CDR2) or SEQ ID NO: 82 (CDR2), and SEQ ID NO: 30 (CDR3) or SEQ ID NO: 83 (CDR3).

    [0135] In one preferred embodiment, the V.sub.L of the anti-CD3-Fab of the present invention comprises SEQ ID NO: 22 (CDR1), SEQ ID NO: 26 (CDR2), and SEQ ID NO: 30 (CDR3).

    [0136] In one embodiment, the V.sub.L of the anti-CD3-Fab of the present invention comprises the amino acid sequence of SEQ ID NO: 11 or SEQ IN NO: 12.

    [0137] In one preferred embodiment, the V.sub.L of the anti-CD3-Fab of the present invention comprises the amino acid sequence of SEQ ID NO: 11.

    [0138] In one embodiment, the CH1 of the anti-CD3-Fab of the present invention comprises the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 35.

    [0139] In one preferred embodiment, the CH1 of the anti-CD3-Fab of the present invention comprises the amino acid sequence of SEQ ID NO: 18.

    [0140] In one embodiment, the C.sub.κ of the anti-CD3-Fab of the present invention comprises the amino acid sequence of SEQ ID NO: 17.

    [0141] In one embodiment, the anti-CD3-Fab of the present invention comprises: [0142] (1) a heavy chain region comprising a heavy chain variable (V.sub.H) comprising SEQ ID NO: 21 (CDR1) or SEQ ID NO: 32 (CDR1), SEQ ID NO: 25 (CDR2) or SEQ ID NO: 33 (CDR2), and SEQ ID NO: 29 (CDR3) or SEQ ID NO: 34 (CDR3); and a heavy chain constant domain 1 (CH1) comprising the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 35; and [0143] (2) a light chain region comprising a light chain variable (V.sub.L) comprising SEQ ID NO: 22 (CDR1) or SEQ ID NO: 81 (CDR1), SEQ ID NO: 26 (CDR2) or SEQ ID NO: 82 (CDR2), and SEQ ID NO: 30 (CDR3) or SEQ ID NO: 83 (CDR3); and a kappa constant light chain (C.sub.κ) comprising the amino acid sequence of SEQ ID NO: 17.

    [0144] In one preferred embodiment, the anti-CD3-Fab of the present invention comprises: [0145] (1) a heavy chain region comprising a heavy chain variable (V.sub.H) comprising SEQ ID NO: 21 (CDR1), SEQ ID NO: 25 (CDR2), and SEQ ID NO: 29 (CDR3); and a heavy chain constant domain 1 (CH1) comprising the amino acid sequence of SEQ ID NO: 18; and [0146] (2) a light chain region comprising a light chain variable (V.sub.L) comprising SEQ ID NO: 22 (CDR1), SEQ ID NO: 26 (CDR2), and SEQ ID NO: 30 (CDR3); and a kappa constant light chain (C.sub.κ) comprising the amino acid sequence of SEQ ID NO: 17.

    [0147] In one embodiment, the anti-CD3-Fab of the present invention comprises: [0148] (1) a heavy chain region comprising a heavy chain variable (V.sub.H) comprising the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10 and a heavy chain constant domain 1 (CH1) comprising the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 35, and [0149] (2) a light chain region comprising a light chain variable (V.sub.L) comprising the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12 and a kappa constant light chain (C.sub.κ) comprising the amino acid sequence of SEQ ID NO: 17.

    [0150] In one embodiment, the anti-CD3-Fab of the present invention comprises: [0151] (1) a heavy chain region comprising a heavy chain variable (V.sub.H) comprising the amino acid sequence of SEQ ID NO: 9 and a heavy chain constant domain 1 (CH1) comprising the amino acid sequence of SEQ ID NO: 18, [0152] (2) a light chain region comprising a light chain variable (V.sub.L) comprising the amino acid sequence of SEQ ID NO: 11 and a kappa constant light chain (C.sub.κ) comprising the amino acid sequence of SEQ ID NO: 17.

    [0153] The Fc Region

    [0154] In one embodiment, the bispecific molecule of the present invention further comprises a fragment crystallizable region (Fc).

    [0155] The term “Fc” or “Fc region” is a term well known to the skilled artisan and is involved in complement activation, Clq binding, C3 activation and Fc receptor binding.

    [0156] In one embodiment, the Fc region of the present invention is derived from human origin.

    [0157] In one embodiment, the Fc region of the present invention is a human IgG1 Fc region or derived from a human IgG1 Fc region.

    [0158] In one embodiment, the Fc region of the present invention comprises a first CH2CH3 region comprising a first CH2 domain and a first CH3 domain.

    [0159] In one embodiment, the Fc region of the present invention comprises a second CH2′CH3′ region comprising a second CH2 domain (CH2′) and a second CH3 domain (CH3′).

    [0160] In one embodiment, the Fc region of the present invention comprises a first constant region comprising a first constant domain 2 (CH2) and a first constant domain 3 (CH3).

    [0161] In one embodiment, the Fc region of the present invention comprises a second constant region comprising a second constant domain 2 (CH2′) and a second constant domain 3 (CH3′).

    [0162] In embodiments, the Fc region comprises a first CH2CH3 region and a second CH2′CH3′ region, wherein the first CH2CH3 region is covalently bound by two disulfide bridges to the second CH2′CH3′ region.

    [0163] In one embodiment, the Fc region of the present invention comprises a hinge region. The term “hinge region” refers to a flexible amino acid stretch in the central part of the heavy chains of immunoglobulin antibodies, which links these 2 chains by disulfide bonds. Various hinge regions can be used in the bispecific molecules of the present invention, for example, to optimize certain characteristics. In an illustrative example, one or more amino acid substitutions, insertions, and/or deletions within a hinge region of a human IgG.sub.1, IgG.sub.2, IgG.sub.3 or IgG.sub.4 can be introduced to reduce the level or rate of fragmentation and/or aggregation.

    [0164] In one embodiment, the Fc region of the present invention is engineered to comprise at least one amino acid substitution in the human IgG1 Fc region in its constant heavy chain domain 3 (CH3) to promote the heterodimerization through “knob-in-hole” technology (KiH). In this technique, through gene manipulation, a mutation is induced in a CH3 domain of two different Ig heavy chains, a hole structure is made in a CH3 domain of one Ig heavy chain, a knob structure is made the CH3 domain of the other Ig heavy chain, and two Ig heavy chains are induced to form a heterodimer (e.g., Carter, P., J. Immunol. Meth. 248 (2001) 7-15; Merchant, A. M., et al., Nat. Biotechnol. 16 (1998) 677-681; Zhu, Z., et al., Prot. Sci. 6 (1997) 781-788; Ridgway, J. B., et al., Prot. Eng. 9 (1996) 617-621; Atwell, S., et al., J. Mol. Biol. 270 (1997) 26-35).

    [0165] For example, amino acid residues included in a hydrophobic core contributing to formation of the homodimer between human IgG1 heavy chain CH3 domains are Leu351, Thr366, Leu368, and Tyr407 according to EU numbering of the amino acid number of the antibody chain (Cunningham, Pflumm et al. 1969). In the knob-into-hole technique, with respect to residues positioned at a hydrophobic core in a CH3 domain interface, a hole structure is made in one heavy chain CH3 domain such that hydrophobic amino acid residues having a large side chain are substituted with hydrophobic amino acids having a small side chain (Thr366Ser, Leu368Ala, Tyr407Val), a knob structure is made in the other heavy chain CH3 domain such that hydrophobic amino acid residues having a small side chain are substituted with hydrophobic amino acids having a large side chain (Thr366Trp). When two mutation pairs, heavy chain constant region mutation pairs in which the first CH3 (Thr366Ser, Leu368Ala, and Tyr407Val) and the second CH3′ (Thr366Trp) are introduced to form the heterodimeric Fc.

    [0166] In one embodiment, the Fc region of the present invention is derived from the human IgG1 Fc region and comprises the amino acid sequence of SEQ ID NO: 13 comprising in the first CH3 domain at least one of the following amino acid substitutions: Thr128 with serine, Leu130 with alanine, and Tyr169 with valine, which are corresponding to Thr366Ser, Leu368Ala and Tyr407Val of the human IgG1 heavy chain respectively according to EU numbering of the amino acid number of the antibody chain.

    [0167] In one embodiment, the Fc region of the present invention is derived from the human IgG1 Fc region and comprises the amino acid sequence of SEQ ID NO: 13 comprising in the first CH3 domain of the human IgG1 Fc region, amino acid substitutions of Thr128 with serine, Leu130 with alanine, and Tyr169 with valine, which are corresponding to Thr366, Leu368 and Tyr407 of the human IgG1 heavy chain respectively according to EU numbering of the amino acid number of the antibody chain.

    [0168] In one embodiment, the Fc region of the present invention is derived from the human IgG1 Fc region and comprises the amino acid sequence of SEQ ID NO: 16 in the second CH3 domain (CH3′) an amino acid substitution of Thr146 with tryptophan, which corresponds to Thr366 of the human IgG1 heavy chain respectively according to EU numbering of the amino acid number of the antibody chain.

    [0169] In one embodiment, the Fc region of the present invention comprises one or more mutations to modulate Fc receptor-based function of the Fc region. In one embodiment, the Fc region of the present invention comprises one or more mutations to modulate FcγR-based effector function of the Fc region. In one embodiment, the Fc region of the present invention is derived from the human IgG1 Fc region and comprises the amino acid sequence of SEQ ID NO: 13 comprising in the first CH2 domain an amino acid substitution of Asn59 with alanine, which corresponds to Asn297 of the human IgG1 heavy chain respectively according to EU numbering of the amino acid number of the antibody chain. In one embodiment, the Fc region of the present invention is derived from the human IgG1 Fc region and comprises the amino acid sequence of SEQ ID NO: 16 comprising in the second CH2 domain (CH2′) an amino acid substitution of Asn77 with alanine, which corresponds to Asn297 of the human IgG1 heavy chain respectively according to EU numbering of the amino acid number of the antibody chain.

    [0170] In one embodiment, the Fc region of the present invention comprises a first constant region (CH2CH3) comprising a first constant domain 2 (CH2) and a first constant domain 3 (CH3).

    [0171] In one embodiment, the Fc region of the present invention comprises: [0172] (1) a first constant region CH2CH3 comprising a first constant domain 2 (CH2) and a first constant domain 3 (CH3); and [0173] (2) a second constant region CH2′CH3′ comprising a second constant domain 2 (CH2′) and a second constant domain 3 (CH3′).

    [0174] In one embodiment, the CH2CH3 of the Fc region of the present invention comprises the amino acid sequence selected from a group consisting of SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15.

    [0175] In one embodiment, the CH2CH3 of the Fc region of the present invention comprises the amino acid sequence of SEQ ID NO: 13.

    [0176] In one embodiment, the CH2′ CH3′ of the Fc region of the present invention comprises the amino acid sequence of SEQ ID NO: 16.

    [0177] In one embodiment, the Fc region of the present invention comprises: [0178] (1) a first constant region comprising a first constant domain 2 (CH2) and a first constant domain 3 (CH3), wherein the CH2CH3 comprises the amino acid sequence selected from a group consisting of SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15; and [0179] (2) a second constant region comprising a second constant domain 2 (CH2′) and a second constant domain 3 (CH3′), wherein the CH2′CH3′ comprises the amino acid sequence of SEQ ID NO: 16.

    [0180] In one embodiment, the Fc region of the present invention comprises: [0181] (1) a first constant region comprising a first constant domain 2 (CH2) and a first constant domain 3 (CH3), wherein the CH2CH3 comprises the amino acid sequence of SEQ ID NO: 13; and [0182] (2) a second constant region comprising a second constant domain 2 (CH2′) and a second constant domain 3 (CH3′), wherein the CH2′CH3′ comprises the amino acid sequence of SEQ ID NO: 16.

    [0183] In one embodiment, the Fc region of the present invention is connected to the anti-CD3-Fab of the present invention between the CH1 of the anti-CD3-Fab and the CH2 of the Fc region to form a CH1CH2CH3 domain.

    [0184] In one embodiment, the CH1CH2CH3 of the present invention comprises the amino acid sequence selected from a group consisting of SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39.

    [0185] In one preferred embodiment, the CH1CH2CH3 of the present invention comprises the amino acid sequence of SEQ ID NO: 37.

    [0186] Formats of the Bispecific Molecules

    [0187] According to the present invention, the CD3 binding part, the Survivin binding part and the Fc region can be formatted in various orientations. To assist understanding, five exemplary embodiments of bispecific molecules are illustrated in FIGS. 1A-1E.

    [0188] With reference to FIG. 1A, one exemplary embodiment of a bispecific molecule comprises (1) a single-chain soluble T cell receptor (sTCR) which binds to a complex of the Survivin peptide and the HLA-A2 molecule and (2) an antigen binding fragment (Fab) which binds to human CD3 (anti-CD3-Fab). The sTCR comprises a sTCR variable beta region (V.sub.α) and a sTCR variable alpha region (V.sub.β), where the V.sub.α and the V.sub.β are connected via a first peptide linker (L1). The anti-CD3-Fab comprises a heavy chain variable (V.sub.H), a heavy chain constant domain 1 (CH1), a light chain variable (V.sub.L) and a kappa constant light chain (C.sub.κ) The sTCR and the anti-CD3-Fab are connected via a second peptide linker (L2) connecting the V.sub.β of the sTCR and the V.sub.H of the anti-CD3-Fab. The bispecific molecule is in the form of V.sub.α-L1-V.sub.β-L2-anti-CD3-Fab.

    [0189] With reference to FIG. 1B, one exemplary embodiment of a bispecific molecule comprises (1) a single-chain soluble T cell receptor (sTCR) which binds to a complex of the Survivin peptide and the HLA-A2 molecule, (2) an antigen binding fragment (Fab) which binds to human CD3 (anti-CD3-Fab) and (3) a fragment crystallizable region (Fc). The sTCR, the anti-CD3-Fab and the Fc region are connected in the order of sTCR-anti-CD3-Fab-Fc. The sTCR comprises a sTCR variable beta region (V.sub.β) and a sTCR variable alpha region (V.sub.α), where the V.sub.β and the V.sub.α are connected via a first peptide linker (L1). The anti-CD3-Fab comprises a heavy chain variable (V.sub.H), a heavy chain constant domain 1 (CH1), a light chain variable (V.sub.L) and a kappa constant light chain (C.sub.κ). The sTCR and the anti-CD3-Fab are connected via a second linker (L2) between the V.sub.α of the sTCR and V.sub.H of the anti-CD3-Fab. The Fc region comprises a constant domain 2 (CH2) and a constant domain 3 (CH3). The anti-CD3-Fab and the Fc region are enclosed in a format of a half-body of an antibody. The bispecific molecule is in the form of V.sub.β-L1-V.sub.α-L2-anti-CD3-Fab-Fc.

    [0190] With reference to FIG. 1C, one exemplary embodiment of a bispecific molecule comprises (1) a single-chain soluble T cell receptor (sTCR) which binds to a complex of the Survivin peptide and the HLA-A2 molecule, (2) an antigen binding fragment (Fab) which binds to human CD3 (anti-CD3-Fab) and (3) a fragment crystallizable region (Fc). The sTCR, the anti-CD3-Fab and the Fc region are connected in the order of sTCR-anti-CD3-Fab-Fc. The sTCR comprises a sTCR variable beta region (V.sub.β) and a sTCR variable alpha region (V.sub.α), where the V.sub.β and the V.sub.α are connected via a first peptide linker (L1). The anti-CD3-Fab comprises a heavy chain variable (V.sub.H), a heavy chain constant domain 1 (CH1), a light chain variable (V.sub.L) and a kappa constant light chain (C.sub.κ). The sTCR and the anti-CD3-Fab are connected via a second peptide linker (L2) between the V.sub.α and V.sub.H. The Fc region comprises a first constant region comprising a first constant domain 2 (CH2) and a first constant domain 3 (CH3) and a second constant region comprising a second constant domain 2 (CH2′) and a second constant domain 3 (CH3′). The first CH3 and the second CH3 are engineered for heterodimerization through “knob-in-hole” technology. The bispecific molecule is in the form of V.sub.β-L1-V.sub.α-L2-anti-CD3-Fab-Fc.

    [0191] With reference to FIG. 1D, one exemplary embodiment of a bispecific molecule comprises (1) a single-chain soluble T cell receptor (sTCR) which binds to a complex of the Survivin peptide and the HLA-A2 molecule and (2) an antigen binding fragment (Fab) which binds to human CD3 (anti-CD3-Fab). The sTCR comprises a sTCR variable beta region (V.sub.β) and a sTCR variable alpha region (V.sub.α), where the V.sub.β and the V.sub.α are connected via a first peptide linker (L1). The anti-CD3-Fab comprises a heavy chain variable (V.sub.H), a heavy chain constant domain 1 (CH1), a light chain variable (V.sub.L) and a kappa constant light chain (C.sub.κ). The sTCR and the anti-CD3-Fab are connected via a second peptide linker (L2) connecting the V.sub.α of the sTCR and the V.sub.H of the anti-CD3-Fab. The bispecific molecule is in the form of V.sub.β-L1-V.sub.α-L2-anti-CD3-Fab.

    [0192] With reference to FIG. 1E, one exemplary embodiment of a bispecific molecule comprises (1) a single-chain soluble T cell receptor (sTCR) which binds to a complex of the Survivin peptide and the HLA-A2 molecule, (2) an antigen binding fragment (Fab) which binds to human CD3 (anti-CD3-Fab) and (3) a fragment crystallizable region (Fc). The sTCR, the anti-CD3-Fab and the Fc region are connected in the order of sTCR-anti-CD3-Fab-Fc. The sTCR comprises a sTCR variable beta region (V.sub.α) and a sTCR variable alpha region (V.sub.β), where the V.sub.α and the V.sub.β are connected via a first peptide linker (L1). The anti-CD3-Fab comprises a heavy chain variable (V.sub.H), a heavy chain constant domain 1 (CH1), a light chain variable (V.sub.L) and a kappa constant light chain (C.sub.κ) The sTCR and the anti-CD3-Fab are connected via a second linker (L2) between the V.sub.β of the sTCR and V.sub.H of the anti-CD3-Fab. The Fc region comprises a constant domain 2 (CH2) and a constant domain 3 (CH3). The anti-CD3-Fab and the Fc region are enclosed in a format of a half-body of an antibody. The bispecific molecule is in the form of V.sub.α-L1-V.sub.β-L2-anti-CD3-Fab-Fc.

    [0193] The following specific embodiments of the present invention are listed:

    [0194] In one embodiment, the present invention provides a bispecific molecule comprising: [0195] a) a single-chain soluble T cell receptor (sTCR), which binds to a complex of the peptide Survivin, wherein the complex comprises the amino acid sequence of SEQ ID NO: 40 and the HLA-A2 molecule, wherein the sTCR comprises: [0196] (1) a sTCR variable beta region (V.sub.β) comprising SEQ ID NO: 20 (CDR1), SEQ ID NO: 24 (CDR2), and SEQ ID NO: 28 (CDR3) or SEQ ID NO: 31 (CDR3), and [0197] (2) a sTCR variable alpha region (V.sub.α) comprising SEQ ID NO: 19 (CDR1), SEQ ID NO: 23 (CDR2), and SEQ ID NO: 27 (CDR3); [0198] wherein the V.sub.α and V.sub.β regions of the sTCR are connected via a first peptide linker (L1); [0199] b) an antigen binding fragment (Fab) which binds to human CD3 (anti-CD3-Fab), wherein the anti-CD3-Fab comprises: [0200] (1) a heavy chain region comprising a heavy chain variable (V.sub.H) comprising SEQ ID NO: 21 (CDR1) or SEQ ID NO: 32 (CDR1), SEQ ID 25 (CDR2) or SEQ ID NO: 33 (CDR2), and SEQ ID NO: 29 (CDR3) or SEQ ID NO: 34 (CDR3); and a heavy chain constant domain 1 (CH1) comprising the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 35 and, [0201] (2) a light chain region comprising a light chain variable (V.sub.L) comprising SEQ ID NO: 22 (CDR1) or SEQ ID NO: 81 (CDR1), SEQ ID NO: 26 (CDR2) or SEQ ID NO: 82 (CDR2), and SEQ ID NO: 30 (CDR3) or SEQ ID NO: 83 (CDR3); and a kappa constant light chain (C.sub.κ) comprising the amino acid sequence of SEQ ID NO: 17, [0202] wherein the V.sub.β of the sTCR and the V.sub.H of the anti-CD3-Fab are connected via a second peptide linker (L2);

    [0203] wherein the bispecific molecule is in the form of


    V.sub.β-L1-V.sub.β-L2-V.sub.H-CH1 V.sub.L-Cκ.

    [0204] In one embodiment, the present invention provides a bispecific molecule comprising: [0205] a) a single-chain soluble T cell receptor (sTCR), which binds to a complex of the peptide Survivin, wherein the complex comprises the amino acid sequence of SEQ ID NO: 40 and the HLA-A2 molecule, wherein the sTCR comprises: [0206] (1) a sTCR variable beta region (V.sub.β) comprising the amino acid sequence selected from a group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, and [0207] (2) a sTCR variable alpha region (V.sub.α) comprising the amino acid sequence selected from a group consisting of SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, [0208] wherein the V.sub.α and V.sub.β regions of the sTCR are connected via a first peptide linker (L1); [0209] b) an antigen binding fragment (Fab) which binds to human CD3 (anti-CD3-Fab), wherein the anti-CD3-Fab comprises: [0210] (1) a heavy chain region comprising a heavy chain variable (V.sub.H) comprising the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10; and a heavy chain constant domain 1 (CH1) comprising the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 35, and [0211] (2) a light chain region comprising a light chain variable (V.sub.L) comprising the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12; and a kappa constant light chain (C.sub.κ) comprising the amino acid sequence of SEQ ID NO: 17, [0212] wherein the V.sub.β of the sTCR and the V.sub.H of the anti-CD3-Fab are connected via a second peptide linker (L2),

    [0213] wherein the bispecific molecule is in the form of


    V.sub.α-L1-V.sub.β-L2-V.sub.H-CH1 V.sub.L-Cκ.

    [0214] In one embodiment, the present invention provides a bispecific molecule comprising: [0215] a) a single-chain soluble T cell receptor (sTCR), which binds to a complex of the peptide Survivin, wherein the complex comprises the amino acid sequence of SEQ ID NO: 40 and the HLA-A2 molecule, wherein the sTCR comprises: [0216] (1) a sTCR variable beta region (V.sub.β) comprising SEQ ID NO: 20 (CDR1), SEQ ID NO: 24 (CDR2), and SEQ ID NO: 28 (CDR3) or SEQ ID NO: 31 (CDR3), and [0217] (2) a sTCR variable alpha region (V.sub.α) comprising SEQ ID NO: 19 (CDR1), SEQ ID NO: 23 (CDR2), and SEQ ID NO: 27 (CDR3); [0218] wherein the V.sub.β and V.sub.α regions of the sTCR are connected via a first peptide linker (L1); [0219] b) an antigen binding fragment (Fab) which binds to human CD3 (anti-CD3-Fab), wherein the anti-CD3-Fab comprises: [0220] (1) a heavy chain region comprising a heavy chain variable (V.sub.H) comprising SEQ ID NO: 21 (CDR1) or SEQ ID NO: 32 (CDR1), SEQ ID NO: 25 (CDR2) or SEQ ID NO: 33 (CDR2), and SEQ ID NO: 29 (CDR3) or SEQ ID NO: 34 (CDR3); and a heavy chain constant domain 1 (CH1) comprising the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 35, and [0221] (2) a light chain region comprising a light chain variable (V.sub.L) comprising SEQ ID NO: 22 (CDR1) or SEQ ID NO: 81 (CDR1), SEQ ID NO: 26 (CDR2) or SEQ ID NO: 82 (CDR2), and SEQ ID NO: 30 (CDR3) or SEQ ID NO: 83 (CDR3); and a kappa constant light chain (C.sub.κ) comprising the amino acid sequence of SEQ ID NO: 17, [0222] wherein the V.sub.α of the sTCR and the V.sub.H of the anti-CD3-Fab are connected via a second peptide linker (L2);

    [0223] wherein the bispecific molecule is in the form of


    V.sub.β-L1-V.sub.α-L2-V.sub.H-CH1 V.sub.L-Cκ.

    [0224] In one embodiment, the present invention provides a bispecific molecule comprising: [0225] a) a single-chain soluble T cell receptor (sTCR), which binds to a complex of the peptide Survivin, wherein the complex comprises the amino acid sequence of SEQ ID NO: 40 and the HLA-A2 molecule, wherein the sTCR comprises: [0226] (1) a sTCR variable beta region (V.sub.β) comprising the amino acid sequence selected from a group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, and [0227] (2) a sTCR variable alpha region (V.sub.α) comprising the amino acid sequence selected from a group consisting of SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, [0228] wherein the V.sub.β and V.sub.α regions of the sTCR are connected via a first peptide linker (L1); [0229] b) an antigen binding fragment (Fab) which binds to human CD3 (anti-CD3-Fab), wherein the anti-CD3-Fab comprises: [0230] (1) a heavy chain region comprising a heavy chain variable (V.sub.H) comprising the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10; and a heavy chain constant domain 1 (CH1) comprising the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 35, and [0231] (2) a light chain region comprising a light chain variable (V.sub.L) comprising the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12; and a kappa constant light chain (C.sub.κ) comprising the amino acid sequence of SEQ ID NO: 17, [0232] wherein the V.sub.α of the sTCR and the V.sub.H of the anti-CD3-Fab are connected via a second peptide linker (L2),

    [0233] wherein the bispecific molecule is in the form of


    V.sub.β-L1-V.sub.α-L2-V.sub.H-CH1 V.sub.L-Cκ.

    [0234] In one embodiment, the present invention provides a bispecific molecule comprising: [0235] a) a single-chain soluble T cell receptor (sTCR), which binds to a complex of the peptide Survivin, wherein the complex comprises the amino acid sequence of SEQ ID NO: 40 and the HLA-A2 molecule, wherein the sTCR comprises: [0236] (1) a sTCR variable beta region (V.sub.β) comprising SEQ ID NO: 20 (CDR1), SEQ ID NO: 24 (CDR2), and SEQ ID NO: 28 (CDR3) or SEQ ID NO: 31 (CDR3), and [0237] (2) a sTCR variable alpha region (V.sub.α) comprising SEQ ID NO: 19 (CDR1), SEQ ID NO: 23 (CDR2), and SEQ ID NO: 27 (CDR3), [0238] wherein the V.sub.β and V.sub.α regions of the sTCR are connected via a first peptide linker (L1); [0239] b) an antigen binding fragment (Fab) which binds to human CD3 (anti-CD3-Fab), wherein the anti-CD3-Fab comprises: [0240] (1) a heavy chain region comprising a heavy chain variable (V.sub.H) comprising SEQ ID NO: 21 (CDR1) or SEQ ID NO: 32 (CDR1), SEQ ID NIO: 25 (CDR2) or SEQ ID NO: 33 (CDR2), and SEQ ID NO: 29 (CDR3) or SEQ ID NO: 34 (CDR3); and a heavy chain constant domain 1 (CH1) comprising the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 35, and [0241] (2) a light chain region comprising a light chain variable (V.sub.L) comprising SEQ ID NO: 22 (CDR1) or SEQ ID NO: 81 (CDR1), SEQ ID NO: 26 (CDR2) or SEQ ID NO: 82 (CDR2), and SEQ ID NO: 30 (CDR3) or SEQ ID NO: 83 (CDR3); and a kappa constant light chain (C.sub.κ) comprising the amino acid sequence of SEQ ID NO: 17, [0242] wherein the V.sub.α of the sTCR and the V.sub.H of the anti-CD3-Fab are connected via a second peptide linker (L2); and [0243] c) a fragment crystallizable region (Fc) comprising a constant domain 2 (CH2) and a constant domain 3 (CH3), wherein the CH2CH3 comprises the amino acid sequence selected from a group consisting of SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15;

    [0244] wherein the bispecific molecule is in the form of


    V.sub.β-L1-V.sub.α-L2-V.sub.H-CH1CH2CH3V.sub.L-Cκ.

    [0245] In one embodiment, the present invention provides a bispecific molecule comprising: [0246] a) a single-chain soluble T cell receptor (sTCR), which binds to a complex of the peptide Survivin, wherein the complex comprises the amino acid sequence of SEQ ID NO: 40 and the HLA-A2 molecule, wherein the sTCR comprises: [0247] (1) a sTCR variable beta region (V.sub.β) comprising the amino acid sequence selected from a group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, and [0248] (2) a sTCR variable alpha region (V.sub.α) comprising the amino acid sequence selected from a group consisting of SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, [0249] wherein the V.sub.β and V.sub.α regions of the sTCR are connected via a first peptide linker (L1); [0250] b) an antigen binding fragment (Fab) which binds to human CD3 (anti-CD3-Fab), wherein the anti-CD3-Fab comprises: [0251] (1) a heavy chain region comprising a heavy chain variable (V.sub.H) comprising the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10; a heavy chain constant domain 1 (CH1) comprising the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 35, and [0252] (2) a light chain region comprising a light chain variable (V.sub.L) comprising the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12; and a kappa constant light chain (C.sub.κ) comprising the amino acid sequence of SEQ ID NO: 17, [0253] wherein the V.sub.α of the sTCR and the V.sub.H of the anti-CD3-Fab are connected via a second peptide linker (L2); and [0254] c) a fragment crystallizable region (Fc) comprising a constant domain 2 (CH2) and a constant domain 3 (CH3), wherein the CH2CH3 comprises the amino acid sequence selected from a group consisting of SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15; [0255] wherein the bispecific molecule is in the form of


    V.sub.β-L1-V.sub.α-L2-V.sub.H-CH1CH2CH3V.sub.L-Cκ.

    [0256] In one embodiment, the present invention provides a bispecific molecule comprising: [0257] a) a single-chain soluble T cell receptor (sTCR), which binds to a complex of the peptide Survivin, wherein the complex comprises the amino acid sequence of SEQ ID NO: 40 and the HLA-A2 molecule, wherein the sTCR comprises: [0258] (1) a sTCR variable beta region (V.sub.β) comprising SEQ ID NO: 20 (CDR1), SEQ ID NO: 24 (CDR2), and SEQ ID NO: 28 (CDR3) or SEQ ID NO: 31 (CDR3), and [0259] (2) a sTCR variable alpha region (V.sub.α) comprising SEQ ID NO: 19 (CDR1), SEQ ID NO: 23 (CDR2), and SEQ ID NO: 27 (CDR3), [0260] wherein the V.sub.α and V.sub.β regions of the sTCR are connected via a first peptide linker (L1); [0261] b) an antigen binding fragment (Fab) which binds to human CD3 (anti-CD3-Fab), wherein the anti-CD3-Fab comprises: [0262] (1) a heavy chain region comprising a heavy chain variable (V.sub.H) comprising SEQ ID NO: 21 (CDR1) or SEQ ID NO: 32 (CDR1), SEQ ID NIO: 25 (CDR2) or SEQ ID NO: 33 (CDR2), and SEQ ID NO: 29 (CDR3) or SEQ ID NO: 34 (CDR3); and a heavy chain constant domain 1 (CH1) comprising the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 35, and [0263] (2) a light chain region comprising a light chain variable (V.sub.L) comprising SEQ ID NO: 22 (CDR1) or SEQ ID NO: 81 (CDR1), SEQ ID NO: 26 (CDR2) or SEQ ID NO: 82 (CDR2), and SEQ ID NO: 30 (CDR3) or SEQ ID NO: 83 (CDR3); and a kappa constant light chain (C.sub.κ) comprising the amino acid sequence of SEQ ID NO: 17, [0264] wherein the V.sub.β of the sTCR and the V.sub.H of the anti-CD3-Fab are connected via a second peptide linker (L2); and [0265] c) a fragment crystallizable region (Fc) comprising a constant domain 2 (CH2) and a constant domain 3 (CH3), wherein the CH2CH3 comprises the amino acid sequence selected from a group consisting of SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15;

    [0266] wherein the bispecific molecule is in the form of


    V.sub.α-L1-V.sub.β-L2-V.sub.H-CH1CH2CH3V.sub.L-Cκ.

    [0267] In one embodiment, the present invention provides a bispecific molecule comprising: [0268] a) a single-chain soluble T cell receptor (sTCR), which binds to a complex of the peptide Survivin, wherein the complex comprises the amino acid sequence of SEQ ID NO: 40 and the HLA-A2 molecule, wherein the sTCR comprises: [0269] (1) a sTCR variable beta region (V.sub.β) comprising the amino acid sequence selected from a group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, and [0270] (2) a sTCR variable alpha region (V.sub.α) comprising the amino acid sequence selected from a group consisting of SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, [0271] wherein the V.sub.α and V.sub.β regions of the sTCR are connected via a first peptide linker (L1); [0272] b) an antigen binding fragment (Fab) which binds to human CD3 (anti-CD3-Fab), wherein the anti-CD3-Fab comprises: [0273] (1) a heavy chain region comprising a heavy chain variable (V.sub.H) comprising the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10; and a heavy chain constant domain 1 (CH1) comprising the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 35, and [0274] (2) a light chain region comprising a light chain variable (V.sub.L) comprising the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12; and a kappa constant light chain (C.sub.κ) comprising the amino acid sequence of SEQ ID NO: 17, [0275] wherein the V.sub.β of the sTCR and the V.sub.H of the anti-CD3-Fab are connected via a second peptide linker (L2); and [0276] c) a fragment crystallizable region (Fc) comprising a constant domain 2 (CH2) and a constant domain 3 (CH3), wherein the CH2CH3 comprises the amino acid sequence selected from a group consisting of SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15; [0277] wherein the bispecific molecule is in the form of


    V.sub.α-L1-V.sub.β-L2-V.sub.H-CH1CH2CH3V.sub.L-Cκ.

    [0278] In one embodiment, the present invention provides a bispecific molecule comprising: [0279] a) a single-chain soluble T cell receptor (sTCR), which binds to a complex of the peptide Survivin, wherein the complex comprises the amino acid sequence of SEQ ID NO: 40 and the HLA-A2 molecule, wherein the sTCR comprises: [0280] (1) a sTCR variable beta region (V.sub.β) comprising SEQ ID NO: 20 (CDR1), SEQ ID NO: 24 (CDR2), and SEQ ID NO: 28 (CDR3) or SEQ ID NO: 31 (CDR3), and [0281] (2) a sTCR variable alpha region (V.sub.α) comprising SEQ ID NO: 19 (CDR1), SEQ ID NO: 23 (CDR2), and SEQ ID NO: 27 (CDR3), [0282] wherein the V.sub.β and V.sub.α regions of the sTCR are connected via a first peptide linker (L1); [0283] b) an antigen binding fragment (Fab) which binds to human CD3 (anti-CD3-Fab), wherein the anti-CD3-Fab comprises: [0284] (1) a heavy chain variable (V.sub.H) comprising SEQ ID NO: 21 (CDR1) or SEQ ID NO: 32 (CDR1), SEQ ID NO: 25 (CDR2) or SEQ ID NO: 33 (CDR2), and SEQ ID NO: 29 (CDR3) or SEQ ID NO: 34 (CDR3); and a heavy chain constant domain 1 (CH1) comprising the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 35, and [0285] (2) a light chain region comprising a light chain variable (V.sub.L) comprising SEQ ID NO: 22 (CDR1) or SEQ ID NO: 81 (CDR1), SEQ ID NO: 26 (CDR2) or SEQ ID NO: 82 (CDR2), and SEQ ID NO: 30 (CDR3) or SEQ ID NO: 83 (CDR3); and a kappa constant light chain (C.sub.κ) comprising the amino acid sequence of SEQ ID NO: 17, [0286] wherein the V.sub.α of the sTCR and the V.sub.H of the anti-CD3-Fab are connected via a second peptide linker (L2); and [0287] c) a fragment crystallizable region (Fc) comprising: [0288] (1) a first constant region comprising a first constant domain 2 (CH2) and a first constant domain 3 (CH3), wherein the first constant region (CH2CH3) comprises the amino acid sequence selected from a group consisting of SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, and [0289] (2) a second constant region comprising a second constant domain 2 (CH2′) and a second constant domain 3 (CH3′), wherein the second constant region (CH2′CH3′) comprises the amino acid sequence of SEQ ID NO: 16,

    [0290] wherein the bispecific molecule is in the form of


    V.sub.β-L1-V.sub.α-L2-V.sub.H-CH1CH2CH3V.sub.L-CκCH2′CH3′.

    [0291] In one embodiment, the present invention provides a bispecific molecule comprising: [0292] a) a single-chain soluble T cell receptor (sTCR), which binds to a complex of the peptide Survivin, wherein the complex comprises the amino acid sequence of SEQ ID NO: 40 and the HLA-A2 molecule, wherein the sTCR comprises: [0293] (1) a sTCR variable beta region (V.sub.β) comprising SEQ ID NO: 20 (CDR1), SEQ ID NO: 24 (CDR2), and SEQ ID NO: 28 (CDR3), and [0294] (2) a sTCR variable alpha region (V.sub.α) comprising SEQ ID NO: 19 (CDR1), SEQ ID NO: 23 (CDR2), and SEQ ID NO: 27 (CDR3), [0295] wherein the V.sub.β and V.sub.α regions of the sTCR are connected via a first peptide linker (L1); [0296] b) an antigen binding fragment (Fab) which binds to human CD3 (anti-CD3-Fab), wherein the anti-CD3-Fab comprises: [0297] (1) a heavy chain variable (V.sub.H) comprising SEQ ID NO: 21 (CDR1), SEQ ID NO: 25 (CDR2), and SEQ ID NO: 29 (CDR3); and a heavy chain constant domain 1 (CH1) comprising the amino acid sequence of SEQ ID NO: 18, and [0298] (2) a light chain region comprising a light chain variable (V.sub.L) comprising SEQ ID NO: 22 (CDR1), SEQ ID NO: 26 (CDR2), and SEQ ID NO: 30 (CDR3); and a kappa constant light chain (C.sub.κ) comprising the amino acid sequence of SEQ ID NO: 17, [0299] wherein the V.sub.α of the sTCR and the V.sub.H of the anti-CD3-Fab are connected via a second peptide linker (L2); [0300] c) a fragment crystallizable region (Fc) comprising: [0301] (1) a first constant region comprising a first constant domain 2 (CH2) and a first constant domain 3 (CH3), wherein the first constant region (CH2CH3) comprises the amino acid sequence of SEQ ID NO: 13; and [0302] (2) a second constant region comprising a second constant domain 2 (CH2′) and a second constant domain 3 (CH3′), wherein the second constant region (CH2′CH3′) comprises the amino acid sequence of SEQ ID NO: 16;

    [0303] wherein the bispecific molecule is in the form of


    V.sub.β-L1-V.sub.α-L2-V.sub.H-CH1CH2CH3V.sub.L-CκCH2′CH3′.

    [0304] In one embodiment, the present invention provides a bispecific molecule comprising: [0305] a) a single-chain soluble T cell receptor (sTCR), which binds to a complex of the peptide Survivin, wherein the complex comprises the amino acid sequence of SEQ ID NO: 40 and the HLA-A2 molecule, wherein the sTCR comprises: [0306] (1) a sTCR variable beta region (V.sub.β) comprising the amino acid sequence selected from a group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, and [0307] (2) a sTCR variable alpha region (V.sub.α) comprising the amino acid sequence selected from a group consisting of SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, [0308] wherein the V.sub.β and V.sub.α regions of the sTCR are connected via a first peptide linker (L1); [0309] b) an antigen binding fragment (Fab) which binds to human CD3 (anti-CD3-Fab), wherein the anti-CD3-Fab comprises: [0310] (1) a heavy chain variable (V.sub.H) comprising the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10, and a heavy chain constant domain 1 (CH1) comprising the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 35, and [0311] (2) a light chain region comprising a light chain variable (V.sub.L) comprising the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12; and a kappa constant light chain (C.sub.κ) comprising the amino acid sequence of SEQ ID NO: 17, [0312] wherein the V.sub.α of the sTCR and the V.sub.H of the anti-CD3-Fab are connected via a second peptide linker (L2); [0313] c) a fragment crystallizable region (Fc) comprising: [0314] (1) a first constant region comprising a first constant domain 2 (CH2) and a first constant domain 3 (CH3), wherein the first constant region (CH2CH3) comprises the amino acid sequence selected from a group consisting of SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15; and [0315] (2) a second constant region comprising a second constant domain 2 (CH2′) and a second constant domain 3 (CH3′), wherein the second constant region (CH2′CH3′) comprises the amino acid sequence of SEQ ID NO: 16;

    [0316] wherein the bispecific molecule is in the form of


    V.sub.β-L1-V.sub.α-L2-V.sub.H-CH1CH2CH3V.sub.L-CκCH2′CH3′.

    [0317] In one embodiment, the present invention provides a bispecific molecule comprising: [0318] a) a single-chain soluble T cell receptor (sTCR), which binds to a complex of the peptide Survivin, wherein the complex comprises the amino acid sequence of SEQ ID NO: 40 and the HLA-A2 molecule, wherein the sTCR comprises: [0319] (1) a sTCR variable beta region (V.sub.β) comprising the amino acid sequence of SEQ ID NO: 2, and [0320] (2) a sTCR variable alpha region (V.sub.α) comprising the amino acid sequence of SEQ ID NO: 6, [0321] wherein the V.sub.β and V.sub.α regions of the sTCR are connected via a first peptide linker (L1); [0322] b) an antigen binding fragment (Fab) which binds to human CD3 (anti-CD3-Fab), wherein the anti-CD3-Fab comprises: [0323] (1) a heavy chain variable (V.sub.H) comprising the amino acid sequence of SEQ ID NO: 9, and a heavy chain constant domain 1 (CH1) comprising the amino acid sequence of SEQ ID NO: 18, and [0324] (2) a light chain region comprising a light chain variable (V.sub.L) comprising the amino acid sequence of SEQ ID NO: 11; and a kappa constant light chain (C.sub.κ) comprising the amino acid sequence of SEQ ID NO: 17, [0325] wherein the V.sub.α of the sTCR and the V.sub.H of the anti-CD3-Fab are connected via a second peptide linker (L2); [0326] c) a fragment crystallizable region (Fc) comprising: [0327] (1) a first constant region comprising a first constant domain 2 (CH2) and a first constant domain 3 (CH3), wherein the first constant region (CH2CH3) comprises the amino acid sequence of SEQ ID NO: 13; and [0328] (2) a second constant region comprising a second constant domain 2 (CH2′) and a second constant domain 3 (CH3′), wherein the second constant region (CH2′CH3′) comprises the amino acid sequence of SEQ ID NO: 16;

    [0329] wherein the bispecific molecule is in the form of


    V.sub.β-L1-V.sub.α-L2-V.sub.H-CH1CH2CH3V.sub.L-CκCH2′CH3′.

    [0330] In one embodiment, the present invention provides a bispecific molecule which binds to both human CD3 and a complex of the peptide Survivin, wherein the bispecific molecule comprises: [0331] a) a first heavy chain region comprising [0332] 1) a single-chain soluble T cell receptor (sTCR) which binds to a complex of the peptide Survivin, wherein the complex comprises the amino acid sequence of SEQ ID NO: 40 and the HLA-A2 molecule, wherein the sTCR comprises: [0333] i. a sTCR variable beta region (V.sub.β) comprising SEQ ID NO: 20 (CDR1), SEQ ID NO: 24 (CDR2), and SEQ ID NO: 28 (CDR3) or SEQ ID NO: 31 (CDR3); and [0334] ii. a sTCR variable alpha region (V.sub.α) comprising SEQ ID NO: 19 (CDR1), SEQ ID NO: 23 (CDR2), and SEQ ID NO: 27 (CDR3); wherein the V.sub.β and the V.sub.α are connected via a first peptide linker (L1); [0335] 2) a heavy chain variable (V.sub.H) comprising SEQ ID NO: 21 (CDR1) or SEQ ID NO: 32 (CDR1), SEQ ID NO: 25 (CDR2) or SEQ ID NO: 33 (CDR2), and SEQ ID NO: 29 (CDR3) or SEQ ID NO: 34 (CDR3); and [0336] 3) a first heavy chain constant region (CH1CH2CH3) comprising the amino acid sequence selected from a group consisting of SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39; [0337] wherein the V.sub.α of the sTCR and the V.sub.H are connected via a second peptide linker (L2), and [0338] wherein the first heavy chain region is in the form of V.sub.β-L1-V.sub.α-L2-V.sub.H-CH1CH2CH3; [0339] b) a second heavy chain region (CH2′CH3′) comprising the amino acid sequence of SEQ ID NO: 16; and [0340] c) a light chain comprising: [0341] 1) a light chain variable (V.sub.L) comprising SEQ ID NO: 22 (CDR1) or SEQ ID NO: 81 (CDR1), SEQ ID NO: 26 (CDR2) or SEQ ID NO: 82 (CDR2), and SEQ ID NO: 30 (CDR3) or SEQ ID NO: 83 (CDR3); and [0342] 2) a kappa constant light chain (C.sub.κ) having the amino acid sequence of SEQ ID NO: 17;

    [0343] wherein the CH2CH3 of the first heavy chain and CH2′CH3′ of the second heavy chain form a dimeric Fc region, to form a bispecific molecule with the following form:


    V.sub.β-L1-V.sub.α-L2-V.sub.H-CH1CH2CH3V.sub.L-CκCH2′CH3′.

    [0344] In one embodiment, the present invention provides a bispecific molecule which binds to both human CD3 and a complex of the peptide Survivin, wherein the bispecific molecule comprises: [0345] a) a first heavy chain region comprising [0346] 1) a single-chain soluble T cell receptor (sTCR) which binds to a complex of the peptide Survivin, wherein the complex comprises the amino acid sequence of SEQ ID NO: 40 and the HLA-A2 molecule, wherein the sTCR comprises: [0347] i. a sTCR variable beta region (V.sub.β) comprising SEQ ID NO: 20 (CDR1), SEQ ID NO: 24 (CDR2), and SEQ ID NO: 28 (CDR3); and [0348] ii. a sTCR variable alpha region (V.sub.α) comprising SEQ ID NO: 19 (CDR1), SEQ ID NO: 23 (CDR2), and SEQ ID NO: 27 (CDR3); wherein the V.sub.β and the V.sub.α are connected via a first peptide linker (L1) comprising the amino acid sequence of SEQ ID NO: 1; [0349] 2) a heavy chain variable (V.sub.H) comprising SEQ ID NO: 21 (CDR1), SEQ ID NO: 25 (CDR2), and SEQ ID NO: 29 (CDR3); and [0350] 3) a first heavy chain constant region (CH1CH2CH3) comprising the amino acid sequence of SEQ ID NO: 37; [0351] wherein the V.sub.α of the sTCR and the V.sub.H are connected via a second peptide linker (L2), and [0352] wherein the first heavy chain region is in the form of V.sub.β-L1-V.sub.α-L2-V.sub.H-CH1CH2CH3; [0353] b) a second heavy chain region (CH2′CH3′) comprising the amino acid sequence of SEQ ID NO: 16; and [0354] c) a light chain comprising: [0355] 1) a light chain variable (V.sub.L) comprising SEQ ID NO: 22 (CDR1), SEQ ID NO: 26 (CDR2), and SEQ ID NO: 30 (CDR3); and [0356] 2) a kappa constant light chain (C.sub.κ) having the amino acid sequence of SEQ ID NO: 17;

    [0357] wherein the CH2CH3 of the first heavy chain and CH2′CH3′ of the second heavy chain form a dimeric Fc region, to form a bispecific molecule with the following form


    V.sub.β-L1-V.sub.α-L2-V.sub.H-CH1CH2CH3V.sub.L-CκCH2′CH3′.

    [0358] In one embodiment, the present invention provides a bispecific molecule which binds to both human CD3 and a complex of the peptide Survivin, wherein the bispecific molecule comprises: [0359] a) a first heavy chain region comprising [0360] 1) a single-chain soluble T cell receptor (sTCR) which binds to a complex of the peptide Survivin, wherein the complex comprises the amino acid sequence of SEQ ID NO: 40 and the HLA-A2 molecule, wherein the sTCR comprises: [0361] i. a sTCR variable beta region (V.sub.β) comprising the amino acid sequence selected from a group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5; and [0362] ii. a sTCR variable alpha region (V.sub.α) comprising the amino acid sequence selected from a group consisting of SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8; [0363] wherein the V.sub.β and the V.sub.α are connected via a first peptide linker (L1); [0364] 2) a heavy chain variable (V.sub.H) comprising the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10; and [0365] 3) a first heavy chain constant region (CH1CH2CH3) comprising the amino acid sequence selected from a group consisting of SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39; [0366] wherein the V.sub.α of the sTCR and the V.sub.H are connected via a second peptide linker (L2), and [0367] wherein the first heavy chain region is in the form of V.sub.β-L1-V.sub.α-L2-V.sub.H-CH1CH2CH3; [0368] b) a second heavy chain region (CH2′CH3′) comprising the amino acid sequence of SEQ ID NO: 16; and [0369] c) a light chain comprising: [0370] 1) a light chain variable (V.sub.L) comprising the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12; and [0371] 2) a kappa constant light chain (C.sub.κ) having the amino acid sequence of SEQ ID NO: 17;

    [0372] wherein the CH2CH3 of the first heavy chain and CH2′CH3′ of the second heavy chain form a dimeric Fc region, to form a bispecific molecule with the following form:


    V.sub.β-L1-V.sub.α-L2-V.sub.H-CH1CH2CH3V.sub.L-CκCH2′CH3′.

    [0373] In one embodiment, the present invention provides a bispecific molecule which binds to both human CD3 and a complex of the peptide Survivin, wherein the bispecific molecule comprises: [0374] a) a first heavy chain region comprising: [0375] 1) a single-chain soluble T cell receptor (sTCR) which binds to a complex of the peptide Survivin, wherein the complex comprises the amino acid sequence of SEQ ID NO: 40 and the HLA-A2 molecule, wherein the sTCR comprises: [0376] i. a sTCR variable beta region (V.sub.β) comprising the amino acid sequence of SEQ ID NO: 2; and [0377] ii. a sTCR variable alpha region (V.sub.α) comprising the amino acid sequence of SEQ ID NO: 6; [0378] wherein the V.sub.β and the V.sub.α are connected via a first peptide linker (L1); [0379] 2) a heavy chain variable (V.sub.H) comprising the amino acid sequence of SEQ ID NO: 9; and [0380] 3) a first heavy chain constant region (CH1CH2CH3) comprising the amino acid sequence of SEQ ID NO: 37; [0381] wherein the V.sub.α of the sTCR and the V.sub.H are connected via a second peptide linker (L2), and [0382] wherein the first heavy chain region is in the form of V.sub.β-L1-V.sub.α-L2-V.sub.H-CH1CH2CH3; [0383] b) a second heavy chain region (CH2′CH3′) comprising the amino acid sequence of SEQ ID NO: 16; and [0384] c) a light chain comprising: [0385] 1) a light chain variable (V.sub.L) comprising the amino acid sequence of SEQ ID NO: 11; and [0386] 2) a kappa constant light chain (C.sub.κ) having the amino acid sequence of SEQ ID NO: 17;

    [0387] wherein the CH2CH3 of the first heavy chain and CH2′CH3′ of the second heavy chain form a dimeric Fc region, to form a bispecific molecule with the following form:


    V.sub.β-L1-V.sub.α-L2-V.sub.H-CH1CH2CH3V.sub.L-CκCH2′CH3′.

    [0388] In one embodiment, the present invention provides a bispecific molecule which binds to human CD3 and a complex of the peptide Survivin, wherein the complex comprises the amino acid sequence of SEQ ID NO: 40 and the HLA-A2 molecule, wherein the bispecific molecule comprises: [0389] a) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 36, [0390] b) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 16 and [0391] c) a light chain comprising the amino acid sequence of SEQ ID NO: 76.

    [0392] In one embodiment, the present invention provides a bispecific molecule which binds to human CD3 and a complex of the peptide Survivin, wherein the complex comprises the amino acid sequence of SEQ ID NO: 40 and the HLA-A2 molecule, wherein the bispecific molecule comprises: [0393] a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 88, [0394] b) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 16 and [0395] c) a light chain comprising the amino acid sequence of SEQ ID NO: 76.

    [0396] In one embodiment, the present invention provides a bispecific molecule comprising: [0397] a) a single-chain soluble T cell receptor (sTCR), which binds to a complex of the peptide Survivin, wherein the complex comprises the amino acid sequence of SEQ ID NO: 40 and the HLA-A2 molecule, wherein the sTCR comprises: [0398] (1) a sTCR variable beta region (V.sub.β) comprising the amino acid sequence of SEQ ID NO: 5, and [0399] (2) a sTCR variable alpha region (V.sub.α) comprising the amino acid sequence of SEQ ID NO: 6, [0400] wherein the V.sub.β and V.sub.α regions of the sTCR are connected via a first peptide linker (L1); [0401] b) an antigen binding fragment (Fab) which binds to human CD3 (anti-CD3-Fab), wherein the anti-CD3-Fab comprises: [0402] (1) a heavy chain region comprising a heavy chain variable (V.sub.H) comprising the amino acid sequence of SEQ ID NO: 9; and a heavy chain constant domain 1 (CH1) comprising the amino acid sequence of SEQ ID NO: 18, and [0403] (2) a light chain region comprising a light chain variable (V.sub.L) comprising the amino acid sequence of SEQ ID NO: 11; and a kappa constant light chain (C.sub.κ) comprising the amino acid sequence of SEQ ID NO: 17, [0404] wherein the V.sub.α of the sTCR and the V.sub.H of the anti-CD3-Fab are connected via a second peptide linker (L2); and [0405] c) a fragment crystallizable region (Fc) comprising: [0406] (1) a first constant region comprising a first constant domain 2 (CH2) and a first constant domain 3 (CH3), wherein the first constant region (CH2CH3) comprises the amino acid sequence of SEQ ID NO: 13, and [0407] (2) a second constant region comprising a second constant domain 2 (CH2′) and a second constant domain 3 (CH3′), wherein the second constant region (CH2′CH3′) comprises the amino acid sequence of SEQ ID NO: 16,

    [0408] wherein the bispecific molecule is in the form of


    V.sub.β-L1-V.sub.α-L2-V.sub.H-CH1CH2CH3V.sub.L-CκCH2′CH3′.

    [0409] In another aspect, the present invention provides a bispecific molecule which binds to both human CD3 and a complex of the peptide Survivin, wherein the bispecific molecule comprises: [0410] a) a first heavy chain region comprising [0411] 1) a single-chain soluble T cell receptor (sTCR) which binds to a complex of the peptide Survivin, wherein the complex comprises the amino acid sequence of SEQ ID NO: 40 and the HLA-A2 molecule, wherein the sTCR comprises: [0412] i. a sTCR variable beta region (V.sub.β) comprising the amino acid sequence of SEQ ID NO: 5; and [0413] ii. a sTCR variable alpha region (V.sub.α) comprising the amino acid sequence of SEQ ID NO: 6; [0414] wherein the V.sub.β and the V.sub.α are connected via a first peptide linker (L1); [0415] 2) a heavy chain variable (V.sub.H) comprising the amino acid sequence of SEQ ID NO: 9; and [0416] 3) a first heavy chain constant region (CH1CH2CH3) comprising the amino acid sequence of SEQ ID NO: 37; [0417] wherein the V.sub.α of the sTCR and the V.sub.H are connected via a second peptide linker (L2), and [0418] wherein the first heavy chain region is in the form of V.sub.β-L1-V.sub.α-L2-V.sub.H-CH1CH2CH3; [0419] b) a second heavy chain region (CH2′CH3′) comprising the amino acid sequence of SEQ ID NO: 16; and [0420] c) a light chain comprising: [0421] 1) a light chain variable (V.sub.L) comprising the amino acid sequence of SEQ ID NO: 11; and [0422] 2) a kappa constant light chain (C.sub.κ) having the amino acid sequence of SEQ ID NO: 17;

    [0423] wherein the CH2CH3 of the first heavy chain and CH2′CH3′ of the second heavy chain form a dimeric Fc region, to form a bispecific molecule with the following form:


    V.sub.β-L1-V.sub.α-L2-V.sub.H-CH1CH2CH3V.sub.L-CκCH2′CH3′.

    [0424] In one embodiment, the present invention provides a bispecific molecule comprising: [0425] a) a single-chain soluble T cell receptor (sTCR), which binds to a complex of the peptide Survivin, wherein the complex comprises the amino acid sequence of SEQ ID NO: 40 and the HLA-A2 molecule, wherein the sTCR comprises: [0426] (1) a sTCR variable beta region (V.sub.β) comprising the amino acid sequence of SEQ ID NO: 3, and [0427] (2) a sTCR variable alpha region (V.sub.α) comprising the amino acid sequence of SEQ ID NO: 7, [0428] wherein the V.sub.β and V.sub.α regions of the sTCR are connected via a first peptide linker (L1); [0429] b) an antigen binding fragment (Fab) which binds to human CD3 (anti-CD3-Fab), wherein the anti-CD3-Fab comprises: [0430] (1) a heavy chain region comprising a heavy chain variable (V.sub.H) comprising the amino acid sequence of SEQ ID NO: 9; and a heavy chain constant domain 1 (CH1) comprising the amino acid sequence of SEQ ID NO: 35, and [0431] (2) a light chain region comprising a light chain variable (V.sub.L) comprising the amino acid sequence of SEQ ID NO: 11; and a kappa constant light chain (C.sub.κ) comprising the amino acid sequence of SEQ ID NO: 17, [0432] wherein the V.sub.α of the sTCR and the V.sub.H of the anti-CD3-Fab are connected via a second peptide linker (L2); and [0433] c) a fragment crystallizable region (Fc) comprising: [0434] (1) a constant region comprising a constant domain 2 (CH2) and a constant domain 3 (CH3), wherein the constant region (CH2CH3) comprises the amino acid sequence of SEQ ID NO: 14,

    [0435] wherein the bispecific molecule is in the form of


    V.sub.β-L1-V.sub.α-L2-V.sub.H-CH1CH2CH3V.sub.L-Cκ.

    [0436] In another aspect, the present invention provides a bispecific molecule which binds to both human CD3 and a complex of the peptide Survivin, wherein the bispecific molecule comprises: [0437] a) a heavy chain region comprising [0438] 1) a single-chain soluble T cell receptor (sTCR) which binds to a complex of the peptide Survivin, wherein the complex comprises the amino acid sequence of SEQ ID NO: 40 and the HLA-A2 molecule, wherein the sTCR comprises: [0439] i. a sTCR variable beta region (V.sub.β) comprising the amino acid sequence of SEQ ID NO: 3; and [0440] ii. a sTCR variable alpha region (V.sub.α) comprising the amino acid sequence of SEQ ID NO: 7; [0441] wherein the V.sub.β and the V.sub.α are connected via a first peptide linker (L1); [0442] 2) a heavy chain variable (V.sub.H) comprising the amino acid sequence of SEQ ID NO: 9; and [0443] 3) a heavy chain constant region (CH1CH2CH3) comprising the amino acid sequence of SEQ ID NO: 38; [0444] wherein the V.sub.α of the sTCR and the V.sub.H are connected via a second peptide linker (L2), and [0445] wherein the heavy chain region is in the form of V.sub.β-L1-V.sub.α-L2-V.sub.H-CH1CH2CH3; and [0446] b) a light chain comprising: [0447] 1) a light chain variable (V.sub.L) comprising the amino acid sequence of SEQ ID NO: 11; and [0448] 2) a kappa constant light chain (C.sub.κ) having the amino acid sequence of SEQ ID NO: 17;

    [0449] to form a bispecific molecule with the following form:


    V.sub.β-L1-V.sub.α-L2-V.sub.H-CH1CH2CH3V.sub.L-Cκ.

    [0450] In one embodiment, the present invention provides a bispecific molecule comprising: [0451] a) a single-chain soluble T cell receptor (sTCR), which binds to a complex of the peptide Survivin, wherein the complex comprises the amino acid sequence of SEQ ID NO: 40 and the HLA-A2 molecule, wherein the sTCR comprises: [0452] (1) a sTCR variable alpha region (V.sub.α) comprising the amino acid sequence of SEQ ID NO: 7, and [0453] (2) a sTCR variable beta region (V.sub.β) comprising the amino acid sequence of SEQ ID NO: 3, [0454] wherein the V.sub.β and V.sub.α regions of the sTCR are connected via a first peptide linker (L1); [0455] b) an antigen binding fragment (Fab) which binds to human CD3 (anti-CD3-Fab), wherein the anti-CD3-Fab comprises: [0456] (1) a heavy chain region comprising a heavy chain variable (V.sub.H) comprising the amino acid sequence of SEQ ID NO: 9; and a heavy chain constant domain 1 (CH1) comprising the amino acid sequence of SEQ ID NO: 35, and [0457] (2) a light chain region comprising a light chain variable (V.sub.L) comprising the amino acid sequence of SEQ ID NO: 11; and a kappa constant light chain (C.sub.κ) comprising the amino acid sequence of SEQ ID NO: 17, [0458] wherein the V.sub.β of the sTCR and the V.sub.H of the anti-CD3-Fab are connected via a second peptide linker (L2); and [0459] c) a fragment crystallizable region (Fc) comprising: [0460] (1) a constant region comprising a constant domain 2 (CH2) and a constant domain 3 (CH3), wherein the constant region (CH2CH3) comprises the amino acid sequence of SEQ ID NO: 14,

    [0461] wherein the bispecific molecule is in the form of


    V.sub.β-L1-V.sub.α-L2-V.sub.H-CH1CH2CH3V.sub.L-Cκ.

    [0462] In another aspect, the present invention provides a bispecific molecule which binds to both human CD3 and a complex of the peptide Survivin, wherein the bispecific molecule comprises: [0463] a) a heavy chain region comprising [0464] 1) a single-chain soluble T cell receptor (sTCR) which binds to a complex of the peptide Survivin, wherein the complex comprises the amino acid sequence of SEQ ID NO: 40 and the HLA-A2 molecule, wherein the sTCR comprises: [0465] i. a sTCR variable alpha region (V.sub.α) comprising the amino acid sequence of SEQ ID NO: 7; and [0466] ii. a sTCR variable beta region (V.sub.β) comprising the amino acid sequence of SEQ ID NO: 3; [0467] wherein the V.sub.β and the V.sub.α are connected via a first peptide linker (L1); [0468] 2) a heavy chain variable (V.sub.H) comprising the amino acid sequence of SEQ ID NO: 9; and [0469] 3) a heavy chain constant region (CH1CH2CH3) comprising the amino acid sequence of SEQ ID NO: 38; [0470] wherein the V.sub.β of the sTCR and the V.sub.H are connected via a second peptide linker (L2), and [0471] wherein the heavy chain region is in the form of V.sub.α-L1-V.sub.β-L2-V.sub.H-CH1CH2CH3; and [0472] b) a light chain comprising: [0473] 1) a light chain variable (V.sub.L) comprising the amino acid sequence of SEQ ID NO: 11; and [0474] 2) a kappa constant light chain (C.sub.κ) having the amino acid sequence of SEQ ID NO: 17;

    [0475] to form a bispecific molecule with the following form:


    V.sub.β-L1-V.sub.α-L2-V.sub.H-CH1CH2CH3V.sub.L-Cκ.

    [0476] In one embodiment, the present invention provides a bispecific molecule comprising: [0477] a) a single-chain soluble T cell receptor (sTCR), which binds to a complex of the peptide Survivin, wherein the complex comprises the amino acid sequence of SEQ ID NO: 40 and the HLA-A2 molecule, wherein the sTCR comprises: [0478] (1) a sTCR variable alpha region (V.sub.α) comprising the amino acid sequence of SEQ ID NO: 7, and [0479] (2) a sTCR variable beta region (V.sub.β) comprising the amino acid sequence of SEQ ID NO: 3, [0480] wherein the V.sub.β and V.sub.α regions of the sTCR are connected via a first peptide linker (L1); [0481] b) an antigen binding fragment (Fab) which binds to human CD3 (anti-CD3-Fab), wherein the anti-CD3-Fab comprises: [0482] (1) a heavy chain region comprising a heavy chain variable (V.sub.H) comprising the amino acid sequence of SEQ ID NO: 9; and a heavy chain constant domain 1 (CH1) comprising the amino acid sequence of SEQ ID NO: 18, and [0483] (2) a light chain region comprising a light chain variable (V.sub.L) comprising the amino acid sequence of SEQ ID NO: 11; and a kappa constant light chain (C.sub.κ) comprising the amino acid sequence of SEQ ID NO: 17, and [0484] wherein the V.sub.β of the sTCR and the V.sub.H of the anti-CD3-Fab are connected via a second peptide linker (L2);

    [0485] wherein the bispecific molecule is in the form of


    V.sub.β-L1-V.sub.α-L2-V.sub.H-CH1CH2CH3V.sub.L-Cκ.

    [0486] In another aspect, the present invention provides a bispecific molecule which binds to both human CD3 and a complex of the peptide Survivin, wherein the bispecific molecule comprises: [0487] a) a heavy chain region comprising [0488] 1) a single-chain soluble T cell receptor (sTCR) which binds to a complex of the peptide Survivin, wherein the complex comprises the amino acid sequence of SEQ ID NO: 40 and the HLA-A2 molecule, wherein the sTCR comprises: [0489] i. a sTCR variable alpha region (V.sub.α) comprising the amino acid sequence of SEQ ID NO: 7; and [0490] ii. a sTCR variable beta region (V.sub.β) comprising the amino acid sequence of SEQ ID NO: 3; [0491] wherein the V.sub.β and the V.sub.α are connected via a first peptide linker (L1); [0492] 2) a heavy chain variable (V.sub.H) comprising the amino acid sequence of SEQ ID NO: 9; and [0493] 3) a heavy chain constant region (CH1) comprising the amino acid sequence of SEQ ID NO: 18; [0494] wherein the V.sub.β of the sTCR and the V.sub.H are connected via a second peptide linker (L2), and [0495] wherein the heavy chain region is in the form of V.sub.α-L1-V.sub.β-L2-V.sub.H—CH1; and [0496] b) a light chain comprising: [0497] 1) a light chain variable (V.sub.L) comprising the amino acid sequence of SEQ ID NO: 11; and [0498] 2) a kappa constant light chain (C.sub.κ) having the amino acid sequence of SEQ ID NO: 17;

    [0499] to form a bispecific molecule with the following form:


    V.sub.β-L1-V.sub.α-L2-V.sub.H-CH1CH2CH3V.sub.L-Cκ.

    [0500] In one embodiment, the present invention provides a bispecific molecule comprising: [0501] a) a single-chain soluble T cell receptor (sTCR), which binds to a complex of the peptide Survivin, wherein the complex comprises the amino acid sequence of SEQ ID NO: 40 and the HLA-A2 molecule, wherein the sTCR comprises: [0502] (1) a sTCR variable beta region (V.sub.β) comprising the amino acid sequence of SEQ ID NO: 3, and [0503] (2) a sTCR variable alpha region (V.sub.α) comprising the amino acid sequence of SEQ ID NO: 7, [0504] wherein the V.sub.β and V.sub.α regions of the sTCR are connected via a first peptide linker (L1); [0505] b) an antigen binding fragment (Fab) which binds to human CD3 (anti-CD3-Fab), wherein the anti-CD3-Fab comprises: [0506] (1) a heavy chain region comprising a heavy chain variable (V.sub.H) comprising the amino acid sequence of SEQ ID NO: 9; and a heavy chain constant domain 1 (CH1) comprising the amino acid sequence of SEQ ID NO: 18, and [0507] (2) a light chain region comprising a light chain variable (V.sub.L) comprising the amino acid sequence of SEQ ID NO: 11; and a kappa constant light chain (C.sub.κ) comprising the amino acid sequence of SEQ ID NO: 17, and [0508] wherein the V.sub.α of the sTCR and the V.sub.H of the anti-CD3-Fab are connected via a second peptide linker (L2);

    [0509] wherein the bispecific molecule is in the form of


    V.sub.β-L1-V.sub.α-L2-V.sub.H-CH1CH2CH3V.sub.L-Cκ.

    [0510] In another aspect, the present invention provides a bispecific molecule which binds to both human CD3 and a complex of the peptide Survivin, wherein the bispecific molecule comprises: [0511] a) a heavy chain region comprising [0512] 1) a single-chain soluble T cell receptor (sTCR) which binds to a complex of the peptide Survivin, wherein the complex comprises the amino acid sequence of SEQ ID NO: 40 and the HLA-A2 molecule, wherein the sTCR comprises: [0513] i. a sTCR variable beta region (V.sub.β) comprising the amino acid sequence of SEQ ID NO: 3; and [0514] ii. a sTCR variable alpha region (V.sub.α) comprising the amino acid sequence of SEQ ID NO: 7; [0515] wherein the V.sub.β and the V.sub.α are connected via a first peptide linker (L1); [0516] 2) a heavy chain variable (V.sub.H) comprising the amino acid sequence of SEQ ID NO: 9; and [0517] 3) a heavy chain constant region (CH1) comprising the amino acid sequence of SEQ ID NO: 18; [0518] wherein the V.sub.α of the sTCR and the V.sub.H are connected via a second peptide linker (L2), and [0519] wherein the heavy chain region is in the form of V.sub.β-L1-V.sub.α-L2-V.sub.H—CH1; and [0520] b) a light chain comprising: [0521] 1) a light chain variable (V.sub.L) comprising the amino acid sequence of SEQ ID NO: 11; and [0522] 2) a kappa constant light chain (C.sub.κ) having the amino acid sequence of SEQ ID NO: 17; [0523] to form a bispecific molecule with the following form:


    V.sub.β-L1-V.sub.α-L2-V.sub.H-CH1CH2CH3V.sub.L-Cκ.

    [0524] In one embodiment, the present invention provides a bispecific molecule comprising: [0525] a) a single-chain soluble T cell receptor (sTCR), which binds to a complex of the peptide Survivin, wherein the complex comprises the amino acid sequence of SEQ ID NO: 40 and the HLA-A2 molecule, wherein the sTCR comprises: [0526] (1) a sTCR variable beta region (V.sub.β) comprising the amino acid sequence of SEQ ID NO: 4, and [0527] (2) a sTCR variable alpha region (V.sub.α) comprising the amino acid sequence of SEQ ID NO: 8, [0528] wherein the V.sub.β and V.sub.α regions of the sTCR are connected via a first peptide linker (L1); [0529] b) an antigen binding fragment (Fab) which binds to human CD3 (anti-CD3-Fab), wherein the anti-CD3-Fab comprises: [0530] (1) a heavy chain region comprising a heavy chain variable (V.sub.H) comprising the amino acid sequence of SEQ ID NO: 9; and a heavy chain constant domain 1 (CH1) comprising the amino acid sequence of SEQ ID NO: 35, and [0531] (2) a light chain region comprising a light chain variable (V.sub.L) comprising the amino acid sequence of SEQ ID NO: 11; and a kappa constant light chain (C.sub.κ) comprising the amino acid sequence of SEQ ID NO: 17, [0532] wherein the V.sub.α of the sTCR and the V.sub.H of the anti-CD3-Fab are connected via a second peptide linker (L2); and [0533] c) a fragment crystallizable region (Fc) comprising: [0534] (1) a constant region comprising a constant domain 2 (CH2) and a constant domain 3 (CH3), wherein the constant region (CH2CH3) comprises the amino acid sequence of SEQ ID NO: 14,

    [0535] wherein the bispecific molecule is in the form of


    V.sub.β-L1-V.sub.α-L2-V.sub.H-CH1CH2CH3V.sub.L-Cκ.

    [0536] In another aspect, the present invention provides a bispecific molecule which binds to both human CD3 and a complex of the peptide Survivin, wherein the bispecific molecule comprises: [0537] a) a heavy chain region comprising [0538] 1) a single-chain soluble T cell receptor (sTCR) which binds to a complex of the peptide Survivin, wherein the complex comprises the amino acid sequence of SEQ ID NO: 40 and the HLA-A2 molecule, wherein the sTCR comprises: [0539] i. a sTCR variable beta region (V.sub.β) comprising the amino acid sequence of SEQ ID NO: 4; and [0540] ii. a sTCR variable alpha region (V.sub.α) comprising the amino acid sequence of SEQ ID NO: 8; [0541] wherein the V.sub.β and the V.sub.α are connected via a first peptide linker (L1); [0542] 2) a heavy chain variable (V.sub.H) comprising the amino acid sequence of SEQ ID NO: 9; and [0543] 3) a heavy chain constant region (CH1CH2CH3) comprising the amino acid sequence of SEQ ID NO: 38; [0544] wherein the V.sub.α of the sTCR and the V.sub.H are connected via a second peptide linker (L2), and [0545] wherein the heavy chain region is in the form of V.sub.β-L1-V.sub.α-L2-V.sub.H-CH1CH2CH3; and [0546] b) a light chain comprising: [0547] 1) a light chain variable (V.sub.L) comprising the amino acid sequence of SEQ ID NO: 11; and [0548] 2) a kappa constant light chain (C.sub.κ) having the amino acid sequence of SEQ ID NO: 17;

    [0549] to form a bispecific molecule with the following form:


    V.sub.β-L1-V.sub.α-L2-V.sub.H-CH1CH2CH3V.sub.L-Cκ.

    [0550] In one embodiment, the present invention provides a bispecific molecule comprising: [0551] a) a single-chain soluble T cell receptor (sTCR), which binds to a complex of the peptide Survivin, wherein the complex comprises the amino acid sequence of SEQ ID NO: 40 and the HLA-A2 molecule, wherein the sTCR comprises: [0552] (1) a sTCR variable beta region (V.sub.β) comprising the amino acid sequence of SEQ ID NO: 5, and [0553] (2) a sTCR variable alpha region (V.sub.α) comprising the amino acid sequence of SEQ ID NO: 6, [0554] wherein the V.sub.β and V.sub.α regions of the sTCR are connected via a first peptide linker (L1); [0555] b) an antigen binding fragment (Fab) which binds to human CD3 (anti-CD3-Fab), wherein the anti-CD3-Fab comprises: [0556] (1) a heavy chain region comprising a heavy chain variable (V.sub.H) comprising the amino acid sequence of SEQ ID NO: 9; and a heavy chain constant domain 1 (CH1) comprising the amino acid sequence of SEQ ID NO: 35, and [0557] (2) a light chain region comprising a light chain variable (V.sub.L) comprising the amino acid sequence of SEQ ID NO: 11; and a kappa constant light chain (C.sub.κ) comprising the amino acid sequence of SEQ ID NO: 17, [0558] wherein the V.sub.α of the sTCR and the V.sub.H of the anti-CD3-Fab are connected via a second peptide linker (L2); and [0559] c) a fragment crystallizable region (Fc) comprising: [0560] (1) a constant region comprising a constant domain 2 (CH2) and a constant domain 3 (CH3), wherein the constant region (CH2CH3) comprises the amino acid sequence of SEQ ID NO: 14,

    [0561] wherein the bispecific molecule is in the form of


    V.sub.β-L1-V.sub.α-L2-V.sub.H-CH1CH2CH3V.sub.L-Cκ.

    [0562] In another aspect, the present invention provides a bispecific molecule which binds to both human CD3 and a complex of the peptide Survivin, wherein the bispecific molecule comprises: [0563] a) a heavy chain region comprising [0564] 1) a single-chain soluble T cell receptor (sTCR) which binds to a complex of the peptide Survivin, wherein the complex comprises the amino acid sequence of SEQ ID NO: 40 and the HLA-A2 molecule, wherein the sTCR comprises: [0565] i. a sTCR variable beta region (V.sub.β) comprising the amino acid sequence of SEQ ID NO: 5; and [0566] ii. a sTCR variable alpha region (V.sub.α) comprising the amino acid sequence of SEQ ID NO: 6; [0567] wherein the V.sub.β and the V.sub.α are connected via a first peptide linker (L1); [0568] 2) a heavy chain variable (V.sub.H) comprising the amino acid sequence of SEQ ID NO: 9; and [0569] 3) a heavy chain constant region (CH1CH2CH3) comprising the amino acid sequence of SEQ ID NO: 38; [0570] wherein the V.sub.α of the sTCR and the V.sub.H are connected via a second peptide linker (L2), and [0571] wherein the heavy chain region is in the form of V.sub.β-L1-V.sub.α-L2-V.sub.H-CH1CH2CH3; and [0572] b) a light chain comprising: [0573] 1) a light chain variable (V.sub.L) comprising the amino acid sequence of SEQ ID NO: 11; and [0574] 2) a kappa constant light chain (C.sub.κ) having the amino acid sequence of SEQ ID NO: 17;

    [0575] to form a bispecific molecule with the following form:


    V.sub.β-L1-V.sub.α-L2-V.sub.H-CH1CH2CH3V.sub.L-Cκ.

    [0576] In one embodiment, the present invention provides a bispecific molecule comprising: [0577] a) a single-chain soluble T cell receptor (sTCR), which binds to a complex of the peptide Survivin, wherein the complex comprises the amino acid sequence of SEQ ID NO: 40 and the HLA-A2 molecule, wherein the sTCR comprises: [0578] (1) a sTCR variable beta region (V.sub.β) comprising the amino acid sequence of SEQ ID NO: 5, and [0579] (2) a sTCR variable alpha region (V.sub.α) comprising the amino acid sequence of SEQ ID NO: 6, [0580] wherein the V.sub.β and V.sub.α regions of the sTCR are connected via a first peptide linker (L1); [0581] b) an antigen binding fragment (Fab) which binds to human CD3 (anti-CD3-Fab), wherein the anti-CD3-Fab comprises: [0582] (1) a heavy chain region comprising a heavy chain variable (V.sub.H) comprising the amino acid sequence of SEQ ID NO: 10; and a heavy chain constant domain 1 (CH1) comprising the amino acid sequence of SEQ ID NO: 35, and [0583] (2) a light chain region comprising a light chain variable (V.sub.L) comprising the amino acid sequence of SEQ ID NO: 12; and a kappa constant light chain (C.sub.κ) comprising the amino acid sequence of SEQ ID NO: 17, [0584] wherein the V.sub.α of the sTCR and the V.sub.H of the anti-CD3-Fab are connected via a second peptide linker (L2); and [0585] c) a fragment crystallizable region (Fc) comprising: [0586] (2) a constant region comprising at constant domain 2 (CH2) and a constant domain 3 (CH3), wherein the constant region (CH2CH3) comprises the amino acid sequence of SEQ ID NO: 14,

    [0587] wherein the bispecific molecule is in the form of


    V.sub.β-L1-V.sub.α-L2-V.sub.H-CH1CH2CH3V.sub.L-Cκ.

    [0588] In another aspect, the present invention provides a bispecific molecule which binds to both human CD3 and a complex of the peptide Survivin, wherein the bispecific molecule comprises: [0589] a) a heavy chain region comprising [0590] 1) a single-chain soluble T cell receptor (sTCR) which binds to a complex of the peptide Survivin, wherein the complex comprises the amino acid sequence of SEQ ID NO: 40 and the HLA-A2 molecule, wherein the sTCR comprises: [0591] i. a sTCR variable beta region (V.sub.β) comprising the amino acid sequence of SEQ ID NO: 5; and [0592] ii. a sTCR variable alpha region (V.sub.α) comprising the amino acid sequence of SEQ ID NO: 6; [0593] wherein the V.sub.β and the V.sub.α are connected via a first peptide linker (L1); [0594] 2) a heavy chain variable (V.sub.H) comprising the amino acid sequence of SEQ ID NO: 10; and [0595] 3) a heavy chain constant region (CH1CH2CH3) comprising the amino acid sequence of SEQ ID NO: 38; [0596] wherein the V.sub.α of the sTCR and the V.sub.H are connected via a second peptide linker (L2), and [0597] wherein the heavy chain region is in the form of V.sub.β-L1-V.sub.α-L2-V.sub.H-CH1CH2CH3; and [0598] b) a light chain comprising: [0599] 1) a light chain variable (V.sub.L) comprising the amino acid sequence of SEQ ID NO: 12; and [0600] 2) a kappa constant light chain (C.sub.κ) having the amino acid sequence of SEQ ID NO: 17;

    [0601] to form a bispecific molecule with the following form:


    V.sub.β-L1-V.sub.α-L2-V.sub.H-CH1CH2CH3V.sub.L-Cκ.

    [0602] In one embodiment, the present invention provides a bispecific molecule comprising: [0603] a) a single-chain soluble T cell receptor (sTCR), which binds to a complex of the peptide Survivin, wherein the complex comprises the amino acid sequence of SEQ ID NO: 40 and the HLA-A2 molecule, wherein the sTCR comprises: [0604] (1) a sTCR variable beta region (V.sub.β) comprising the amino acid sequence of SEQ ID NO: 5, and [0605] (2) a sTCR variable alpha region (V.sub.α) comprising the amino acid sequence of SEQ ID NO: 6, [0606] wherein the V.sub.β and V.sub.α regions of the sTCR are connected via a first peptide linker (L1); [0607] b) an antigen binding fragment (Fab) which binds to human CD3 (anti-CD3-Fab), wherein the anti-CD3-Fab comprises: [0608] (1) a heavy chain region comprising a heavy chain variable (V.sub.H) comprising the amino acid sequence of SEQ ID NO: 9; and a heavy chain constant domain 1 (CH1) comprising the amino acid sequence of SEQ ID NO: 18, and [0609] (2) a light chain region comprising a light chain variable (V.sub.L) comprising the amino acid sequence of SEQ ID NO: 11; and a kappa constant light chain (C.sub.κ) comprising the amino acid sequence of SEQ ID NO: 17, and [0610] wherein the V.sub.α of the sTCR and the V.sub.H of the anti-CD3-Fab are connected via a second peptide linker (L2);

    [0611] wherein the bispecific molecule is in the form of


    V.sub.β-L1-V.sub.α-L2-V.sub.H-CH1CH2CH3V.sub.L-Cκ.

    [0612] In another aspect, the present invention provides a bispecific molecule which binds to both human CD3 and a complex of the peptide Survivin, wherein the bispecific molecule comprises: [0613] a) a heavy chain region comprising [0614] 1) a single-chain soluble T cell receptor (sTCR) which binds to a complex of the peptide Survivin, wherein the complex comprises the amino acid sequence of SEQ ID NO: 40 and the HLA-A2 molecule, wherein the sTCR comprises: [0615] i. a sTCR variable beta region (V.sub.β) comprising the amino acid sequence of SEQ ID NO: 5; and [0616] ii. a sTCR variable alpha region (V.sub.α) comprising the amino acid sequence of SEQ ID NO: 6; [0617] wherein the V.sub.β and the V.sub.α are connected via a first peptide linker (L1); [0618] 2) a heavy chain variable (V.sub.H) comprising the amino acid sequence of SEQ ID NO: 9; and [0619] 3) a heavy chain constant region (CH1) comprising the amino acid sequence of SEQ ID NO: 18; [0620] wherein the V.sub.α of the sTCR and the V.sub.H are connected via a second peptide linker (L2), and [0621] wherein the heavy chain region is in the form of V.sub.β-L1-V.sub.α-L2-V.sub.H—CH1; and [0622] b) a light chain comprising: [0623] 1) a light chain variable (V.sub.L) comprising the amino acid sequence of SEQ ID NO: 11; and [0624] 2) a kappa constant light chain (C.sub.κ) having the amino acid sequence of SEQ ID NO: 17;

    [0625] to form a bispecific molecule with the following form:


    V.sub.β-L1-V.sub.α-L2-V.sub.H-CH1CH2CH3V.sub.L-Cκ.

    [0626] In one embodiment, the present invention provides a bispecific molecule comprising: [0627] a) a single-chain soluble T cell receptor (sTCR), which binds to a complex of the peptide Survivin, wherein the complex comprises the amino acid sequence of SEQ ID NO: 40 and the HLA-A2 molecule, wherein the sTCR comprises: [0628] (1) a sTCR variable beta region (V.sub.β) comprising the amino acid sequence of SEQ ID NO: 5, and [0629] (2) a sTCR variable alpha region (V.sub.α) comprising the amino acid sequence of SEQ ID NO: 6, [0630] wherein the V.sub.β and V.sub.α regions of the sTCR are connected via a first peptide linker (L1); [0631] b) an antigen binding fragment (Fab) which binds to human CD3 (anti-CD3-Fab), wherein the anti-CD3-Fab comprises: [0632] (1) a heavy chain region comprising a heavy chain variable (V.sub.H) comprising the amino acid sequence of SEQ ID NO: 10; and a heavy chain constant domain 1 (CH1) comprising the amino acid sequence of SEQ ID NO: 18, and [0633] (2) a light chain region comprising a light chain variable (V.sub.L) comprising the amino acid sequence of SEQ ID NO: 12; and a kappa constant light chain (C.sub.κ) comprising the amino acid sequence of SEQ ID NO: 17, and [0634] wherein the V.sub.α of the sTCR and the V.sub.H of the anti-CD3-Fab are connected via a second peptide linker (L2); [0635] wherein the bispecific molecule is in the form of


    V.sub.β-L1-V.sub.α-L2-V.sub.H-CH1CH2CH3V.sub.L-Cκ.

    [0636] In another aspect, the present invention provides a bispecific molecule which binds to both human CD3 and a complex of the peptide Survivin, wherein the bispecific molecule comprises: [0637] a) a heavy chain region comprising [0638] 1) a single-chain soluble T cell receptor (sTCR) which binds to a complex of the peptide Survivin, wherein the complex comprises the amino acid sequence of SEQ ID NO: 40 and the HLA-A2 molecule, wherein the sTCR comprises: [0639] i. a sTCR variable beta region (V.sub.β) comprising the amino acid sequence of SEQ ID NO: 5; and [0640] ii. a sTCR variable alpha region (V.sub.α) comprising the amino acid sequence of SEQ ID NO: 6; [0641] wherein the V.sub.β and the V.sub.α are connected via a first peptide linker (L1); [0642] 2) a heavy chain variable (V.sub.H) comprising the amino acid sequence of SEQ ID NO: 10; and [0643] 3) a heavy chain constant region (CH1) comprising the amino acid sequence of SEQ ID NO: 18; [0644] wherein the V.sub.α of the sTCR and the V.sub.H are connected via a second peptide linker (L2), and [0645] wherein the heavy chain region is in the form of V.sub.β-L1-V.sub.α-L2-V.sub.H—CH1; and [0646] b) a light chain comprising: [0647] 1) a light chain variable (V.sub.L) comprising the amino acid sequence of SEQ ID NO: 12; and [0648] 2) a kappa constant light chain (C.sub.κ) having the amino acid sequence of SEQ ID NO: 17;

    [0649] to form a bispecific molecule with the following form:


    V.sub.β-L1-V.sub.α-L2-V.sub.H-CH1CH2CH3V.sub.L-Cκ.

    [0650] In one embodiment, the present invention provides a bispecific molecule comprising: [0651] a) a single-chain soluble T cell receptor (sTCR), which binds to a complex of the peptide Survivin, wherein the complex comprises the amino acid sequence of SEQ ID NO: 40 and the HLA-A2 molecule, wherein the sTCR comprises: [0652] (1) a sTCR variable beta region (V.sub.β) comprising the amino acid sequence of SEQ ID NO: 5, and [0653] (2) a sTCR variable alpha region (V.sub.α) comprising the amino acid sequence of SEQ ID NO: 6, [0654] wherein the V.sub.β and V.sub.α regions of the sTCR are connected via a first peptide linker (L1); [0655] b) an antigen binding fragment (Fab) which binds to human CD3 (anti-CD3-Fab), wherein the anti-CD3-Fab comprises: [0656] (1) a heavy chain region comprising a heavy chain variable (V.sub.H) comprising the amino acid sequence of SEQ ID NO: 9; and a heavy chain constant domain 1 (CH1) comprising the amino acid sequence of SEQ ID NO: 35, and [0657] (2) a light chain region comprising a light chain variable (V.sub.L) comprising the amino acid sequence of SEQ ID NO: 11; and a kappa constant light chain (C.sub.κ) comprising the amino acid sequence of SEQ ID NO: 17, [0658] wherein the V.sub.α of the sTCR and the V.sub.H of the anti-CD3-Fab are connected via a second peptide linker (L2); and [0659] c) a fragment crystallizable region (Fc) comprising: [0660] a constant region comprising at constant domain 2 (CH2) and a constant domain 3 (CH3), wherein the constant region (CH2CH3) comprises the amino acid sequence of SEQ ID NO: 15,

    [0661] wherein the bispecific molecule is in the form of


    V.sub.β-L1-V.sub.α-L2-V.sub.H-CH1CH2CH3V.sub.L-Cκ.

    [0662] In another aspect, the present invention provides a bispecific molecule which binds to both human CD3 and a complex of the peptide Survivin, wherein the bispecific molecule comprises: [0663] a) a heavy chain region comprising [0664] 1) a single-chain soluble T cell receptor (sTCR) which binds to a complex of the peptide Survivin, wherein the complex comprises the amino acid sequence of SEQ ID NO: 40 and the HLA-A2 molecule, wherein the sTCR comprises: [0665] i. a sTCR variable beta region (V.sub.β) comprising the amino acid sequence of SEQ ID NO: 5; and [0666] ii. a sTCR variable alpha region (V.sub.α) comprising the amino acid sequence of SEQ ID NO: 6; [0667] wherein the V.sub.β and the V.sub.α are connected via a first peptide linker (L1); [0668] 2) a heavy chain variable (V.sub.H) comprising the amino acid sequence of SEQ ID NO: 9; and [0669] 3) a heavy chain constant region (CH1CH2CH3) comprising the amino acid sequence of SEQ ID NO: 39; [0670] wherein the V.sub.α of the sTCR and the V.sub.H are connected via a second peptide linker (L2), and [0671] wherein the heavy chain region is in the form of V.sub.β-L1-V.sub.α-L2-V.sub.H-CH1CH2CH3; and [0672] b) a light chain comprising: [0673] 1) a light chain variable (V.sub.L) comprising the amino acid sequence of SEQ ID NO: 11; and [0674] 2) a kappa constant light chain (C.sub.κ) having the amino acid sequence of SEQ ID NO: 17;

    [0675] to form a bispecific molecule with the following form:


    V.sub.β-L1-V.sub.α-L2-V.sub.H-CH1CH2CH3V.sub.L-Cκ.

    [0676] In one embodiment, the sTCR of the bispecific molecule binds to a peptide derived from human Survivin.

    [0677] In one embodiment, the sTCR of the bispecific molecule binds to a peptide derived from human Survivin in complex with HLA-A2.

    [0678] In one embodiment, the sTCR of the bispecific molecule binds to a peptide comprising the amino acid sequence of SEQ ID NO: 40.

    [0679] In one embodiment, the sTCR of the bispecific molecule binds to a peptide comprising the amino acid sequence of SEQ ID NO: 40, which is derived from human Survivin in complex with HLA-A2.

    [0680] In one embodiment, the V.sub.β and V.sub.α regions of the sTCR are connected via a first peptide linker (L1) comprising the amino acid sequence of SEQ ID NO: 1.

    [0681] In one embodiment, the V.sub.α and V.sub.β regions of the sTCR are connected via a first peptide linker (L1) comprising the amino acid sequence of SEQ ID NO: 1.

    [0682] In one embodiment, the V.sub.α of the sTCR and V.sub.H of the anti-CD3-Fab are connected via a second peptide linker comprising the amino acid sequence of SEQ ID NO: 1.

    [0683] In one embodiment, the V.sub.β of the sTCR and V.sub.H of the anti-CD3-Fab are connected via a second peptide linker comprising the amino acid sequence of SEQ ID NO: 1.

    [0684] As described in the examples below, several unexpected aspects of the molecules of the present invention have been identified. For example, the molecules of the present invention have a high affinity to Survivin as well as to human CD3. The Survivin TCR part of the molecules exhibit an apparent affinity of about 2 nM to Survivin, particularly remarkable high specificity directed towards a Survivin-derived peptide (SEQ ID NO: 40) complexed to HLA-A2, at the same time the molecules inhibit tumor growth and induce T cell activation and proliferation. Further, the molecules with KiH have a serum half-life of about 5 days, which is a significant improvement compared to the 0.5 hour half-life of the molecules that do not contain KiH.

    [0685] In another aspect, the present disclosure pertains to a pharmaceutical composition comprising a bispecific molecule of the present invention.

    [0686] In another aspect, the present disclosure pertains to a method of treating acute myeloid leukemia or B-cell non-Hodgkin's lymphoma, comprising administering to a patient in need thereof, a bispecific molecule of the present invention, or a pharmaceutical composition thereof.

    [0687] In another aspect, the present disclosure pertains to nucleic acid molecules encoding the bispecific molecules of the present invention,

    [0688] In another aspect, the present disclosure pertains to vectors comprising nucleic acid molecules encoding the bispecific molecules of the present invention.

    [0689] In another aspect, the present disclosure pertains to host cells capable of producing the bispecific molecules of the present invention.

    EXAMPLES

    [0690] The following Examples are provided for purposes of illustration, and not limitation.

    Example 1: TCR-CD3 Bispecific Molecule Generation

    [0691] Bispecific molecules were generated. The polypeptide sequence of each component of the bispecific molecules is listed in Table 1, and the DNA sequence encoding such polypeptide is identified. CDRs within such polypeptides are underlined and their sequences are separately identified.

    [0692] In some embodiments, the polypeptide sequence of CH2CH3, CH2′CH3′, CH1CH2CH3, Heavy Chain 1 and/or Heavy Chain 2 components of the bispecific molecules listed in Table 1 lack the C-terminal lysine, resulting in a C-terminal glycine residue.

    [0693] In some embodiments, the polypeptide sequence of CH1 component of V.sub.αV.sub.β-FTab, V.sub.β V.sub.α-FTab-1, V.sub.β V.sub.α-FTab-2, or V.sub.β V.sub.α-FTab-3 listed in Table 1 further includes a 6-His tag (HHHHHH, SEQ ID NO: 90) placed at the C-terminus of the CH1 domain for these bispecific molecules.

    TABLE-US-00002 TABLE 1 Bispecific Component Amino Acid Sequence DNA Sequence V.sub.βV.sub.α-FTab-KiH V.sub.β - V.sub.α SEQ ID NO: 1 SEQ ID NO: 41 Linker GGGGSGGGGSGGGGSGGGGS V.sub.α - V.sub.H SEQ ID NO: 1 SEQ ID NO: 41 Linker V.sub.β SEQ ID NO: 2 SEQ ID NO: 42 SQTIHQWPATLVQPVGSPLSLECTVEcustom-character LYWYRQAAGRCLELLFYcustom-character QISSEVPQN                       (CDR1; SEQ ID NO: 20)  (CDR2; SEQ ID NO: 24) LSASRPQDRQFILSSKKLLLSDSGFYLCcustom-character FGPGTRLTVLEDLKD                          (CDR3; SEQ ID NO: 28) V.sub.α SEQ ID NO: 6 SEQ ID NO: 46 QKEVEQNSGPLSVPEGAIASLNCTYScustom-character FFWYRQYPGKSPELIMScustom-character KEDGRFTA                        (CDR1; SEQ ID NO: 19)  (CDR2; SEQ ID NO: 23) QLNKASQYVSLLIRDSQPSDSATYLCcustom-character FGCGTQLVVKPNIR                        (CDR3; SEQ ID NO: 27) V.sub.H SEQ ID NO: 9 SEQ ID NO: 49 QVQLVQSGAEVKKPGASVKVSCKASGYTFcustom-character WVRQAPGQGLEWMGcustom-character                      (CDR1; SEQ ID NO: 21)    (CDR2; SEQ ID NO: 25) custom-character KATLTADKSASTAYMELSSLRSEDTAVYYCARcustom-character WGQGTLVTVSS                                       (CDR3; SEQ ID NO: 29) V.sub.L SEQ ID NO: 11 SEQ ID NO: 51 DIQMTQSPSSLSASVGDRVTITCcustom-character WYQQKPGKAPKRLIYcustom-character GVPSRFSGS                      (CDR1; SEQ ID NO: 22)   (CDR2; SEQ ID NO: 26) GSGTDFTLTISSLQPEDFATYYCcustom-character FGGGTKVEIKR                      (CDR3; SEQ ID NO: 30) CH2CH3 SEQ ID NO: 13 SEQ ID NO: 53 SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK CH2′CH3′ SEQ ID NO: 16 SEQ ID NO: 56 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK C.sub.κ SEQ ID NO: 17 SEQ ID NO: 57 TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC CH1 SEQ ID NO: 18 SEQ ID NO: 58 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGP CH1CH2CH3 SEQ ID NO: 37 SEQ ID NO: 77 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK Heavy SEQ ID NO: 36 SEQ ID NO: 80 Chain 1 SQTIHQWPATLVQPVGSPLSLECTVEGTSNPNLYWYRQAAGRCLELLFYSVGIGQISSEVPQNL SASRPQDRQFILSSKKLLLSDSGFYLCAWSIGAEMFFGPGTRLTVLEDLKDGGGGSGGGGSGG GGSGGGGSQKEVEQNSGPLSVPEGAIASLNCTYSDRYAQNFFWYRQYPGKSPELIMSIYSNGD KEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVSKGYKVFGCGTQLVVKPNIRGGGGSG GGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFISYTMHWVRQAPGQGLE WMGYINPRSGYTHYNQKLKDKATLTADKSASTAYMELSSLRSEDTAVYYCARSAYYDYDGF AYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPA PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK Heavy SEQ ID NO: 16 SEQ ID NO: 56 Chain 2 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Light SEQ ID NO: 76 SEQ ID NO: 87 Chain DIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQKPGKAPKRLIYDTSKLASGVPSRFSGS GSGTDFTLTISSLQPEDFATYYCQQWSSNPPTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTA SVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC V.sub.βV.sub.α-FTab-KiH-2 V.sub.β -V.sub.α SEQ ID NO: 1 SEQ ID NO: 41 Linker V.sub.α - V.sub.H SEQ ID NO: 1 SEQ ID NO: 41 Linker V.sub.β SEQ ID NO: 5 SEQ ID NO: 45 SQTIHQWPATLVQPVGSPLSLECTVEcustom-character LYWYRQAAGRCLELLFYcustom-character QISSEVPQN                      (CDR1; SEQ ID NO: 20)   (CDR2; SEQ ID NO: 24) LSASRPQDRQFILSSKKLLLSDSGFYLCcustom-character FGPGTRLTVLEDLKN                          (CDR3; SEQ ID NO: 28) V.sub.α SEQ ID NO: 6 SEQ ID NO: 46 V.sub.H SEQ ID NO: 9 SEQ ID NO: 49 V.sub.L SEQ ID NO: 11 SEQ ID NO: 51 CH2CH3 SEQ ID NO: 13 SEQ ID NO: 53 CH2′CH3′ SEQ ID NO: 16 SEQ ID NO: 56 C.sub.κ SEQ ID NO: 17 SEQ ID NO: 57 CH1 SEQ ID NO: 18 SEQ ID NO: 58 CH1CH2CH3 SEQ ID NO: 37 SEQ ID NO: 77 Heavy SEQ ID NO: 88 SEQ ID NO: 89 Chain 1 SQTIHQWPATLVQPVGSPLSLECTVEGTSNPNLYWYRQAAGRCLELLFYSVGIGQISSEVPQNL SASRPQDRQFILSSKKLLLSDSGFYLCAWSIGAEMFFGPGTRLTVLEDLKNGGGGSGGGGSGG GGSGGGGSQKEVEQNSGPLSVPEGAIASLNCTYSDRYAQNFFWYRQYPGKSPELIMSIYSNGD KEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVSKGYKVFGCGTQLVVKPNIRGGGGSG GGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFISYTMHWVRQAPGQGLE WMGYINPRSGYTHYNQKLKDKATLTADKSASTAYMELSSLRSEDTAVYYCARSAYYDYDGF AYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPA PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK Heavy SEQ ID NO: 16 SEQ ID NO: 56 Chain 2 Light SEQ ID NO: 76 SEQ ID NO: 87 Chain V.sub.βV.sub.α-FTab-hb-1 V.sub.β - V.sub.α SEQ ID NO: 1 SEQ ID NO: 41 Linker V.sub.α - V.sub.H SEQ ID NO: 1 SEQ ID NO: 41 Linker V.sub.β SEQ ID NO: 3 SEQ ID NO: 43 SQTIHQWPATLVQPVGSPLSLECTVEcustom-character LYWYRQAAGRGPELLFYcustom-character QISSEVPQN                      (CDR1; SEQ ID NO: 20)   (CDR2; SEQ ID NO: 24) LFASRPQDRQFILSSKKLLLSDSGFYLCcustom-character FGPGTRLTVLEDLKN                           (CDR3; SEQ ID NO: 31) V.sub.α SEQ ID NO: 7 SEQ ID NO: 47 QKEVEQNSGPLSVPEGAIASLNCTYScustom-character FFWYRQYSGKSPELIMScustom-character KEDGRFTA                       (CDR1; SEQ ID NO: 19)  (CDR2; SEQ ID NO: 23) QLNKASQYVSLLIRDSQPSDSATYLCcustom-character FGDGTQLVVKPNIR                         (CDR3; SEQ ID NO: 27)) V.sub.H SEQ ID NO: 9 SEQ ID NO: 49 V.sub.L SEQ ID NO: 11 SEQ ID NO: 51 CH2CH3 SEQ ID NO: 14 SEQ ID NO: 54 CVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTAPVLDSDGSFRLRSDLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK C.sub.κ SEQ ID NO: 17 SEQ ID NO: 57 CH1 SEQ ID NO: 35 SEQ ID NO: 75 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTSPPCPAPELLGGP CH1CH2CH3 SEQ ID NO: 38 SEQ ID NO: 78 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTSPPCPAPELLGGPCVFLFPPK PKDTLMISRTPEVTCVVVDVSHEDPEVKFNVVYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTAPVLDSDGSFRLRSDLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK V.sub.αV.sub.β-FTab-hb-1 V.sub.α - V.sub.β SEQ ID NO: 1 SEQ ID NO: 41 Linker V.sub.β - V.sub.H SEQ ID NO: 1 SEQ ID NO: 41 Linker V.sub.β SEQ ID NO: 3 SEQ ID NO: 43 V.sub.α SEQ ID NO: 7 SEQ ID NO: 47 V.sub.H SEQ ID NO: 9 SEQ ID NO: 49 V.sub.L SEQ ID NO: 11 SEQ ID NO: 51 CH2CH3 SEQ ID NO: 14 SEQ ID NO: 54 C.sub.κ SEQ ID NO: 17 SEQ ID NO: 57 CH1 SEQ ID NO: 35 SEQ ID NO: 75 CH1CH2CH3 SEQ ID NO: 38 SEQ ID NO: 78 V.sub.αV.sub.β-FTab V.sub.α - V.sub.β SEQ ID NO: 1 SEQ ID NO: 41 Linker V.sub.β - V.sub.H SEQ ID NO: 1 SEQ ID NO: 41 Linker V.sub.β SEQ ID NO: 3 SEQ ID NO: 43 V.sub.α SEQ ID NO: 7 SEQ ID NO: 47 V.sub.H SEQ ID NO: 9 SEQ ID NO: 49 V.sub.L SEQ ID NO: 11 SEQ ID NO: 51 C.sub.κ SEQ ID NO: 17 SEQ ID NO: 57 CH1 SEQ ID NO: 18 SEQ ID NO: 58 V.sub.βV.sub.α-FTab-1 V.sub.β - V.sub.α SEQ ID NO: 1 SEQ ID NO: 41 Linker V.sub.α - V.sub.H SEQ ID NO: 1 SEQ ID NO: 41 Linker V.sub.β SEQ ID NO: 3 SEQ ID NO: 43 V.sub.α SEQ ID NO: 7 SEQ ID NO: 47 V.sub.H SEQ ID NO: 9 SEQ ID NO: 49 V.sub.L SEQ ID NO: 11 SEQ ID NO: 51 C.sub.κ SEQ ID NO: 17 SEQ ID NO: 57 CH1 SEQ ID NO: 18 SEQ ID NO: 58 V.sub.βV.sub.α-FTab-hb-2 V.sub.β - V.sub.α SEQ ID NO: 1 SEQ ID NO: 41 Linker V.sub.α - V.sub.H SEQ ID NO: 1 SEQ ID NO: 41 Linker V.sub.β SEQ ID NO: 4 SEQ ID NO: 44 SQTIHQWPATLVQPVGSPLSLECTVEcustom-character LYWYRQAAGRCLELLFYcustom-character QISSEVPQN                      (CDR1; SEQ ID NO: 20)   (CDR2; SEQ ID NO: 24) LFASRPQDRQFILSSKKLLLSDSGFYLCcustom-character FGPGTRLTVLEDLKN                           (CDR3; SEQ ID NO: 31) V.sub.α SEQ ID NO: 8 SEQ ID NO: 48 QKEVEQNSGPLSVPEGAIASLNCTYScustom-character FFWYRQYSGKSPELIMScustom-character KEDGRETA                       (CDR1; SEQ ID NO: 19)  (CDR2; SEQ ID NO: 23) QLNKASQYVSLLIRDSQPSDSATYLCcustom-character FGCGTQLVVKPNIR                         (CDR3; SEQ ID NO: 27) V.sub.H SEQ ID NO: 9 SEQ ID NO: 49 V.sub.L SEQ ID NO: 11 SEQ ID NO: 51 CH2CH3 SEQ ID NO: 14 SEQ ID NO: 54 C.sub.κ SEQ ID NO: 17 SEQ ID NO: 57 CH1 SEQ ID NO: 35 SEQ ID NO: 75 CH1CH2CH3 SEQ ID NO: 38 SEQ ID NO: 78 V.sub.βV.sub.α-FTab-hb-3 V.sub.β - V.sub.α SEQ ID NO: 1 SEQ ID NO: 41 Linker V.sub.α - V.sub.H SEQ ID NO: 1 SEQ ID NO: 41 Linker V.sub.β SEQ ID NO: 5 SEQ ID NO: 45 V.sub.α SEQ ID NO: 6 SEQ ID NO: 46 V.sub.H SEQ ID NO: 9 SEQ ID NO: 49 V.sub.L SEQ ID NO: 11 SEQ ID NO: 51 CH2CH3 SEQ ID NO: 14 SEQ ID NO: 54 C.sub.κ SEQ ID NO: 17 SEQ ID NO: 57 CH1 SEQ ID NO: 35 SEQ ID NO: 75 CH1CH2CH3 SEQ ID NO: 38 SEQ ID NO: 78 V.sub.βV.sub.α-FTab-hb-4 V.sub.β - V.sub.α SEQ ID NO: 1 SEQ ID NO: 41 Linker V.sub.α - V.sub.H SEQ ID NO: 1 SEQ ID NO: 41 Linker V.sub.β SEQ ID NO: 5 SEQ ID NO: 45 V.sub.α SEQ ID NO: 6 SEQ ID NO: 46 V.sub.H SEQ ID NO: 10 SEQ ID NO: 50 EVQLVESGGGLVQPGGSLRLSCAAScustom-character WVRQAPGKGLEWVAcustom-character TY ORF Start: 1                    (CDR1; SEQ ID NO: 32)   (CDR2; SEQ ID NO: 33) ORF Stop: 366 ADSVKGRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARcustom-character WGQGTLVTV                                      (CDR3; SEQ ID NO: 34) SS V.sub.L SEQ ID NO: 12 SEQ ID NO: 52 DIQMTQSPSSLSASVGDRVTITCcustom-character WYQQKPGKAPKLLIYcustom-character GVPSRFS                     (CDR1; SEQ ID NO: 81)    (CDR2; SEQ ID NO: 82) GSGSGTDYTLTISSLQPEDFATYYCcustom-character FGQGTKVEIKR                        (CDR3; SEQ ID NO: 83) CH2CH3 SEQ ID NO: 14 SEQ ID NO: 54 C.sub.κ SEQ ID NO: 17 SEQ ID NO: 57 CH1 SEQ ID NO: 35 SEQ ID NO: 75 CH1CH2CH3 SEQ ID NO: 38 SEQ ID NO: 78 V.sub.βV.sub.α-FTab-2 V.sub.β - V.sub.α SEQ ID NO: 1 SEQ ID NO: 41 Linker V.sub.α - V.sub.H SEQ ID NO: 1 SEQ ID NO: 41 Linker V.sub.β SEQ ID NO: 5 SEQ ID NO: 45 V.sub.α SEQ ID NO: 6 SEQ ID NO: 46 V.sub.H SEQ ID NO: 9 SEQ ID NO: 49 V.sub.L SEQ ID NO: 11 SEQ ID NO: 51 C.sub.κ SEQ ID NO: 17 SEQ ID NO: 57 CH1 SEQ ID NO: 18 SEQ ID NO: 58 V.sub.βV.sub.α-FTab-3 V.sub.β - V.sub.α SEQ ID NO: 1 SEQ ID NO: 41 Linker V.sub.α - V.sub.H SEQ ID NO: 1 SEQ ID NO: 41 Linker V.sub.β SEQ ID NO: 5 SEQ ID NO: 45 V.sub.α SEQ ID NO: 6 SEQ ID NO: 46 V.sub.H SEQ ID NO: 10 SEQ ID NO: 50 V.sub.L SEQ ID NO: 12 SEQ ID NO: 52 C.sub.κ SEQ ID NO: 17 SEQ ID NO: 57 CH1 SEQ ID NO: 18 SEQ ID NO: 58 V.sub.βV.sub.α-FTab-hb-5 V.sub.β - V.sub.α SEQ ID NO: 1 SEQ ID NO: 41 Linker V.sub.α - V.sub.H SEQ ID NO: 1 SEQ ID NO: 41 Linker V.sub.β SEQ ID NO: 5 SEQ ID NO: 45 V.sub.α SEQ ID NO: 6 SEQ ID NO: 46 V.sub.H SEQ ID NO: 9 SEQ ID NO: 49 V.sub.L SEQ ID NO: 11 SEQ ID NO: 51 CH2CH3 SEQ ID NO: 15 SEQ ID NO: 55 SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFRLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK C.sub.κ SEQ ID NO: 17 SEQ ID NO: 57 CH1 SEQ ID NO: 35 SEQ ID NO: 75 CH1CH2CH3 SEQ ID NO: 39 SEQ ID NO: 79 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTSPPCPAPELLGGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSHEDPEVKFNVVYVDGVEVHNAKTKPREEQYASTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFRLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK

    Example 2: Expression and Purification

    [0694] Plasmid DNA was provided internally, and protein was expressed in HEK293-6E cells using a transient transfection method. 0.5 mg DNA per liter cell culture was transfected into HEK293-6E cells at a density of 1.4×10.sup.6 cells/mL using Polyethylenimine Max (PEI Max, Polysciences Inc) at a PEI:DNA ratio of 4:1 and Light Chain:Heavy Chain DNA ratio of 3:2. HEK293-6E cells were grown in FreeStyle™ 293 medium (Invitrogen) in suspension with 5% CO.sub.2 at 37° C., in 2.8 L shaking flasks (125 RPM). Cells were fed with 0.5% Tryptone N1 one day after transfection. On day 7 post-transfection, the transfected cell cultures were cleared by centrifugation followed by filtration through 0.2 μm PES filter (Corning).

    [0695] Expression of bispecific molecules: All bispecific proteins of Example 1 were expressed in HEK293-6E cells using a transient transfection method. 0.5 mg DNA per liter cell culture was transfected into HEK293-6E cells at a density of 1.4×10.sup.6 cells/mL using Polyethylenimine Max (PEI Max, Polysciences Inc) at a PEI:DNA ratio of 4:1 and Light Chain:Heavy Chain 1:Heavy Chain 2 DNA ratio of 1:1:1. HEK293-6E cells were grown in FreeStyle™ 293 medium (Invitrogen) in suspension with 5% CO.sub.2 at 37° C., in a 10 L Wave bag (28 RPM, 7Angle). Cells were fed with 0.5% Tryptone N1 one day after transfection. On day 7 post-transfection, the transfected cell culture was cleared by centrifugation followed by filtration through 0.45/0.2 μm filter (Sartorius Stedim).

    [0696] Purification of V.sub.βV.sub.α-FTab-hb-1, V.sub.βV.sub.α-FTab-hb-2, V.sub.βV.sub.α-FTab-hb-3, V.sub.βV.sub.α-FTab-hb-4, V.sub.βV.sub.α-FTab-hb-5, V.sub.βV.sub.α-FTab-KiH and V.sub.βV.sub.α-FTab-KiH-2: Cleared medium was loaded on a MabSelect SuRe™ column (GE Healthcare) equilibrated with PBS, pH 7.4. The column was washed with PBS, pH 7.4 and bound protein was eluted with 0.1M acetic acid pH 2.7, 0.15M NaCl. Fractions were neutralized with 1M Tris pH 9.0 at a ratio of 1:10. Neutralized protein was further purified by SEC on a Superdex 200 column (GE Healthcare) equilibrated and run with PBS, pH 7.4. Fractions containing protein were pooled, concentration was measured by absorbance at 280 nm, and samples were analyzed by SEC, SDS-PAGE, and mass spectrometry. The final material was stored in aliquots at −80° C.

    [0697] Purification of V.sub.βV.sub.α-FTab-1, V.sub.βV.sub.α-FTab-2, V.sub.βV.sub.α-FTab-3 and V.sub.αV.sub.β-FTab: Cleared medium was buffer exchanged to PBS, pH 7.4 using a Kvick™ TFF system equipped with 10 kDa membranes (GE Healthcare) and loaded on a HisTrap™ FF column (GE Healthcare) equilibrated with PBS, pH 7.4. The column was washed with 25 mm imidazole in PBS, pH 7.4 and bound protein was eluted with 250 mM imidazole in PBS, pH 7.4. Eluted protein was further purified by SEC on a Superdex® 200 column (GE Healthcare), equilibrated, and run with PBS, pH 7.4. Fractions containing anti-CD3-Fab were pooled, concentration was measured by absorbance at 280 nm, and samples were analyzed by SEC, SDS-PAGE, and mass spectrometry. Final material was stored in aliquots at −80° C.

    Example 3: Assays and Characterization

    [0698] The bispecific molecules with a Fc region that is either a dimeric knob-in-hole (e.g., V.sub.βV.sub.α-FTab KiH′ which was also designated as V.sub.βV.sub.α-FTab KiH-2) or a halfbody (e.g., V.sub.βV.sub.α-FTab-hb-1) exhibited improved pharmacokinetics and serum stability properties while maintaining potency and specificity through monovalent binding to both SURV/HLA-A2 and CD3. As shown in FIG. 9, in a single dose comparison study in the HCT-116 CRC ES model, it was unexpectedly discovered that at molar equivalent doses, the bispecific molecule containing KiH (V.sub.βV.sub.α-FTab-KiH-2) exhibited greater anti-tumor efficacy than V.sub.βV.sub.α-FTab-hb-5. V.sub.βV.sub.α-FTab-KiH-2 is almost identical to V.sub.βV.sub.α-FTab-KiH except for one amino acid substitution to mitigate deamidation, which is not expected to have any impact on potency.

    Example 4: Target Cell Labeling for AML Cell Line Functional Assays

    [0699] Cell lines OCI-AML2 (ACC-99), OCI-AML3 (ACC-582), and OCI-Ly19 (ACC-528) were purchased from DSMZ and were cultured in α-MEM supplemented with 20% FBS and incubated at 37° C. and 5% CO.sub.2. OCI-M1 (ACC-529) was also purchased from DSMZ and cultured in IMDM supplemented with 10% FBS and incubated at 37° C. and 5% CO.sub.2. Cells were stained with CellVue™ Burgundy (Invitrogen) prior to co-culture with T cells. Target cells were pelleted and washed with PBS once. Cells were resuspended in Diluent C per manufacturer instructions and incubated with a final concentration of 2 μM Burgundy CellVue™ dye for 5 minutes at room temperature in the dark. The reaction was stopped by adding equal volume of FBS (Sigma). Samples were washed 3 times with cell-line specific complete medium. Cells were counted and checked for efficiency of labeling by FACS, prior to seeding into functional assays. The APC-Cy7 channel was used to detect CellVue™ Burgundy signal.

    Example 5: Effector T Cell Labeling for Functional Assays

    [0700] Effector T cells were isolated from donor PBMC stocks by negative selection using a T cell isolation kit (Miltenyi) on LS columns (Miltenyi). MACS™ buffer (PBS supplemented with 0.1% BSA and 2 mM EDTA) was used for isolation of CD3+ T cells. Isolated CD3+ T cells were cultured in AIM V™ media supplemented with 5% AB serum and incubated at 37° C. and 5% CO.sub.2 overnight. The following day, cells were counted and labeled with CellTrace™ Violet (Invitrogen). Effector cells were pelleted and washed once with PBS. Effector cells were aliquoted 10′ per 50 mL tube in 10 mL PBS. CellTrace™ stock solution was prepared immediately prior to use by adding the 20 μL volume of DMSO (Component B) to one vial of CellTrace™ reagent (Component A) and mixing well. Ten microliters of CellTrace™ reagent was added to each 50 mL tube containing effector cells. Effector cells were stained for 20 minutes at 37° C. and 5% CO.sub.2 and shaken sporadically to ensure efficient staining. To stop the reaction, 40 mL of AIM V™ supplemented with 10% FBS was added to each 50 mL tube. Reaction blocking took 5 minutes at room temperature in the dark; cells were pelleted and resuspended with AIM V™ media supplemented with 5% AB serum. Cells were counted and checked for efficiency of labeling by FACS, prior to seeding into functional assays. The Pacific Blue channel was used to detect CellTrace™ Violet signal.

    Example 6: Redirected T Cell Cytotoxicity and Activation Assays

    [0701] CellVue™ Burgundy-labeled target cells were seeded at 20,000 cells per well into a round bottom 96-well plate (BD) in 50 μl volume per well. CellTrace™ Violet-labeled effector T cells were added to appropriate wells (in duplicate) at 200,000 cells per well in 50 μl volume, for approximate Effector T-cell/Target ratio (E:T) of 10:1. Serially diluted Survivin TCR/CD3 bispecific molecule was added to appropriate wells in a 50 μl volume, starting at 6 nM per well and titrated in a 3-fold dilution across 9 wells (in duplicate). The mixed cultures were placed at 37° C. and 5% CO.sub.2 for 48 hours. Target cytotoxicity and T cell activation parameters were found to be optimal at 48 hours. At the time of the harvest, the culture supernatant was collected for cytokine release analysis while the cells were pelleted and stained with FACS antibodies to detect target cytotoxicity, T cell activation, and T cell proliferation. Briefly, the 96-well plates containing samples were palleted and washed twice with FACS buffer (PBS supplemented with 0.5% BSA and 2 mM EDTA). Antibodies against T cell activation markers CD25-PE (Biolegend), CD69-APC (Biolegend), and CD3-PE-Cy7 (Biolegend) were mixed at 7.5 μl/ml FACS buffer and 25 μl were added per well. Samples were allowed to incubate for 25 minutes at 4° C. in the dark. Samples were washed twice with FACS buffer. Viability dyes Annexin-FITC (Biolegend) and 7AAD (Biolegend) were mixed in Annexin V binding buffer (Biolegend) at 7.5 μl/ml and 15 μl/ml, respectively, and added to wells at 25 μl/well for 15 minutes at room temperature in the dark. At the end of the incubation, 75 μl of Annexin V binding buffer was added to each well. Data was acquired on FACSCanto II™ and analyzed using FlowJo™ V10 analysis software. The dose-response data for target cytotoxicity, T cell activation and T cell proliferation were fitted to a sigmoidal curve using nonlinear regression, and the EC.sub.50 values calculated with the aid of GraphPad 5.0 Software.

    [0702] As shown in FIG. 2, V.sub.βV.sub.α-FTab-KiH was evaluated for its ability to redirect killing by CD3+ T cells against the HLA-A2, Survivin-positive AML cell line OCI-AML2. V.sub.βV.sub.α-FTab-KiH induced potent killing of OCI-AML2 across 4 healthy CD3+ T cell donors, while no activity was observed with a negative control (irrelevant TCR/CD3 bispecific) (Neg Ctrl).

    [0703] As shown in FIG. 3, V.sub.βV.sub.α-FTab-KiH was evaluated for its ability to redirect killing by CD3+ T cells against the HLA-A2, Survivin-positive AML cell line OCI-AML3. V.sub.βV.sub.α-FTab-KiH induced potent killing of OCI-AML3 across 4 healthy CD3+ T cell donors, while no activity was observed with an irrelevant TCR/CD3 bispecific (Neg Ctrl).

    [0704] As shown in FIG. 4, V.sub.βV.sub.α-FTab-KiH was evaluated for its ability to redirect killing by CD3+ T cells against the HLA-A2 negative, Survivin-positive AML cell line OCI-Ly19. V.sub.βV.sub.α-FTab-KiH did not induce killing of OCI-Ly19, due to the lack of HLA-A2 expression by this cell line.

    [0705] As shown in FIG. 5, V.sub.βV.sub.α-FTab-KiH was evaluated for its ability to activate CD3+ T cells against the HLA-A2, Survivin-positive AML cell line OCI-AML2, as measured by CD69 expression. V.sub.βV.sub.α-FTab-KiH induced potent activation of CD3+ T cells across 4 healthy CD3+ T cell donors, against OCI-AML2, while no activity was observed with an irrelevant TCR/CD3 bispecific (Neg Ctrl).

    [0706] As shown in FIG. 6, V.sub.βV.sub.α-FTab-KiH was evaluated for its ability to activate CD3+ T cells against the HLA-A2, Survivin-positive AML cell line OCI-AML3, as measured by CD69 expression. V.sub.βV.sub.α-FTab-KiH induced potent activation of CD3+ T cells across 4 healthy CD3+ T cell donors, against OCI-AML3, while no activity was observed with an irrelevant TCR/CD3 bispecific (Neg Ctrl).

    [0707] As shown in FIG. 7, V.sub.βV.sub.α-FTab-KiH was evaluated for its ability to activate CD3+ T cells against the HLA-A2 negative, Survivin-positive AML cell line OCI-Ly19, as measured by CD69 expression. V.sub.βV.sub.α-FTab-KiH induced minimal activation of CD3+ T cells across 4 healthy CD3+ T cell donors, against OCI-Ly19, due to the lack of HLA-A2 expression by this cell line.

    [0708] As shown in FIG. 12, V.sub.βV.sub.α-FTab-KiH was evaluated for its ability to induce T cell proliferation at varying effector to target ratios. V.sub.βV.sub.α-FTab-KiH induced T cell proliferation at varying effector to target ratios.

    Example 7: Pharmacokinetic Characterization of Survivin TCR/CD3 Bispecific Molecules

    [0709] The pharmacokinetic profiles of Survivin/CD3 bispecific molecules were compared in non-tumor bearing SCID mice using a single 16 milligrams/kilogram (mpk) IV bolus dose. Whole blood samples were collected for both early and later time points (until 168 hours) for V.sub.βV.sub.α-FTab-KiH. Other molecules were analyzed for up to 48 hours. Analyte concentration was determined by a Meso Scale Discovery (MSD)-based assay with goat anti-human IgG-Fc as the capture reagent and goat anti-sulfate as the detection reagent. The half-life (t.sub.1/2), area under the curve (AUC), clearance (CL) and steady state volume (Vss) values for all test molecules are summarized in Table 2. Results indicated that V.sub.βV.sub.α-FTab-KiH exhibited antibody-like pharmacokinetics with a surprisingly longer half-life (˜5 days) and higher exposure as compared to other molecules tested.

    TABLE-US-00003 TABLE 2 Pharmacokinetic properties of bispecifics in SCID mice t.sub.1/2 AUC.sub.0-∞ CL Vss Molecules (hr) (mg*hr/mL) (mL/h/kg) (mL/kg) V.sub.βV.sub.α-FTab-hb-1 24 0.75 22 350 V.sub.βV.sub.α-FTab-hb-2 14 1.1 15 170 V.sub.βV.sub.α-FTab-hb-5 6.4 0.77 21 140 V.sub.βV.sub.α-FTab-KiH 117.6 0.18 2.76 359

    [0710] In addition, serum samples were analyzed for V.sub.βV.sub.α-FTab-KiH concentrations in a total anti-human MSD (Meso Scale Discovery) assay with electrochemiluminescent detection (FIG. 8). In the assay, total antibody was analyzed by employing a Bio anti-id capture reagent and a Sulfo anti-id mAb detection reagent. The linear range of the assay was 0.069-50 μg/mL, with a lower limit of quantitation (LLOQ) of 0.069 μg/mL. The serum concentrations at the first sampling time point (C.sub.0.5h) were read directly from the concentration data for each monkey. Toxicokinetic parameters were calculated using Pharmacokinetics Laboratory Automation Software for Management and Analysis (PLASMA) Version 2.6.12 (SPaRCS, AbbVie) by non-compartmental analysis and the linear trapezoidal method.

    Example 8: Disulfide Bond Structure of Survivin TCR/CD3 Bispecific Molecules

    [0711] V.sub.βV.sub.α-FTab-KiH consists of one heavy chain subunit paired with one kappa light chain subunit and one Fc chain subunit, through disulfide bridges (FIG. 10). The heavy chain (i.e., Heavy Chain 1 of SEQ ID NO: 36) contains seven intrachain disulfide bridges between cysteines in positions 23 and 91, 43 and 235, 158 and 224, 288 and 362, 413 and 469, 530 and 590, and finally in positions 636 and 694. Among these intrachain disulfides, the disulfide bridge between cysteines 43 and 235 is an interdomain link connecting the V.sub.α and V.sub.β domain, whereas the rest are intradomain disulfide links. The light chain (i.e., Light Chain of SEQ ID NO: 76) contains two intrachain disulfide bridges; the first disulfide bridge is between cysteines in positions 23 and 87, and the second is between cysteines in positions 133 and 193. The Fc chain (i.e., Heavy Chain 2 of SEQ ID NO: 16) contains two intrachain disulfide bridges; the first disulfide bridge is between cysteines in positions 41 and 101, and the second is between cysteines in positions 147 and 205. In each molecule, the heavy chain is linked to the light chain by an interchain disulfide bridge between the cysteine in position 489 of the heavy chain and the cysteine in position 213 of the light chain. Each heavy chain is also paired with an Fc chain by two interchain disulfide bridges, one bridge between the cysteine in position 495 of the heavy chain and the cysteine in position 6 of the Fc chain, the other bridge between the cysteine in position 498 of the heavy chain and the cysteine in position 9 of the Fc chain.

    Example 9: Binding Specificity and Affinity Characterization of Survivin TCR/CD3 Bispecific Molecules

    [0712] TCR specificity screen was carried out for V.sub.βV.sub.α-FTab-KiH (FIG. 11). T2 cells were seeded at 50,000 cells per well in a 96-well plate (Falcon #353077) in a volume of 50 μL AIM-V/5% hAB (Gibco #12055-091/Sigma #H4522) per well and the parental Survivin peptide, 43 homologous peptides, and 2 control peptides were added in 50 uL AIM-V/5% hAB to a final concentration of 20 μM with T2 and pre-incubated for 3-4 hours. 100,000 CD3+ cells were seeded in each well in a volume of 50 μL AIM-V/5% hAB per well. V.sub.βV.sub.α-FTab-KiH was diluted in AIM-V/5% hAB so that the final concentration in the co-culture was 1 nM in duplicate for each donor. Plates were incubated for 19 hours at 37° C. 5% CO.sub.2. Supernatants were removed from each plate, transferred to a fresh 96-well plate and frozen at −80° C. until ready to assay for Interferon-γ secretion via ELISA. The TCR part of V.sub.βV.sub.α-FTab-KiH exhibited remarkable high specificity directed towards a Survivin-derived peptide (FIG. 11).

    [0713] The Survivin/CD3 Bispecific Binding Kinetics to Survivin peptide/MHC for all test molecules are summarized in Table 3.

    TABLE-US-00004 TABLE 3 Survivin/CD3 Bispecific Binding Kinetics to Survivin peptide/MHC Bispecific ka (1/Ms) kd (1/s) t½ (s) K.sub.D (M) V.sub.βV.sub.α-FTab-KiH 1.8E+05 2.5E−04 2758 1.4E−09 V.sub.βV.sub.α-FTab-KiH-2 1.4E+05 2.8E−04 2506 2.0E−09

    [0714] V.sub.βV.sub.α-FTab-KiH also has a high affinity to human CD3. The anti-CD3 part was described in Cole M S et al. (1999) Transplantation 68:563-571, the content of which is incorporated by reference herein in its entirety.

    [0715] All publications, patents, patent applications and other documents cited in this application are hereby incorporated by reference in their entireties for all purposes to the same extent as if each individual publication, patent, patent application or other document were individually indicated to be incorporated by reference for all purposes.

    [0716] While various specific embodiments have been illustrated and described, it will be appreciated that various changes can be made without departing from the spirit and scope of the invention(s).