WO2021163364A1

WO2021163364A1 - Trispecific binding molecules

Info

Publication number: WO2021163364A1
Application number: PCT/US2021/017708
Authority: WO
Inventors: Adam S. CHERVIN; Feng Dong; Edward B. Reilly; Jennifer D. Stone; Michael K. WHITE; Holger Wesche; Rick AUSTIN; Bryan LEMON
Original assignee: Abbvie Biotechnology Ltd.; Abbvie Inc.
Priority date: 2020-02-12
Filing date: 2021-02-11
Publication date: 2021-08-19
Also published as: TW202146444A

Abstract

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

Description

TRISPECIFIC BINDING MOLECULES

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Application No. 62/975,396, filed February 12, 2020, and U.S. Provisional Application NO. 62/976,138, filed February 13, 2020, the content of each of which is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

[0002] The present application pertains to, among other things, novel trispecific molecules that bind to both human Survivin and human CD3, compositions comprising the trispecific molecules, nucleic acids encoding the trispecific polypeptides, and methods of making and using the same.

SEQUENCE LISTING

[0003] Incorporated herein by reference in its entirety is a Sequence Listing entitled, “AVR-54125_ST25.txt”, comprising SEQ ID NO: 1 through SEQ ID NO: 51, which includes the amino acid sequences disclosed herein. The Sequence Listing has been submitted herewith in ASCII text format via EFS. The Sequence Listing was first created on February 11, 2021 and is 111,326 bytes in size.

BACKGROUND OF THE INVENTION

[0004] Cancer therapies comprise a wide range of therapeutic approaches including surgery, radiation, and chemotherapy. Many existing therapeutics suffer from disadvantages, such as a lack of selectivity of targeting cancer cells over healthy cells, and the development of resistance by the cancer to the treatment. [0005] Recent approaches based on targeted therapeutics, which preferentially affect cancer cells over normal cells, have led to chemotherapeutic regimens with fewer side effects as compared to non-targeted therapies such as radiation treatment.

[0006] Cancer immunotherapy, including agents that empower patient T-cells to kill cancer cells, has emerged as a promising therapeutic approach. T cell receptors, unlike antibodies, have evolved to recognize intracellular proteins processed as small peptides that are complexed to and presented by a major histocompatibility complex (MHC) molecule, also known as human leukocyte antigen (HLA) complex, on the cell surface.

[0007] Soluble T cell receptors (sTCRs) represent a novel class of therapeutics with the potential to target tumor-selective antigens in both hematological and solid tumors which are not currently accessible using traditional antibody-based therapeutics. However, several challenges have hindered the development of therapeutic sTCRs, including difficulty in expressing soluble, stable, and high affinity TCRs. Survivin is an attractive intracellular target overexpressed in multiple solid and hematological cancers, potentially accessible by sTCRs. Therefore, there is a need to develop a new sTCR-based immunotherapeutic approach for targeting Survivin.

BRIEF SUMMARY OF THE INVENTION

[0008] Described herein are trispecific molecules that bind to human Survivin and human CD3.

[0009] One aspect of the invention pertains to a trispecific molecule that binds to human Survivin, CD3 and albumin, wherein the trispecific molecule comprises: a) a first domain (T) comprising a single-chain T cell receptor (sTCR) comprising: i. a variable beta region (V_p) comprising SEQ ID NO: 37 (CDR1), SEQ ID NO: 38 (CDR2) and SEQ ID NO: 39 (CDR3), and ii. a variable alpha region (V_a) comprising SEQ ID NO: 34 (CDR1), SEQ ID NO: 35 (CDR2) and SEQ ID NO: 36 (CDR3), wherein the V_P and V_a regions of the sTCR are connected via a first peptide linker (LI); b) a second domain (A) comprising a VHH domain comprising SEQ ID NO: 46 (CDR1), SEQ ID NO: 47 (CDR2) and SEQ ID NO: 48 (CDR3); and c) a third domain (C) comprising a single chain variable fragment (scFv) comprising i. a heavy chain variable region (V_H) comprising SEQ ID NO: 40 (CDR1), SEQ ID NO: 41 (CDR2) and SEQ ID NO: 42 (CDR3), and ii. a light chain variable region (V_L) comprising SEQ ID NO: 43 (CDR1), SEQ ID NO: 44 (CDR2) and SEQ ID NO: 45 (CDR3), wherein V_H and VL are connected via a second peptide linker (L2), wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-V_HH-L3-V_H-L2-V_L-L4-V_P-L1- V_a-COOH.

[0010] Another aspect of the invention pertains to a trispecific molecule that binds to human Survivin, CD3 and albumin, wherein the trispecific molecule comprises: a) a first domain (T) comprising a single-chain T cell receptor (sTCR) comprising: i. a variable beta region (V_p) comprising the amino acid sequence of SEQ ID NO: 6, and ii. a variable alpha region (V_a) comprising the amino acid sequence of SEQ ID NO: 8, wherein the V_p and V_a regions of the sTCR are connected via a first peptide linker (LI); b) a second domain (A) comprising a V_HH domain comprising the amino acid sequence of SEQ ID NO: 12; and c) a third domain (C) comprising a single chain variable fragment (scFv) comprising i. a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 10, and ii. a light chain variable region (V_L) comprising the amino acid sequence of SEQ ID NO: 11, wherein V_H and V_L are connected via a second peptide linker (L2), wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-V_HH-L3-V_H-L2-V_I -L4-V_|',-L 1- V_a-COOH.

[0011] One aspect of the invention pertains to a trispecific molecule that binds to human Survivin, CD3 and albumin, wherein the trispecific molecule comprises: a) a first domain (T) comprising a single-chain T cell receptor (sTCR) comprising: i. a variable beta region (V_p) comprising SEQ ID NO: 37 (CDR1), SEQ ID NO: 38 (CDR2) and SEQ ID NO: 39 (CDR3), and ii. a variable alpha region (V_a) comprising SEQ ID NO: 34 (CDR1), SEQ ID NO: 35 (CDR2) and SEQ ID NO: 36 (CDR3), wherein the V_P and V_a regions of the sTCR are connected via a first peptide linker (LI); b) a second domain (A) comprising a VHH domain comprising SEQ ID NO: 46 (CDR1), SEQ ID NO: 47 (CDR2) and SEQ ID NO: 48 (CDR3); and c) a third domain (C) comprising a single chain variable fragment (scFv) comprising: i. a heavy chain variable region (V_H) comprising SEQ ID NO: 40 (CDR1), SEQ ID NO: 41 (CDR2) and SEQ ID NO: 42 (CDR3), and ii. a light chain variable region (V_L) comprising SEQ ID NO: 43 (CDR1), SEQ ID NO: 44 (CDR2) and SEQ ID NO: 45 (CDR3), wherein V_H and V_L are connected via a second peptide linker (L2), wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-V_HH-L3-V_H-L2-V_L-L4-V_P-L1- V_a-COOH, wherein the LI comprises the amino acid sequence of SEQ ID NO: 1.

[0012] Another aspect of the invention pertains to a trispecific molecule that binds to human Survivin, CD3 and albumin, wherein the trispecific molecule comprises: a) a first domain (T) comprising a single-chain T cell receptor (sTCR) comprising: i. a variable beta region (V_p) comprising the amino acid sequence of SEQ ID NO: 6, and ii. a variable alpha region (V_a) comprising the amino acid sequence of SEQ ID NO: 8, wherein the V p, and V_a regions of the sTCR are connected via a first peptide linker (LI); b) a second domain (A) comprising a VHH domain comprising the amino acid sequence of SEQ ID NO: 12; and c) a third domain (C) comprising a single chain variable fragment (scFv) comprising: i. a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 10, and ii. a light chain variable region (V_L) comprising the amino acid sequence of SEQ ID NO: 11, wherein V_H and V_L are connected via a second peptide linker (L2), wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-V_HH-L3-V_H-L2-V_L-L4- V -Ll-V_a-COOH, wherein the LI comprises the amino acid sequence of SEQ ID NO: 1.

[0013] One aspect of the invention pertains to a trispecific molecule that binds to human Survivin, CD3 and albumin, wherein the trispecific molecule comprises: a) a first domain (T) comprising a single-chain T cell receptor (sTCR) comprising: i. a variable beta region (V_p) comprising SEQ ID NO: 37 (CDR1), SEQ ID NO: 38 (CDR2) and SEQ ID NO: 39 (CDR3), and ii. a variable alpha region (V_a) comprising SEQ ID NO: 34 (CDR1), SEQ ID NO: 35 (CDR2) and SEQ ID NO: 36 (CDR3), wherein the V_p and V_a regions of the sTCR are connected via a first peptide linker (LI); b) a second domain (A) comprising a VHH domain which binds to albumin, wherein the VHH domain comprises SEQ ID NO: 46 (CDR1), SEQ ID NO: 47 (CDR2) and SEQ ID NO:

48 (CDR3); and c) a third domain (C) comprising a single chain variable fragment (scFv) comprising i. a heavy chain variable region (V_H) comprising SEQ ID NO: 40 (CDR1), SEQ ID NO: 41 (CDR2) and SEQ ID NO: 42 (CDR3), and ii. a light chain variable region (V_L) comprising SEQ ID NO: 43 (CDR1), SEQ ID NO: 44 (CDR2) and SEQ ID NO: 45 (CDR3), wherein V_H and V_L are connected via a second peptide linker (L2), wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-V_HH-L3-V_H-L2-V_L-L4-V_|',-L 1- V_a-COOH, wherein the L2 comprises the amino acid sequence of SEQ ID NO: 3.

[0014] Another aspect of the invention pertains to a trispecific molecule that binds to human Survivin, CD3 and albumin, wherein the trispecific molecule comprises: a) a first domain (T) comprising a single-chain T cell receptor (sTCR) comprising: i. a sTCR variable beta region ( V _[,) comprising the amino acid sequence of SEQ ID NO: 6, and ii. a sTCR variable alpha region (V_a) comprising the amino acid sequence of SEQ ID NO: 8, wherein the V _f. and V_a regions of the sTCR are connected via a first peptide linker (LI); b) a second domain (A) comprising a V_HH domain comprising the amino acid sequence of SEQ ID NO: 12; and c) a third domain (C) comprising a single chain variable fragment (scFv) comprising i. a heavy chain variable region (V_H) comprising the amino acid sequence of SEQ ID NO: 10, and ii. a light chain variable region (V_L) comprising the amino acid sequence of SEQ ID NO: 11, wherein V_H and V_L are connected via a second peptide linker (L2), wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-V_HH-L3-V_H-L2-V_L-L4-V_|',-L 1- V_a-COOH, wherein the L2 comprises the amino acid sequence of SEQ ID NO: 3.

[0015] One aspect of the invention pertains to a trispecific molecule that binds to human Survivin, CD3 and albumin, wherein the trispecific molecule comprises: a) a first domain (T) comprising a single-chain T cell receptor (sTCR) comprising: i. a variable beta region (V ) comprising SEQ ID NO: 37 (CDR1), SEQ ID NO: 38 (CDR2) and SEQ ID NO: 39 (CDR3), and ii. a variable alpha region (V_a) comprising SEQ ID NO: 34 (CDR1), SEQ ID NO: 35 (CDR2) and SEQ ID NO: 36 (CDR3), wherein the V_p and V_a regions of the sTCR are connected via a first peptide linker (LI); b) a second domain (A) comprising a VHH domain comprising SEQ ID NO: 46 (CDR1), SEQ ID NO: 47 (CDR2) and SEQ ID NO: 48 (CDR3); and c) a third domain (C) comprising a single chain variable fragment (scFv) comprising i. a heavy chain variable region (VH) comprising SEQ ID NO: 40 (CDR1), SEQ ID NO: 41 (CDR2) and SEQ ID NO: 42 (CDR3), and ii. a light chain variable region (V_L) comprising SEQ ID NO: 43 (CDR1), SEQ ID NO: 44 (CDR2) and SEQ ID NO: 45 (CDR3), wherein V_H and V_L are connected via a second peptide linker (L2), wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-V_HH-L3-V_H-L2-V_L-L4-V_[;-L 1- V_a-COOH, wherein the L3 comprises the amino acid sequence of SEQ ID NO: 2.

[0016] Another aspect of the invention pertains to a trispecific molecule that binds to human Survivin, CD3 and albumin, wherein the trispecific molecule comprises: a) a first domain (T) comprising a single-chain T cell receptor (sTCR) comprising: i. a variable beta region (V_p) comprising the amino acid sequence of SEQ ID NO: 6, and ii. a variable alpha region (V_a) comprising the amino acid sequence of SEQ ID NO: 8, wherein the V_p and V_a regions of the sTCR are connected via a first peptide linker (LI); b) a second domain (A) comprising a VHH domain comprising the amino acid sequence of SEQ ID NO: 12; and c) a third domain (C) comprising a single chain variable fragment (scFv) comprising: i. a heavy chain variable region (V_H) comprising the amino acid sequence of SEQ ID NO: 10, and ii. a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 11, wherein VH and VL are connected via a second peptide linker (L2), wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-V_HH-L3-V_H-L2-V_I -L4-V_|',-L 1

V_a-COOH, wherein the L3 comprises the amino acid sequence of SEQ ID NO: 2.

[0017] One aspect of the invention pertains to a trispecific molecule that binds to human Survivin, CD3 and albumin, wherein the trispecific molecule comprises: a) a first domain (T) comprising a single-chain T cell receptor (sTCR) comprising: i. a variable beta region (V_p) comprising SEQ ID NO: 37 (CDR1), SEQ ID NO: 38 (CDR2) and SEQ ID NO: 39 (CDR3), and ii. a variable alpha region (V_a) comprising SEQ ID NO: 34 (CDR1), SEQ ID NO: 35 (CDR2) and SEQ ID NO: 36 (CDR3), wherein the V_p and V_a regions of the sTCR are connected via a first peptide linker (LI); b) a second domain (A) comprising a VHH domain comprising SEQ ID NO: 46 (CDR1), SEQ ID NO: 47 (CDR2) and SEQ ID NO: 48 (CDR3); and c) a third domain (C) comprising a single chain variable fragment (scFv) comprising i. a heavy chain variable region (V_H) comprising SEQ ID NO: 40 (CDR1), SEQ ID NO: 41 (CDR2) and SEQ ID NO: 42 (CDR3), and ii. a light chain variable region (V_L) comprising SEQ ID NO: 43 (CDR1), SEQ ID NO: 44 (CDR2) and SEQ ID NO: 45 (CDR3), wherein V_H and V_L are connected via a second peptide linker (L2), wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-V_HH-L3-V_H-L2-V_L-L4-V_[;-L 1- V_a-COOH, wherein the L4 comprises the amino acid sequence of SEQ ID NO: 2.

[0018] Another aspect of the invention pertains to a trispecific molecule that binds to human Survivin, CD3 and albumin, wherein the trispecific molecule comprises: a) a first domain (T) comprising a single-chain T cell receptor (sTCR) comprising: i. a variable beta region (V_p) comprising the amino acid sequence of SEQ ID NO: 6, and ii. a variable alpha region (V_a) comprising the amino acid sequence of SEQ ID NO: 8, wherein the V_p and V_a regions of the sTCR are connected via a first peptide linker (LI); b) a second domain (A) comprising a V_HH domain comprising the amino acid sequence of SEQ ID NO: 12; and c) a third domain (C) comprising a single chain variable fragment (scFv) comprising i. a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 10, and ii. a light chain variable region (V_L) comprising the amino acid sequence of SEQ ID NO: 11, wherein V_H and V_L are connected via a second peptide linker (L2), wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-V_HH-L3-V_H-L2-V_I -L4-V_|',-L 1- V_a-COOH, wherein the L4 comprises the amino acid sequence of SEQ ID NO: 2.

[0019] Another aspect of the invention pertains to a trispecific molecule that binds to human Survivin, CD3 and albumin, wherein the trispecific molecule comprises: a) a first domain (T) comprising a single-chain T cell receptor (sTCR) comprising the amino acid sequence of SEQ ID NO: 15; b) a second domain (A) comprising a V_HH domain comprising the amino acid sequence of SEQ ID NO: 12; and c) a third domain (C) comprising a single chain variable fragment (scFv) comprising the amino acid sequence of SEQ ID NO: 17, wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-(A)-L3-(C)-L4-(T)-COOH.

[0020] Another aspect of the invention pertains to a trispecific molecule that binds to human Survivin, CD3 and albumin, wherein the trispecific molecule comprises: a) a first domain (T) comprising a single-chain T cell receptor (sTCR) comprising the amino acid sequence of SEQ ID NO: 15; b) a second domain (A) comprising a V_HH domain comprising the amino acid sequence of SEQ ID NO: 12; and c) a third domain (C) comprising a single chain variable fragment (scFv) comprising the amino acid sequence of SEQ ID NO: 17, wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-(A)-L3-(C)-L4-(T)-COOH, wherein the L3 comprises the amino acid sequence of SEQ ID NO: 4.

[0021] Another aspect of the invention pertains to a trispecific molecule that binds to human Survivin, CD3 and albumin, wherein the trispecific molecule comprises: a) a first domain (T) comprising a single-chain T cell receptor (sTCR) comprising the amino acid sequence of SEQ ID NO: 15; b) a second domain (A) comprising a V_HH domain comprising the amino acid sequence of SEQ ID NO: 12; and c) a third domain (C) comprising a single chain variable fragment (scFv) comprising the amino acid sequence of SEQ ID NO: 17, wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-(A)-L3-(C)-L4-(T)-COOH, wherein the L4 comprises the amino acid sequence of SEQ ID NO: 4.

[0022] Another aspect of the invention pertains to a trispecific molecule that binds to human Survivin, CD3 and albumin, wherein the trispecific molecule comprises the amino acid sequence of SEQ ID NO:

30.

BRIEF DESCRIPTION OF THE FIGURES

[0023] FIGURES 1A-1F show illustrations of the trispecific binding proteins of the present invention. [0024] FIGURES 2A-2H shows directed killing of HCT116 cells by T cells from two different donors with SURVIVIN-targeting TriTACs.

[0025] FIGURES 3A-3H shows directed killing of T2 cells by T cells from two different donors with SURVIVIN-targeting TriTACs.

[0026] FIGURES 4A-4D shows directed killing of R03-a2/b2M cells by T cells with SURVIVIN- targeting TriTACs.

[0027] FIGURE 5 shows SDS-PAGE of purified SURVIVIN-targeting TriTAC proteins. The sizes of the molecular weight standards are indicated by the lines and numbers on the right side of the gel.

[0028] FIGURE 6 shows TDCC activity of purified SURVIVIN-targeting TriTAC proteins with PC3- a2/b2M, T2, and HCT116 cells.

[0029] FIGURE 7, top row, shows directed killing by CD3 T cells against HLA-A2, Survivin-positive AML cell lines (OCI-AML2 and OCI-AML3) but not against the HLA-A2 negative, Survivin-positive AML cell line OCI-Lyl9. The bottom row shows the activation of CD3⁺ T cells against HLA-A2, Survivin-positive AML cell lines (OCI-AML2 and OCI-AML3) but not against the HLA-A2 negative, Survivin-positive AML cell line OCI-Lyl9.

[0030] FIGURE 8 shows tumor growth control induced by aALB: :aCD3 : : la5EP-wt and aALB::aCD3::la5EP-wt-l in the presence of T cells. Each point of the graph represents the mean of 10 tumors. Error bars depict the standard error of the mean.

DETAILED DESCRIPTION OF THE INVENTION [0031] Described herein are novel trispecific molecules comprising an anti-CD3 binding domain, an albumin binding domain, and a soluble single chain T cell receptor having a high binding affinity to Survivin. These trispecific molecules exhibit several unexpected properties, including, for example, remarkable high specificity directed towards a Survivin-derived peptide complexed to HLA-A2, and potent induction of T cell activation and proliferation.

[0032] Abbreviations

[0033] The trispecific 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’ 3’ orientation. For polypeptide sequences, the conventional three or one-letter abbreviations for the genetically encoded amino acids are used.

[0034] Embodiments

[0035] Described herein are trispecific molecules that are capable of binding to Survivin as well as CD3 and have a half- life extension domain, such as a domain binding to human albumin (ALB) . [0036] The term “human CD3” as used herein relates to human cluster of differentiation 3 protein (CD3) described under UniProt P07766 (CD3E-HUMAN).

[0037] The term “human Survivin” or “Survivin” as used herein relates to an inhibitor of apoptosis protein (IAP) described under UniProt 015392 (BIRC5 Human) which is a tumor-associated antigen that is expressed in human cancer cells.

[0038] “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.

[0039] In one aspect, the trispecific molecules of the present invention comprise (1) a Survivin binding part, (2) a CD3 binding part and (3) a half-life extension part.

[0040] Survivin Binding Part [0041] In one aspect, the trispecific molecules of the present invention comprise a Survivin binding part. [0042] In one embodiment, the Survivin binding part of the trispecific molecules of the present invention comprises a single-chain soluble T cell receptor (sTCR).

[0043] 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).

[0044] In one embodiment, the sTCR of the present invention is a modified TCR comprising a variable alpha region (V_a) and a variable beta region (Vp) derived from a wild type T cell receptor, wherein the V_a, the V_p, 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 a Survivin peptide, preferably the Survivin peptide LTLGEFLKL (SEQ ID NO: 51)) and a major histocompatibility complex (MHC) class I molecule, HLA-A2.

[0045] In one embodiment, the sTCR of the present invention comprises a Vp and a V_a, wherein the sTCR binds to a complex of the peptide comprising the amino acid sequence of SEQ ID NO: 51 and the HLA-A2 molecule.

[0046] In one embodiment, the sTCR of the present invention comprises a Vp and a V_a, wherein the sTCR binds to a peptide (SEQ ID NO: 51) derived from human Survivin in complex with HLA-A2.

[0047] In one embodiment, the V_a of the sTCR of the present invention comprises SEQ ID NO: 34 (CDR1), SEQ ID NO: 35 (CDR2), and SEQ ID NO: 36 (CDR3). In another embodiment, the V_a of the sTCR of the present invention comprises SEQ ID NO: 49 (CDR1), SEQ ID NO: 35 (CDR2), and SEQ ID NO: 36 (CDR3).

[0048] In one embodiment, the V_a of the sTCR of the present invention comprises the amino acid sequence selected from a group consisting of SEQ ID NO: 8 and SEQ ID NO: 9.

[0049] In one preferred embodiment, the V_a of the sTCR of the present invention comprises the amino acid sequence of SEQ ID NO: 8. In another embodiment, the V_a of the sTCR of the present invention comprises the amino acid sequence of SEQ ID NO: 9.

[0050] In one embodiment, the Vp of the sTCR of the present invention comprises SEQ ID NO: 37 (CDR1), SEQ ID NO: 38 (CDR2) and SEQ ID NO: 39 (CDR3).

[0051] In one embodiment, the Vp of the sTCR of the present invention comprises the amino acid sequence selected from a group consisting of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7.

[0052] In one embodiment, the Vp of the sTCR of the present invention comprises the amino acid sequence of SEQ ID NO: 5. In another embodiment, the Vp of the sTCR of the present invention comprises the amino acid sequence of SEQ ID NO: 6. In another embodiment, the V p, of the sTCR of the present invention comprises the amino acid sequence of SEQ ID NO: 7.

[0053] In one embodiment, the sTCR variable beta region (Vp) and the sTCR variable alpha region (V_a) are connected via a first peptide linker (LI). 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 trispecific molecule of the present invention. [0054] In one embodiment, the sTCR variable beta region (Vp) and the sTCR variable alpha region (V_a) are connected via a first peptide linker (LI) comprising the amino acid sequence of SEQ ID NO: 1.

[0055] In one embodiment, the sTCR variable beta region (Vp) is linked to the sTCR variable alpha region (V_a) via a disulfide bridge. For example, and without being bound by any particular theory, in some embodiments, one or more cysteines of the sTCR chain may form a disulfide bridge connecting the V_a and V_[i domain, e.g., cysteines 43 and 235 of the sTCR chain.

[0056] One embodiment pertains to 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: 51 and the HLA-A2 molecule, wherein the sTCR comprises:

(1) a sTCR variable beta region (V ) comprising SEQ ID NO: 37 (CDR1), SEQ ID NO: 38 (CDR2), and SEQ ID NO: 39 (CDR3), and

(2) a sTCR variable alpha region (V_a) comprising SEQ ID NO: 34 (CDR1), SEQ ID NO: 35 (CDR2), and SEQ ID NO: 36 (CDR3), wherein the V_[i and V_a regions of the sTCR are connected via a first peptide linker (LI).

[0057] One embodiment pertains to a 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: 51 and the HLA-A2 molecule, wherein the sTCR comprises:

(2) a sTCR variable alpha region (V_a) comprising SEQ ID NO: 49 (CDR1), SEQ ID NO: 35 (CDR2), and SEQ ID NO: 36 (CDR3), wherein the V and V_a regions of the sTCR are connected via a first peptide linker (LI).

[0058] One embodiment pertains to a 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: 51 and the HLA-A2 molecule, wherein the sTCR comprises:

(1) a sTCR variable beta region (V ) comprising the amino acid sequence selected from a group consisting of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, and (2) a sTCR variable alpha region (V_a) comprising the amino acid sequence selected from a group consisting of SEQ ID NO: 8 and SEQ ID NO: 9, wherein the V_[i and V_a regions of the sTCR are connected via a first peptide linker (LI).

[0059] One embodiment pertains to a 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: 51 and the HLA-A2 molecule, wherein the sTCR comprises:

(1) a sTCR variable beta region (Vp) comprising the amino acid sequence of SEQ ID NO: 5, and

(2) a sTCR variable alpha region (V_a) comprising the amino acid sequence of SEQ ID

NO: 8, wherein the V_[i and V_a regions of the sTCR are connected via a first peptide linker (LI).

[0060] One embodiment pertains to a 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: 51 and the HLA-A2 molecule, wherein the sTCR comprises:

NO: 9, wherein the V_[i and V_a regions of the sTCR are connected via a first peptide linker (LI).

[0061] One embodiment pertains to a 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: 51 and the HLA-A2 molecule, wherein the sTCR comprises:

(1) a sTCR variable beta region (Vp) comprising the amino acid sequence of SEQ ID NO: 6, and

[0062] One embodiment pertains to a 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: 51 and the HLA-A2 molecule, wherein the sTCR comprises:

(1) a sTCR variable beta region (U_b) comprising the amino acid sequence of SEQ ID

NO: 6, and (2) a sTCR variable alpha region (V_a) comprising the amino acid sequence of SEQ ID

NO: 9, wherein the V and V_a regions of the sTCR are connected via a first peptide linker (LI).

[0063] One embodiment pertains to a 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: 51 and the HLA-A2 molecule, wherein the sTCR comprises:

(1) a sTCR variable beta region (Vp) comprising the amino acid sequence of SEQ ID NO: 7, and

NO: 8, wherein the V and V_a regions of the sTCR are connected via a first peptide linker (LI).

[0064] One embodiment pertains to a 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: 51 and the HLA-A2 molecule, wherein the sTCR comprises:

[0065] In one embodiment, the first peptide linker (LI) of the present invention comprises the amino acid sequence of SEQ ID NO: 1.

[0066] In one embodiment, the sTCR of the present invention comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16. In another embodiment, the sTCR of the present invention comprises the amino acid sequence of SEQ ID NO: 13. In another embodiment, the sTCR of the present invention comprises the amino acid sequence of SEQ ID NO: 14. In another embodiment, the sTCR of the present invention comprises the amino acid sequence of SEQ ID NO: 15. In another embodiment, the sTCR of the present invention comprises the amino acid sequence of SEQ ID NO: 16.

[0067] CD3 Binding Part

[0068] In one aspect, the trispecific molecules of the present invention comprise a CD3 binding part. [0069] In one embodiment, the CD3 binding part of the trispecific molecules of the present invention comprises an anti-CD3 binding domain which binds to human CD3. [0070] In some embodiments, the anti-CD3 binding domain comprises a humanized or human light chain variable region specific to human CD3, wherein the light chain variable region specific to CD3 comprises human or non-human light chain CDRs in a human light chain framework region. In certain instances, the light chain framework region is a lambda light chain framework. In other instances, the light chain framework region is a kappa light chain framework.

[0071] In some embodiments, the anti-CD3 binding domain comprises a humanized or human heavy chain variable region specific to CD3 wherein the heavy chain variable region specific to CD3 comprises human or non-human heavy chain CDRs in a human heavy chain framework region.

[0072] In one embodiment, the anti-CD3 binding domain is a single chain variable fragment (scFv) comprising a light chain variable domain and a heavy chain variable domain of an amino acid sequence provided herein.

[0073] As used herein, “single chain variable fragment” or “scFv” refers to an antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked via a short flexible polypeptide linker, and capable of being expressed as a single polypeptide chain, and wherein the scFv retains the specificity of the intact antibody from which it is derived.

[0074] As used herein, “variable domain” (variable domain of a light chain (VL), variable region of a heavy chain (VH)) 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.

[0075] In one embodiment, the anti-CD3 scFv of the present invention comprises a light chain variable domain (VL) and a heavy chain variable domain (VH) of an amino acid sequence provided herein.

[0076] In one embodiment, the VH of the anti-CD3 scFv of the present invention comprises SEQ ID NO: 40 (CDR1), SEQ ID NO: 41 (CDR2), and SEQ ID NO: 42 (CDR3).

[0077] In one embodiment, the VH of the anti-CD3 scFv of the present invention comprises the amino acid sequence of SEQ ID NO: 10.

[0078] In one embodiment, the VL of the anti-CD3 scFv of the present invention comprises SEQ ID NO: 43 (CDR1), SEQ ID NO: 44 (CDR2), and SEQ ID NO: 45 (CDR3).

[0079] In one embodiment, the VL of the anti-CD3 scFv of the present invention comprises the amino acid sequence of SEQ ID NO: 11.

[0080] In one embodiment, the anti-CD3 scFv of the present invention comprises: (1) a heavy chain variable (VH) comprising SEQ ID NO: 40 (CDR1), SEQ ID NO: 41 (CDR2), and SEQ ID NO: 42 (CDR3); and

(2) a light chain variable (VL) comprising SEQ ID NO: 43 (CDR1), SEQ ID NO: 44 (CDR2), and SEQ ID NO: 45 (CDR3); wherein the VH and the VL are connected via a second peptide linker (L2).

[0081] In one embodiment, the anti-CD3-Fab of the present invention comprises:

(1) a heavy chain variable (VH) comprising the amino acid sequence of SEQ ID NO: 10; and

(2) a light chain variable (VL) comprising the amino acid sequence of SEQ ID NO: 11; wherein the VH and the VL are connected via a second peptide linker (L2).

[0082] In one embodiment, the second peptide linker (L2) comprises the amino acid sequence of SEQ ID NO: 3.

[0083] In one embodiment, the anti-CD3 scFv of the present invention comprises the amino acid sequence of SEQ ID NO: 17.

[0084] Half-Life Extension Domain

[0085] Trispecific molecules of the present invention further comprise a half-life extension domain. Contemplated herein are domains which extend the half-life of an antigen-binding domain.

[0086] In one embodiment, the trispecific molecules of the present invention comprise a half-life extension domain, for example a domain which binds to human albumin (ALB).

[0087] In some embodiments, the ALB binding domain comprises a single heavy chain domain (VHH) that binds to human serum albumin (HSA).

[0088] In some embodiments, the anti-HSA VHH domain comprises CDR1 comprising the amino acid sequence of SEQ ID NO:46, CDR2 comprising the amino acid sequence of SEQ ID NO: 47 and CDR3 comprising the amino acid sequence of SEQ ID NO: 48.

[0089] In some embodiments, the anti-HSA VHH domain comprises the amino acid sequence of SEQ ID NO: 12.

[0090] Formats of the Trispecific Molecules

[0091] In some embodiment, the trispecific molecules of the present invention comprise a domain (T) comprising a single-chain soluble TCR that binds to a complex of the peptide Survivin, a domain (A) which binds to HSA, and a domain (C) which binds to CD3. The three domains in the trispecific molecules can be arranged in any order.

[0092] Thus, it is contemplated that the domain order of the trispecific molecule are:

H2N-(T)-(C)-(A)-COOH, H2N-(T)-(A)-(C)-COOH,

H2N-(C)-(T)-(A)-COOH,

H2N-(C)-(A)-(T)-COOH,

H2N-(A)-(C)-(T)-COOH, and/or H2N-(A)-(T)-(C)-COOH.

[0093] In some embodiments, the trispecific molecules have a domain order of H2N-(T)-(C)-(A)-COOH. In some embodiments, the trispecific molecules have a domain order of H2N-(T)-(A)-(C)-COOH. In some embodiments, the trispecific molecules have a domain order of H2N-(C)-(T)-(A)-COOH. In some embodiments, the trispecific molecules have a domain order of H2N-(C)-(A)-(T)-COOH. In some embodiments, the trispecific molecules have a domain order of H2N-(A)-(C)-(T)-COOH. In some embodiment, the trispecific molecules have a domain order of H2N-(A)-(T)-(C)-COOH.

[0094] Six exemplary embodiments of trispecific molecules are illustrated in FIGS. 1A-1F and described below.

[0095] With reference to FIG. 1A, one exemplary embodiment of a trispecific molecule comprises (1) a first domain (T) comprising a single-chain soluble T cell receptor (sTCR) which binds to a complex of the Survivin peptide and the HLA-A2 molecule, (2) a second domain (A) comprising a single heavy chain domain (VHH) that binds to human serum albumin (anti-HSA VHH) and (3) a third domain (C) comprising a single-chain variable fragment (scFv) which binds to human CD3 (anti-CD3 scFv), wherein the three domains are linked in the order of H2N-(T)-(C)-(A)-COOH. , the sTCR comprises a sTCR variable beta region (V_[i) and a sTCR variable alpha region (V_a), where the V _f. and the V_a are connected via a first peptide linker (LI). The anti-CD3 scFv comprises a heavy chain variable (VH) and a light chain variable (VL), wherein the VH and the VL are connected via a second peptide linker (L2). The sTCR and the anti- CD3 scFv are connected via a third peptide linker (L3) connecting the V_a of the sTCR and the VH of the anti-CD3 scFv. The VL of the anti-CD3 scFv and the anti-HSA VHH are connected via a fourth peptide linker (L4). Thus, from N-terminus to C-terminus, the trispecific molecule is in the form of V_f-L 1-V„- L3-V H-L2-V L-L4-V HH .

[0096] With reference to FIG. IB, one exemplary embodiment of a trispecific molecule comprises (1) a first domain (T) comprising a single-chain soluble T cell receptor (sTCR) which binds to a complex of the Survivin peptide and the HLA-A2 molecule, (2) a second domain (A) comprising a single heavy chain domain (VHH) that binds to human serum albumin (anti-HSA VHH) and (3) a third domain (C) comprising a single-chain variable fragment (scFv) which binds to human CD3 (anti-CD3 scFv), wherein the three domains are linked in the order of H2N-(T)-(A)-(C)-COOH. , the sTCR comprises a sTCR variable beta region (V_[i) and a sTCR variable alpha region (V_a), where the V p, and the V_a are connected via a first peptide linker (LI). The anti-CD3 scFv comprises a heavy chain variable (VH) and a light chain variable (VL), wherein the VH and the VL are connected via a second peptide linker (L2). The sTCR and the anti- HSA VHH are connected via a third peptide linker (L3) connecting the V_a of the sTCR and the VHH· The anti-CD3 scFv and the anti-HSA VHH are connected via a fourth peptide linker (L4) connecting the VH of the anti-CD3 scFv and the VHH. Thus, from N-terminus to C-terminus, the trispecific molecule is in the form of V _b-L 1 -V_a-L3-V HH-L4-V _H-L2-V L .

[0097] With reference to FIG. 1C, one exemplary embodiment of a trispecific molecule comprises (1) a first domain (T) comprising a single-chain soluble T cell receptor (sTCR) which binds to a complex of the Survivin peptide and the HLA-A2 molecule, (2) a second domain (A) comprising a single heavy chain domain (VHH) that binds to human serum albumin (anti-HSA VHH) and (3) a third domain (C) comprising a single-chain variable fragment (scFv) which binds to human CD3 (anti-CD3 scFv), wherein the three domains are linked in the order of H2N-(C)-(T)-(A)-COOH. , the sTCR comprises a sTCR variable beta region (Vp) and a sTCR variable alpha region (V_a), where the V p, and the V_a are connected via a first peptide linker (LI). The anti-CD3 scFv comprises a heavy chain variable (VH) and a light chain variable (VL), wherein the VH and the VL are connected via a second peptide linker (L2). The anti-CD3 scFv and the sTCR are connected via a third peptide linker (L3) connecting the VL of the anti-CD3 scFv and the V _f. of the sTCR. The sTCR and anti-HSA VHH are connecting via a fourth linker (L4) connecting the V_a of the sTCR and the VHH. Thus, from N-terminus to C-terminus, the trispecific molecule is in the form of

V _H-L2- V L-L3 - V _b-L 1 -V _a-L4- V HH .

[0098] With reference to FIG. ID, one exemplary embodiment of a trispecific molecule comprises (1) a first domain (T) comprising a single-chain soluble T cell receptor (sTCR) which binds to a complex of the Survivin peptide and the HLA-A2 molecule, (2) a second domain (A) comprising a single heavy chain domain (VHH) that binds to human serum albumin (anti-HSA VHH) and (3) a third domain (C) comprising a single-chain variable fragment (scFv) which binds to human CD3 (anti-CD3 scFv), wherein the three domains are linked in the order of H2N-(C)-(A)-(T)-COOH. , the sTCR comprises a sTCR variable beta region (Vp) and a sTCR variable alpha region (V_a), where the V _f. and the V_a are connected via a first peptide linker (LI). The anti-CD3 scFv comprises a heavy chain variable (VH) and a light chain variable (VL), wherein the VH and the VL are connected via a second peptide linker (L2). The anti-CD3 scFv and the anti-HSA VHH are connected via a third peptide linker (L3) connecting the VL of the anti-CD3 scFv and the VHH. The anti-HSA VHH and the sTCR are connected via a fourth linker (L4) connecting the VHH and the V _f. of the sTCR. Thus, from N-terminus to C-terminus, the trispecific molecule is in the form of

V H-L2- V _L-L3 - V HH-L4- V _b-L 1 -V _a . [0099] With reference to FIG. IE, one exemplary embodiment of a trispecific molecule comprises (1) a first domain (T) comprising a single-chain soluble T cell receptor (sTCR) which binds to a complex of the Survivin peptide and the HLA-A2 molecule, (2) a second domain (A) comprising a single heavy chain domain (VHH) that binds to human serum albumin (anti-HSA VHH) and (3) a third domain (C) comprising a single-chain variable fragment (scFv) which binds to human CD3 (anti-CD3 scFv), wherein the three domains are linked in the order of H2N-(A)-(C)-(T)-COOH. , the sTCR comprises a sTCR variable beta region (Vp) and a sTCR variable alpha region (V_a), where the V p, and the V_a are connected via a first peptide linker (LI). The anti-CD3 scFv comprises a heavy chain variable (VH) and a light chain variable (VL), wherein the VH and the VL are connected via a second peptide linker (L2). The anti-HSA VHH and the anti-CD3 scFv are connected via a third peptide linker (L3) connecting the VHH and the VH of the anti- CD3 scFv. The anti-CD3 scFv and the sTCR are connected via a fourth linker (L4) connecting the VL of the anti-CD3 scFv and the V _f. of the sTCR. Thus, from N-terminus to C-terminus, the trispecific molecule is in the form of VHH-L3-VH-L2-VL-L4-Vp-Ll-V_a.

[00100] With reference to FIG. IF, one exemplary embodiment of a trispecific molecule comprises (1) a first domain (T) comprising a single-chain soluble T cell receptor (sTCR) which binds to a complex of the Survivin peptide and the HLA-A2 molecule, (2) a second domain (A) comprising a single heavy chain domain (VHH) that binds to human serum albumin (anti-HSA VHH) and (3) a third domain (C) comprising a single-chain variable fragment (scFv) which binds to human CD3 (anti-CD3 scFv), wherein the three domains are linked in the order of H2N-(A)-(T)-(C)-COOH. , the sTCR comprises a sTCR variable beta region (V_[i) and a sTCR variable alpha region (V_a), where the V _f. and the V_a are connected via a first peptide linker (LI). The anti-CD3 scFv comprises a heavy chain variable (VH) and a light chain variable (VL), wherein the VH and the VL are connected via a second peptide linker (L2). The anti-HSA VHH and the sTCR are connected via a third peptide linker (L3) connecting the VHH and the V _f. of the sTCR. The sTCR and anti-CD3 scFv are connected via a fourth linker (L4) connecting the V_a of the sTCR and the VH of the anti-CD3 scFv. Thus, from N-terminus to C-terminus, the trispecific molecule is in the form of V HH-L3-V _b-L 1 -V _0C-L4-V _H-L2-V _L .

[00101] The following specific embodiments of the present invention are listed:

[00102] In some embodiments, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, wherein the sTCR comprises: i. a sTCR variable beta region (Vp), and ii. a sTCR variable alpha region (V_a), wherein the V p, and V_a regions of the sTCR are connected via a first peptide linker (LI); b) a second domain (A) comprising a V_HH domain which binds to albumin; and c) a third domain (C) comprising a single chain variable fragment (scFv) which binds to human CD3, wherein the scFv comprises a heavy chain variable region (V_H) and a light chain variable region (V_L), wherein V_H and V_L are connected via a second peptide linker (L2), wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4)o form the trispecific molecule in the order:

H₂N-(T)-L3-(C)-L4-(A)-COOH,

H₂N-(T)-L3-(A)-L4-(C)-COOH,

H₂N-(C)-L3-(T)-L4-(A)-COOH,

H₂N-(C)-L3-(A)-L4-(T)-COOH,

H₂N-(A)-L3-(C)-L4-(T)-COOH, or

H₂N-(A)-L3-(T)-L4-(C)-COOH.

[00103] In some embodiments, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, wherein the sTCR comprises: i. a sTCR variable beta region (Vp), and ii. a sTCR variable alpha region (V_a), wherein the V_[i and V_a regions of the sTCR are connected via a first peptide linker (LI); b) a second domain (A) comprising a VHH domain which binds to albumin; and c) a third domain (C) comprising a single chain variable fragment (scFv) which binds to human CD3, wherein the scFv comprises a heavy chain variable region (V_H) and a light chain variable region (V_L), wherein V_H and V_L are connected via a second peptide linker (L2), wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-(T)-L3-(C)-L4-(A)-COOH. [00104] In one embodiment, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, wherein the sTCR comprises: i. a sTCR variable beta region (Vp), and ii. a sTCR variable alpha region (V_a), wherein the V_[i and V_a regions of the sTCR are connected via a first peptide linker (LI); b) a second domain (A) comprising a VHH domain which binds to albumin; and c) a third domain (C) which binds to human CD3, wherein the third domain comprises a single chain variable fragment (scFv) comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH and VL are connected via a second peptide linker (L2), wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-Vp-Ll-V_a-L3-V_H-L2-V_L-L4- VHH-COOH.

[00105] In one embodiment, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, wherein the sTCR comprises: i. a sTCR variable beta region (Vp) comprising SEQ ID NO: 37 (CDR1), SEQ ID NO: 38 (CDR2) and SEQ ID NO: 39 (CDR3), and ii. a sTCR variable alpha region (V_a) comprising SEQ ID NO: 34 (CDR1), SEQ ID NO: 35 (CDR2) and SEQ ID NO: 36 (CDR3), wherein the V p, and V_a regions of the sTCR are connected via a first peptide linker (LI); b) a second domain (A) comprising a VHH domain which binds to albumin, wherein the VHH domain comprises SEQ ID NO: 46 (CDR1), SEQ ID NO: 47 (CDR2) and SEQ ID NO: 48 (CDR3); and c) a third domain (C) comprising a single chain variable fragment (scFv) which binds to human CD3, wherein the scFv comprises i. a heavy chain variable region (VH) comprising SEQ ID NO: 40 (CDR1), SEQ ID NO: 41 (CDR2) and SEQ ID NO: 42 (CDR3), and ii. a light chain variable region (V_L) comprising SEQ ID NO: 43 (CDR1), SEQ ID NO: 44 (CDR2) and SEQ ID NO: 45 (CDR3), wherein V_H and V_L are connected via a second peptide linker (L2), wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-V -Ll-V_a-L3-V_H-L2-V_L-L4- V_HH-COOH.

[00106] In one embodiment, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, wherein the sTCR comprises: i. a sTCR variable beta region ( V _[,) comprising the amino acid sequence of SEQ ID NO: 5, and ii. a sTCR variable alpha region (V_a) comprising the amino acid sequence of SEQ ID NO: 8, wherein the V p, and V_a regions of the sTCR are connected via a first peptide linker (LI); b) a second domain (A) comprising a VHH domain which binds to albumin, wherein the VHH domain comprises the amino acid sequence of SEQ ID NO: 12; and c) a third domain (C) comprising a single chain variable fragment (scFv) which binds to human CD3, wherein the scFv comprises i. a heavy chain variable region (V_H) comprising the amino acid sequence of SEQ ID NO: 10, and ii. a light chain variable region (V_L) comprising the amino acid sequence of SEQ ID NO: 11, wherein V_H and V_L are connected via a second peptide linker (L2), wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-Vp-Ll-V_a-L3-V_H-L2-V_L-L4- V_HH-COOH.

[00107] In one embodiment, the first peptide linker (LI) comprises the amino acid sequence of SEQ ID NO: 1.

[00108] In one embodiment, the second peptide linker (L2) comprises the amino acid sequence of SEQ ID NO: 3.

[00109] In one embodiment, the third peptide linker (L3) comprises the amino acid sequence of SEQ ID

NO: 2 or SEQ ID NO: 4. In another embodiment, the third peptide linker (L3) comprises the amino acid sequence of SEQ ID NO: 2. In another embodiment, the third peptide linker (L3) comprises the amino acid sequence of SEQ ID NO: 4.

[00110] In one embodiment, the fourth peptide linker (L4) comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In another embodiment, the fourth peptide linker (L4) comprises the amino acid sequence of SEQ ID NO: 2. In another embodiment, the fourth peptide linker (L4) comprises the amino acid sequence of SEQ ID NO: 4.

[00111] In one embodiment, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, wherein the sTCR comprises the amino acid sequence of SEQ ID NO: 13; b) a second domain (A) comprising a VHH domain which binds to albumin, wherein the VHH domain comprises the amino acid sequence of SEQ ID NO: 12; and c) a third domain (C) comprising a single chain variable fragment (scFv) which binds to human CD3, wherein the scFv comprises the amino acid sequence of SEQ ID NO: 17, wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-(T)-L3-(C)-L4-(A)-COOH.

[00112] In one embodiment, a trispecific molecule that binds to (i) human CD3, (ii) human serum albumin, and (iii) a complex of the peptide Survivin, wherein the complex comprises the amino acid sequence of LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, comprising the amino acid sequence of SEQ ID NO: 19.

[00113] In one embodiment, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, wherein the sTCR comprises: i. a sTCR variable beta region (Vp) comprising SEQ ID NO: 37 (CDR1), SEQ ID NO: 38 (CDR2) and SEQ ID NO: 39 (CDR3), and ii. a sTCR variable alpha region (V_a) comprising SEQ ID NO: 49 (CDR1), SEQ ID NO: 35 (CDR2) and SEQ ID NO: 36 (CDR3), wherein the V and V_a regions of the sTCR are connected via a first peptide linker (LI); b) a second domain (A) comprising a VHH domain which binds to albumin, wherein the VHH domain comprises SEQ ID NO: 46 (CDR1), SEQ ID NO: 47 (CDR2) and SEQ ID NO:

48 (CDR3); and c) a third domain (C) comprising a single chain variable fragment (scFv) which binds to human CD3, wherein the scFv comprises i. a heavy chain variable region (VH) comprising SEQ ID NO: 40 (CDR1), SEQ ID NO: 41 (CDR2) and SEQ ID NO: 42 (CDR3), and ii. a light chain variable region (V_L) comprising SEQ ID NO: 43 (CDR1), SEQ ID NO: 44 (CDR2) and SEQ ID NO: 45 (CDR3), wherein V_H and V_L are connected via a second peptide linker (L2), wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-Vp-Ll-V_a-L3-V_H-L2-V_L-L4- V_HH-COOH.

[00114] In one embodiment, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, wherein the sTCR comprises: i. a sTCR variable beta region ( V p,) comprising the amino acid sequence of SEQ ID NO: 5, and ii. a sTCR variable alpha region (V_a) comprising the amino acid sequence of SEQ ID NO: 9, wherein the V _f. and V_a regions of the sTCR are connected via a first peptide linker (LI); b) a second domain (A) comprising a VHH domain which binds to albumin, wherein the VHH domain comprises the amino acid sequence of SEQ ID NO: 12; and c) a third domain (C) comprising a single chain variable fragment (scFv) which binds to human CD3, wherein the scFv comprises i. a heavy chain variable region (V_H) comprising the amino acid sequence of SEQ ID NO: 10, and ii. a light chain variable region (V_L) comprising the amino acid sequence of SEQ ID NO: 11, wherein V_H and V_L are connected via a second peptide linker (L2), wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-Vp-Ll-V_a-L3-V_H-L2-V_L-L4- V_HH-COOH.

[00115] In one embodiment, the first peptide linker (LI) comprises the amino acid sequence of SEQ ID NO: 1.

[00116] In one embodiment, the second peptide linker (L2) comprises the amino acid sequence of SEQ ID NO: 3.

[00117] In one embodiment, the third peptide linker (L3) comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In another embodiment, the third peptide linker (L3) comprises the amino acid sequence of SEQ ID NO: 2. In another embodiment, the third peptide linker (L3) comprises the amino acid sequence of SEQ ID NO: 4.

[00118] In one embodiment, the fourth peptide linker (L4) comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In another embodiment, the fourth peptide linker (L4) comprises the amino acid sequence of SEQ ID NO: 2. In another embodiment, the fourth peptide linker (L4) comprises the amino acid sequence of SEQ ID NO: 4.

[00119] In one embodiment, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, wherein the sTCR comprises the amino acid sequence of SEQ ID NO: 14; b) a second domain (A) comprising a VHH domain which binds to albumin, wherein the VHH domain comprises the amino acid sequence of SEQ ID NO: 12; and c) a third domain (C) comprising a single chain variable fragment (scFv) which binds to human CD3, wherein the scFv comprises the amino acid sequence of SEQ ID NO: 17, wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-(T)-L3-(C)-L4-(A)-COOH.

[00120] In one embodiment, a trispecific molecule that binds to (i) human CD3, (ii) human serum albumin, and (iii) a complex of the peptide Survivin, wherein the complex comprises the amino acid sequence of LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, comprises the amino acid sequence of SEQ ID NO: 25.

[00121] In some embodiments, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, wherein the sTCR comprises: i. a sTCR variable beta region (Vp), and ii. a sTCR variable alpha region (V_a), wherein the V and V_a regions of the sTCR are connected via a first peptide linker (LI); b) a second domain (A) comprising a V_HH domain which binds to albumin; and c) a third domain (C) comprising a single chain variable fragment (scFv) which binds to human CD3, wherein the scFv comprises a heavy chain variable region (V_H) and a light chain variable region (V_L), wherein V_H and V_L are connected via a second peptide linker (L2), wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-(T)-L3-(A)-L4-(C)-COOH.

[00122] In one embodiment, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, wherein the sTCR comprises: i. a sTCR variable beta region (Vp), and ii. a sTCR variable alpha region (V_a), wherein the V_[i and V_a regions of the sTCR are connected via a first peptide linker (LI); b) a second domain (A) comprising a VHH domain which binds to albumin; and c) a third domain (C) comprising a single chain variable fragment (scFv) which binds to human CD3, wherein the scFv comprises a heavy chain variable region (V_H) and a light chain variable region (V_L), wherein V_H and V_L are connected via a second peptide linker (L2), wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-Vp-Ll-V_a-L3-V_HH-L4-V_H-L2- V_L-COOH.

[00123] In one embodiment, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, wherein the sTCR comprises: i. a sTCR variable beta region (Vp) comprising SEQ ID NO: 37 (CDR1), SEQ ID NO: 38 (CDR2) and SEQ ID NO: 39 (CDR3), and ii. a sTCR variable alpha region (V_a) comprising SEQ ID NO: 34 (CDR1), SEQ ID NO: 35 (CDR2) and SEQ ID NO: 36 (CDR3), wherein the V p, and V_a regions of the sTCR are connected via a first peptide linker (LI); b) a second domain (A) comprising a VHH domain which binds to albumin, wherein the VHH domain comprises SEQ ID NO: 46 (CDR1), SEQ ID NO: 47 (CDR2) and SEQ ID NO:

48 (CDR3); and c) a third domain (C) comprising a single chain variable fragment (scFv) which binds to human CD3, wherein the scFv comprises i. a heavy chain variable region (VH) comprising SEQ ID NO: 40 (CDR1), SEQ ID NO: 41 (CDR2) and SEQ ID NO: 42 (CDR3), and ii. a light chain variable region (V_L) comprising SEQ ID NO: 43 (CDR1), SEQ ID NO: 44 (CDR2) and SEQ ID NO: 45 (CDR3), wherein V_H and V_L are connected via a second peptide linker (L2), wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-Vp-Ll-V_a-L3-V_HH-L4-V_H-L2- V_L-COOH.

[00124] In one embodiment, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, wherein the sTCR comprises: i. a sTCR variable beta region (VQ comprising the amino acid sequence of SEQ ID NO: 5, and ii. a sTCR variable alpha region (V_a) comprising the amino acid sequence of SEQ ID NO: 8, wherein the V f. and V_a regions of the sTCR are connected via a first peptide linker (LI); b) a second domain (A) comprising a VHH domain which binds to albumin, wherein the VHH domain comprises the amino acid sequence of SEQ ID NO: 12; and c) a third domain (C) comprising a single chain variable fragment (scFv) which binds to human CD3, wherein the scFv comprises i. a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 10, and ii. a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 11, wherein VH and VL are connected via a second peptide linker (L2), wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order EbN- V p — L 1 -V„-L3-VHH-L4-V H- L2-VL-COOH.

[00125] In one embodiment, the first peptide linker (LI) comprises the amino acid sequence of SEQ ID NO: 1.

[00126] In one embodiment, the second peptide linker (L2) comprises the amino acid sequence of SEQ ID NO: 3.

[00127] In one embodiment, the third peptide linker (L3) comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In another embodiment, the third peptide linker (L3) comprises the amino acid sequence of SEQ ID NO: 2. In another embodiment, the third peptide linker (L3) comprises the amino acid sequence of SEQ ID NO: 4.

[00128] In one embodiment, the fourth peptide linker (L4) comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In another embodiment, the fourth peptide linker (L4) comprises the amino acid sequence of SEQ ID NO: 2. In another embodiment, the fourth peptide linker (L4) comprises the amino acid sequence of SEQ ID NO: 4.

[00129] In one embodiment, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, wherein the sTCR comprises the amino acid sequence of SEQ ID NO: 13; b) a second domain (A) comprising a VHH domain which binds to albumin, wherein the VHH domain comprises the amino acid sequence of SEQ ID NO: 12; and c) a third domain (C) comprising a single chain variable fragment (scFv) which binds to human CD3, wherein the scFv comprises the amino acid sequence of SEQ ID NO: 17, wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-(T)-L3-(A)-L4-(C)-COOH.

[00130] In one embodiment, a trispecific molecule that binds to (i) human CD3, (ii) human serum albumin, and (iii) a complex of the peptide Survivin, wherein the complex comprises the amino acid sequence of LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, comprising the amino acid sequence of SEQ ID NO: 18.

[00131] In one embodiment, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, wherein the sTCR comprises: i. a sTCR variable beta region (Vp) comprising SEQ ID NO: 37 (CDR1), SEQ ID NO: 38 (CDR2) and SEQ ID NO: 39 (CDR3), and ii. a sTCR variable alpha region (V_a) comprising SEQ ID NO: 49 (CDR1), SEQ ID NO: 35 (CDR2) and SEQ ID NO: 36 (CDR3), wherein the V and V_a regions of the sTCR are connected via a first peptide linker (LI); b) a second domain (A) comprising a VHH domain which binds to albumin, wherein the VHH domain comprises SEQ ID NO: 46 (CDR1), SEQ ID NO: 47 (CDR2) and SEQ ID NO: 48 (CDR3); and c) a third domain (C) comprising a single chain variable fragment (scFv) which binds to human CD3, wherein the scFv comprises i. a heavy chain variable region (VH) comprising SEQ ID NO: 40 (CDR1), SEQ ID NO: 41 (CDR2) and SEQ ID NO: 42 (CDR3), and ii. a light chain variable region (V_L) comprising SEQ ID NO: 43 (CDR1), SEQ ID NO: 44 (CDR2) and SEQ ID NO: 45 (CDR3), wherein V_H and V_L are connected via a second peptide linker (L2), wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-Vp-Ll-V_a-L3-V_HH-L4-V_H-L2- V_L-COOH.

[00132] In one embodiment, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, wherein the sTCR comprises: i. a sTCR variable beta region (VQ comprising the amino acid sequence of SEQ ID NO: 5, and ii. a sTCR variable alpha region (V_a) comprising the amino acid sequence of SEQ ID NO: 9, wherein the V p, and V_a regions of the sTCR are connected via a first peptide linker (LI); b) a second domain (A) comprising a VHH domain which binds to albumin, wherein the VHH domain comprises the amino acid sequence of SEQ ID NO: 12; and c) a third domain (C) comprising a single chain variable fragment (scFv) which binds to human CD3, wherein the scFv comprises i. a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 10, and ii. a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 11, wherein VH and VL are connected via a second peptide linker (L2), wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order FEN- V p — L 1 -V_I -L3-VHH-L4-V H- L2-VL-COOH.

[00133] In one embodiment, the first peptide linker (LI) comprises the amino acid sequence of SEQ ID NO: 1.

[00134] In one embodiment, the second peptide linker (L2) comprises the amino acid sequence of SEQ ID NO: 3.

[00135] In one embodiment, the third peptide linker (L3) comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In another embodiment, the third peptide linker (L3) comprises the amino acid sequence of SEQ ID NO: 2. In another embodiment, the third peptide linker (L3) comprises the amino acid sequence of SEQ ID NO: 4.

[00136] In one embodiment, the fourth peptide linker (L4) comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In another embodiment, the fourth peptide linker (L4) comprises the amino acid sequence of SEQ ID NO: 2. In another embodiment, the fourth peptide linker (L4) comprises the amino acid sequence of SEQ ID NO: 4.

[00137] In one embodiment, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, wherein the sTCR comprises the amino acid sequence of SEQ ID NO: 14; b) a second domain (A) comprising a VHH domain which binds to albumin, wherein the VHH domain comprises the amino acid sequence of SEQ ID NO: 12; and c) a third domain (C) comprising a single chain variable fragment (scFv) which binds to human CD3, wherein the scFv comprises the amino acid sequence of SEQ ID NO: 17, wherein the domains of T, A and C are connected via a third linker (F3) and a fourth linker (F4) to form the trispecific molecule in the order H₂N-(T)-F3-(A)-F4-(C)-COOH. [00138] In one embodiment, a trispecific molecule that binds to (i) human CD3, (ii) human serum albumin, and (iii) a complex of the peptide Survivin, wherein the complex comprises the amino acid sequence of FTFGEFFKF (SEQ ID NO: 51) and the HLA-A2 molecule, comprising the amino acid sequence of SEQ ID NO: 24.

[00139] In some embodiments, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, wherein the sTCR comprises: i. a sTCR variable beta region (Vp), and ii. a sTCR variable alpha region (V_a), wherein the V_[i and V_a regions of the sTCR are connected via a first peptide linker (LI); b) a second domain (A) comprising a V_HH domain which binds to albumin; and c) a third domain (C) comprising a single chain variable fragment (scFv) which binds to human CD3, wherein the scFv comprises a heavy chain variable region (V_H) and a light chain variable region (V_L), wherein V_H and V_L are connected via a second peptide linker (L2), wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-(C)-L3-(T)-L4-(A)-COOH.

[00140] In one embodiment, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, wherein the sTCR comprises: i. a sTCR variable beta region (Vp), and ii. a sTCR variable alpha region (V_a), wherein the V and V_a regions of the sTCR are connected via a first peptide linker (LI); b) a second domain (A) comprising a VHH domain which binds to albumin; and c) a third domain (C) comprising a single chain variable fragment (scFv) which binds to human CD3, wherein the scFv comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH and VL are connected via a second peptide linker (L2), wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order 1¾N-V_H-L2-V_L-L3-Vp-Ll-V_a-L4- VHH-COOH.

[00141] In one embodiment, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, wherein the sTCR comprises: i. a sTCR variable beta region (Vp) comprising SEQ ID NO: 37 (CDR1), SEQ ID NO: 38 (CDR2) and SEQ ID NO: 39 (CDR3), and ii. a sTCR variable alpha region (V_a) comprising SEQ ID NO: 34 (CDR1), SEQ ID NO: 35 (CDR2) and SEQ ID NO: 36 (CDR3), wherein the V p, and V_a regions of the sTCR are connected via a first peptide linker (LI); b) a second domain (A) comprising a VHH domain which binds to albumin, wherein the VHH domain comprises SEQ ID NO: 46 (CDR1), SEQ ID NO: 47 (CDR2) and SEQ ID NO: 48 (CDR3); and c) a third domain (C) comprising a single chain variable fragment (scFv) which binds to human CD3, wherein the scFv comprises i. a heavy chain variable region (VH) comprising SEQ ID NO: 40 (CDR1), SEQ ID NO: 41 (CDR2) and SEQ ID NO: 42 (CDR3), and ii. a light chain variable region (V_L) comprising SEQ ID NO: 43 (CDR1), SEQ ID NO: 44 (CDR2) and SEQ ID NO: 45 (CDR3), wherein V_H and V_L are connected via a second peptide linker (L2), wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-VH-L2-VL-L3-V_| -L 1-V,-L4- VHH-COOH.

[00142] In one embodiment, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, wherein the sTCR comprises: i. a sTCR variable beta region ( V p.) comprising the amino acid sequence of SEQ ID NO: 5, and ii. a sTCR variable alpha region (V_a) comprising the amino acid sequence of SEQ ID NO: 8, wherein the V p, and V_a regions of the sTCR are connected via a first peptide linker (LI); b) a second domain (A) comprising a VHH domain which binds to albumin, wherein the VHH domain comprises the amino acid sequence of SEQ ID NO: 12; and c) a third domain (C) comprising a single chain variable fragment (scFv) which binds to human CD3, wherein the scFv comprises i. a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 10, and ii. a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 11, wherein VH and VL are connected via a second peptide linker (L2), wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-VH-L2-VL-L3-V_|',-L 1-V,-L4- VHH-COOH.

[00143] In one embodiment, the first peptide linker (LI) comprises the amino acid sequence of SEQ ID NO: 1.

[00144] In one embodiment, the second peptide linker (L2) comprises the amino acid sequence of SEQ ID NO: 3.

[00145] In one embodiment, the third peptide linker (L3) comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In another embodiment, the third peptide linker (L3) comprises the amino acid sequence of SEQ ID NO: 2. In another embodiment, the third peptide linker (L3) comprises the amino acid sequence of SEQ ID NO: 4.

[00146] In one embodiment, the fourth peptide linker (L4) comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In another embodiment, the fourth peptide linker (L4) comprises the amino acid sequence of SEQ ID NO: 2. In another embodiment, the fourth peptide linker (L4) comprises the amino acid sequence of SEQ ID NO: 4.

[00147] In one embodiment, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, wherein the sTCR comprises the amino acid sequence of SEQ ID NO: 13; b) a second domain (A) comprising a VHH domain which binds to albumin, wherein the VHH domain comprises the amino acid sequence of SEQ ID NO: 12; and c) a third domain (C) comprising a single chain variable fragment (scFv) which binds to human CD3, wherein the scFv comprises the amino acid sequence of SEQ ID NO: 17, wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order EhN-(C)— L3-(T)-L4-(A)-COOEI. [00148] In one embodiment, a trispecific molecule that binds to (i) human CD3, (ii) human serum albumin, and (iii) a complex of the peptide Survivin, wherein the complex comprises the amino acid sequence of LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, comprising the amino acid sequence of SEQ ID NO: 21.

[00149] In one embodiment, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, wherein the sTCR comprises: i. a sTCR variable beta region (Vp) comprising SEQ ID NO: 37 (CDR1), SEQ ID NO: 38 (CDR2) and SEQ ID NO: 39 (CDR3), and ii. a sTCR variable alpha region (V_a) comprising SEQ ID NO: 49 (CDR1), SEQ ID NO: 35 (CDR2) and SEQ ID NO: 36 (CDR3), wherein the V and V_a regions of the sTCR are connected via a first peptide linker (LI); b) a second domain (A) comprising a VHH domain which binds to albumin, wherein the VHH domain comprises SEQ ID NO: 46 (CDR1), SEQ ID NO: 47 (CDR2) and SEQ ID NO: 48 (CDR3); and c) a third domain (C) comprising a single chain variable fragment (scFv) which binds to human CD3, wherein the scFv comprises i. a heavy chain variable region (V_H) comprising SEQ ID NO: 40 (CDR1), SEQ ID NO: 41 (CDR2) and SEQ ID NO: 42 (CDR3), and ii. a light chain variable region (V_L) comprising SEQ ID NO: 43 (CDR1), SEQ ID NO: 44 (CDR2) and SEQ ID NO: 45 (CDR3), wherein V_H and V_L are connected via a second peptide linker (L2), wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-V_H-L2-V_L-L3-V_|-L 1-V,-L4- V_HH-COOH.

[00150] In one embodiment, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, wherein the sTCR comprises: i. a sTCR variable beta region ( V _[,) comprising the amino acid sequence of SEQ ID NO: 5, and ii. a sTCR variable alpha region (V_a) comprising the amino acid sequence of SEQ ID NO: 9, wherein the V p, and V_a regions of the sTCR are connected via a first peptide linker (LI); b) a second domain (A) comprising a VHH domain which binds to albumin, wherein the VHH domain comprises the amino acid sequence of SEQ ID NO: 12; and c) a third domain (C) comprising a single chain variable fragment (scFv) which binds to human CD3, wherein the scFv comprises i. a heavy chain variable region (V_H) comprising the amino acid sequence of SEQ ID NO: 10, and ii. a light chain variable region (V_L) comprising the amino acid sequence of SEQ ID NO: 11, wherein V_H and V_L are connected via a second peptide linker (L2), wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-V_H-L2-V_L-L3-V_|-L 1-V,-L4- V_HH-COOH.

[00151] In one embodiment, the first peptide linker (LI) comprises the amino acid sequence of SEQ ID NO: 1.

[00152] In one embodiment, the second peptide linker (L2) comprises the amino acid sequence of SEQ ID NO: 3.

[00153] In one embodiment, the third peptide linker (L3) comprises the amino acid sequence of SEQ ID

[00154] In one embodiment, the fourth peptide linker (L4) comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In another embodiment, the fourth peptide linker (L4) comprises the amino acid sequence of SEQ ID NO: 2. In another embodiment, the fourth peptide linker (L4) comprises the amino acid sequence of SEQ ID NO: 4.

[00155] In one embodiment, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, wherein the sTCR comprises the amino acid sequence of SEQ ID NO: 14; b) a second domain (A) comprising a VHH domain which binds to albumin, wherein the VHH domain comprises the amino acid sequence of SEQ ID NO: 12; and c) a third domain (C) comprising a single chain variable fragment (scFv) which binds to human CD3, wherein the scFv comprises the amino acid sequence of SEQ ID NO: 17, wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order EhN-(C)— L3-(T)-L4-(A)-COOEI. [00156] In one embodiment, a trispecific molecule that binds to (i) human CD3, (ii) human serum albumin, and (iii) a complex of the peptide Survivin, wherein the complex comprises the amino acid sequence of LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, comprising the amino acid sequence of SEQ ID NO: 27.

[00157] In one embodiment, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, wherein the sTCR comprises: i. a sTCR variable beta region (Vp) comprising SEQ ID NO: 37 (CDR1), SEQ ID NO: 38 (CDR2) and SEQ ID NO: 39 (CDR3), and ii. a sTCR variable alpha region (V_a) comprising SEQ ID NO: 34 (CDR1), SEQ ID NO: 35 (CDR2) and SEQ ID NO: 36 (CDR3), wherein the V and V_a regions of the sTCR are connected via a first peptide linker (LI); b) a second domain (A) comprising a VHH domain which binds to albumin, wherein the VHH domain comprises SEQ ID NO: 46 (CDR1), SEQ ID NO: 47 (CDR2) and SEQ ID NO:

48 (CDR3); and c) a third domain (C) comprising a single chain variable fragment (scFv) which binds to human CD3, wherein the scFv comprises i. a heavy chain variable region (VH) comprising SEQ ID NO: 40 (CDR1), SEQ ID NO: 41 (CDR2) and SEQ ID NO: 42 (CDR3), and ii. a light chain variable region (V_L) comprising SEQ ID NO: 43 (CDR1), SEQ ID NO: 44 (CDR2) and SEQ ID NO: 45 (CDR3), wherein V_H and V_L are connected via a second peptide linker (L2), wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-V_H-L2-V_L-L3-V_|',-L 1-V,-L4- V_HH-COOH.

[00158] In one embodiment, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, wherein the sTCR comprises: i. a sTCR variable beta region ( V p,) comprising the amino acid sequence of SEQ ID NO: 5, and ii. a sTCR variable alpha region (V_a) comprising the amino acid sequence of SEQ ID NO: 8, wherein the V _f. and V_a regions of the sTCR are connected via a first peptide linker (LI); b) a second domain (A) comprising a VHH domain which binds to albumin, wherein the VHH domain comprises the amino acid sequence of SEQ ID NO: 12; and c) a third domain (C) comprising a single chain variable fragment (scFv) which binds to human CD3, wherein the scFv comprises i. a heavy chain variable region (V_H) comprising the amino acid sequence of SEQ ID NO: 10, and ii. a light chain variable region (V_L) comprising the amino acid sequence of SEQ ID NO: 11, wherein V_H and V_L are connected via a second peptide linker (L2), wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-VH-L2-VI.-L3-V_| -L 1-V„-L4- VHH-COOH.

[00159] In one embodiment, the first peptide linker (LI) comprises the amino acid sequence of SEQ ID NO: 1.

[00160] In one embodiment, the second peptide linker (L2) comprises the amino acid sequence of SEQ ID NO: 3.

[00161] In one embodiment, the third peptide linker (L3) comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In another embodiment, the third peptide linker (L3) comprises the amino acid sequence of SEQ ID NO: 2. In another embodiment, the third peptide linker (L3) comprises the amino acid sequence of SEQ ID NO: 4.

[00162] In one embodiment, the fourth peptide linker (L4) comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In another embodiment, the fourth peptide linker (L4) comprises the amino acid sequence of SEQ ID NO: 2. In another embodiment, the fourth peptide linker (L4) comprises the amino acid sequence of SEQ ID NO: 4.

[00163] In one embodiment, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, wherein the sTCR comprises the amino acid sequence of SEQ ID NO: 13; b) a second domain (A) comprising a VHH domain which binds to albumin, wherein the VHH domain comprises the amino acid sequence of SEQ ID NO: 12; and c) a third domain (C) comprising a single chain variable fragment (scFv) which binds to human CD3, wherein the scFv comprises the amino acid sequence of SEQ ID NO: 17, wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-(C)-L3-(T)-L4-(A)-COOH.

[00164] In one embodiment, a trispecific molecule that binds to (i) human CD3, (ii) human serum albumin, and (iii) a complex of the peptide Survivin, wherein the complex comprises the amino acid sequence of LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, comprising the amino acid sequence of SEQ ID NO: 32.

[00165] In some embodiments, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, wherein the sTCR comprises: i. a sTCR variable beta region (Vp), and ii. a sTCR variable alpha region (V_a), wherein the V and V_a regions of the sTCR are connected via a first peptide linker (LI); b) a second domain (A) comprising a VHH domain which binds to albumin; and c) a third domain (C) comprising a single chain variable fragment (scFv) which binds to human CD3, wherein the scFv comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH and VL are connected via a second peptide linker (L2), wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4)o form the trispecific molecule in the order H₂N-(C)-L3-(A)-L4-(T)-COOH.

[00166] In one embodiment, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, wherein the sTCR comprises: i. a sTCR variable beta region (Vp), and ii. a sTCR variable alpha region (V_a), wherein the V_[i and V_a regions of the sTCR are connected via a first peptide linker (LI); b) a second domain (A) comprising a VHH domain which binds to albumin; and c) a third domain (C) comprising a single chain variable fragment (scFv) which binds to human CD3, wherein the scFv comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH and VL are connected via a second peptide linker (L2), wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-VH-L2-VI -L3-VHH-L4-V_[I-L 1- V_a-COOH.

[00167] In one embodiment, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, wherein the sTCR comprises: i. a sTCR variable beta region (Vp) comprising SEQ ID NO: 37 (CDR1), SEQ ID NO: 38 (CDR2) and SEQ ID NO: 39 (CDR3), and ii. a sTCR variable alpha region (V_a) comprising SEQ ID NO: 34 (CDR1), SEQ ID NO: 35 (CDR2) and SEQ ID NO: 36 (CDR3), wherein the V p, and V_a regions of the sTCR are connected via a first peptide linker (LI); b) a second domain (A) comprising a VHH domain which binds to albumin, wherein the VHH domain comprises SEQ ID NO: 46 (CDR1), SEQ ID NO: 47 (CDR2) and SEQ ID NO:

48 (CDR3); and c) a third domain (C) comprising a single chain variable fragment (scFv) which binds to human CD3, wherein the scFv comprises i. a heavy chain variable region (VH) comprising SEQ ID NO: 40 (CDR1), SEQ ID NO: 41 (CDR2) and SEQ ID NO: 42 (CDR3), and ii. a light chain variable region (VL) comprising SEQ ID NO: 43 (CDR1), SEQ ID NO: 44 (CDR2) and SEQ ID NO: 45 (CDR3), wherein V_H and VL are connected via a second peptide linker (L2), wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-VH-L2-VI -L3-VHH-L4-V_|',-L 1- V_a-COOH.

[00168] In one embodiment, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, wherein the sTCR comprises: i. a sTCR variable beta region ( V p,) comprising the amino acid sequence of SEQ ID NO: 5, and ii. a sTCR variable alpha region (V_a) comprising the amino acid sequence of SEQ ID NO: 8, wherein the V _f. and V_a regions of the sTCR are connected via a first peptide linker (LI); b) a second domain (A) comprising a VHH domain which binds to albumin, wherein the VHH domain comprises the amino acid sequence of SEQ ID NO: 12; and c) a third domain (C) comprising a single chain variable fragment (scFv) which binds to human CD3, wherein the scFv comprises i. a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 10, and ii. a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 11, wherein VH and VL are connected via a second peptide linker (L2), wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-V_H-L2-V_L-L3-V_HH-L4-V_[;-L 1- V_a-COOH.

[00169] In one embodiment, the first peptide linker (LI) comprises the amino acid sequence of SEQ ID NO: 1.

[00170] In one embodiment, the second peptide linker (L2) comprises the amino acid sequence of SEQ ID NO: 3.

[00171] In one embodiment, the third peptide linker (L3) comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In another embodiment, the third peptide linker (L3) comprises the amino acid sequence of SEQ ID NO: 2. In another embodiment, the third peptide linker (L3) comprises the amino acid sequence of SEQ ID NO: 4.

[00172] In one embodiment, the fourth peptide linker (L4) comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In another embodiment, the fourth peptide linker (L4) comprises the amino acid sequence of SEQ ID NO: 2. In another embodiment, the fourth peptide linker (L4) comprises the amino acid sequence of SEQ ID NO: 4.

[00173] In one embodiment, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, wherein the sTCR comprises the amino acid sequence of SEQ ID NO: 13; b) a second domain (A) comprising a VHH domain which binds to albumin, wherein the VHH domain comprises the amino acid sequence of SEQ ID NO: 12; and c) a third domain (C) comprising a single chain variable fragment (scFv) which binds to human CD3, wherein the scFv comprises the amino acid sequence of SEQ ID NO: 17, wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-(C)-L3-(A)-L4-(T)-COOH.

[00174] In one embodiment, a trispecific molecule that binds to (i) human CD3, (ii) human serum albumin, and (iii) a complex of the peptide Survivin, wherein the complex comprises the amino acid sequence of LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, comprising the amino acid sequence of SEQ ID NO: 23.

[00175] In one embodiment, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, wherein the sTCR comprises: i. a sTCR variable beta region (Vp) comprising SEQ ID NO: 37 (CDR1), SEQ ID NO: 38 (CDR2) and SEQ ID NO: 39 (CDR3), and ii. a sTCR variable alpha region (V_a) comprising SEQ ID NO: 49 (CDR1), SEQ ID NO: 35 (CDR2) and SEQ ID NO: 36 (CDR3), wherein the V and V_a regions of the sTCR are connected via a first peptide linker (LI); b) a second domain (A) comprising a VHH domain which binds to albumin, wherein the VHH domain comprises SEQ ID NO: 46 (CDR1), SEQ ID NO: 47 (CDR2) and SEQ ID NO: 48 (CDR3); and c) a third domain (C) comprising a single chain variable fragment (scFv) which binds to human CD3, wherein the scFv comprises i. a heavy chain variable region (VH) comprising SEQ ID NO: 40 (CDR1), SEQ ID NO: 41 (CDR2) and SEQ ID NO: 42 (CDR3), and ii. a light chain variable region (V_L) comprising SEQ ID NO: 43 (CDR1), SEQ ID NO: 44 (CDR2) and SEQ ID NO: 45 (CDR3), wherein V_H and V_L are connected via a second peptide linker (L2), wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-VH-L2-VI -L3-VHH-L4-V_|',-L 1- V_a-COOH.

[00176] In one embodiment, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, wherein the sTCR comprises: i. a sTCR variable beta region (VQ comprising the amino acid sequence of SEQ ID NO: 5, and ii. a sTCR variable alpha region (V_a) comprising the amino acid sequence of SEQ ID NO: 9, wherein the V p, and V_a regions of the sTCR are connected via a first peptide linker (LI); b) a second domain (A) comprising a VHH domain which binds to albumin, wherein the VHH domain comprises the amino acid sequence of SEQ ID NO: 12; and c) a third domain (C) comprising a single chain variable fragment (scFv) which binds to human CD3, wherein the scFv comprises i. a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 10, and ii. a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 11, wherein VH and VL are connected via a second peptide linker (L2), wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-VH-L2-VI -L3-VHH-L4-V_|',-L 1- V_a-COOH.

[00177] In one embodiment, the first peptide linker (LI) comprises the amino acid sequence of SEQ ID NO: 1.

[00178] In one embodiment, the second peptide linker (L2) comprises the amino acid sequence of SEQ ID NO: 3.

[00179] In one embodiment, the third peptide linker (L3) comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In another embodiment, the third peptide linker (L3) comprises the amino acid sequence of SEQ ID NO: 2. In another embodiment, the third peptide linker (L3) comprises the amino acid sequence of SEQ ID NO: 4.

[00180] In one embodiment, the fourth peptide linker (L4) comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In another embodiment, the fourth peptide linker (L4) comprises the amino acid sequence of SEQ ID NO: 2. In another embodiment, the fourth peptide linker (L4) comprises the amino acid sequence of SEQ ID NO: 4.

[00181] In one embodiment, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, wherein the sTCR comprises the amino acid sequence of SEQ ID NO: 14; b) a second domain (A) comprising a VHH domain which binds to albumin, wherein the VHH domain comprises the amino acid sequence of SEQ ID NO: 12; and c) a third domain (C) comprising a single chain variable fragment (scFv) which binds to human CD3, wherein the scFv comprises the amino acid sequence of SEQ ID NO: 17, wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-(C)-L3-(A)-L4-(T)-COOH. [00182] In one embodiment, a trispecific molecule that binds to (i) human CD3, (ii) human serum albumin, and (iii) a complex of the peptide Survivin, wherein the complex comprises the amino acid sequence of LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, comprising the amino acid sequence of SEQ ID NO: 29.

[00183] In some embodiments, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, wherein the sTCR comprises: i. a sTCR variable beta region (Vp), and ii. a sTCR variable alpha region (V_a), wherein the V_[i and V_a regions of the sTCR are connected via a first peptide linker (LI); b) a second domain (A) comprising a V_HH domain which binds to albumin; and c) a third domain (C) comprising a single chain variable fragment (scFv) which binds to human CD3, wherein the scFv comprises a heavy chain variable region (V_H) and a light chain variable region (V_L), wherein V_H and V_L are connected via a second peptide linker (L2), wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-(A)-L3-(C)-L4-(T)-COOH. [00184] In one embodiment, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, wherein the sTCR comprises: i. a sTCR variable beta region (Vp), and ii. a sTCR variable alpha region (V_a), wherein the V and V_a regions of the sTCR are connected via a first peptide linker (LI); b) a second domain (A) comprising a V_HH domain which binds to albumin; and c) a third domain (C) comprising a single chain variable fragment (scFv) which binds to human CD3, wherein the scFv comprises a heavy chain variable region (V_H) and a light chain variable region (VL), wherein VH and VL are connected via a second peptide linker

(L2), wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-VHH-L3-VH-L2-VI -L4-V_[;-L 1- V_a-COOH.

[00185] In one embodiment, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, wherein the sTCR comprises: i. a sTCR variable beta region (Vp) comprising SEQ ID NO: 37 (CDR1), SEQ ID NO: 38 (CDR2) and SEQ ID NO: 39 (CDR3), and ii. a sTCR variable alpha region (V_a) comprising SEQ ID NO: 34 (CDR1), SEQ ID NO: 35 (CDR2) and SEQ ID NO: 36 (CDR3), wherein the V p, and V_a regions of the sTCR are connected via a first peptide linker (LI); b) a second domain (A) comprising a VHH domain which binds to albumin, wherein the VHH domain comprises SEQ ID NO: 46 (CDR1), SEQ ID NO: 47 (CDR2) and SEQ ID NO: 48 (CDR3); and c) a third domain (C) comprising a single chain variable fragment (scFv) which binds to human CD3, wherein the scFv comprises i. a heavy chain variable region (VH) comprising SEQ ID NO: 40 (CDR1), SEQ ID NO: 41 (CDR2) and SEQ ID NO: 42 (CDR3), and ii. a light chain variable region (VL) comprising SEQ ID NO: 43 (CDR1), SEQ ID NO: 44 (CDR2) and SEQ ID NO: 45 (CDR3), wherein V_H and VL are connected via a second peptide linker (L2), wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-VHH-L3-VH-L2-VL-L4-V_|',-L 1- V_a-COOH.

[00186] In one embodiment, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, wherein the sTCR comprises: i. a sTCR variable beta region ( V _[,) comprising the amino acid sequence of SEQ ID NO: 5, and ii. a sTCR variable alpha region (V_a) comprising the amino acid sequence of SEQ ID NO: 8, wherein the V p, and V_a regions of the sTCR are connected via a first peptide linker (LI); b) a second domain (A) comprising a VHH domain which binds to albumin, wherein the VHH domain comprises the amino acid sequence of SEQ ID NO: 12; and c) a third domain (C) comprising a single chain variable fragment (scFv) which binds to human CD3, wherein the scFv comprises i. a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 10, and ii. a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 11, wherein VH and VL are connected via a second peptide linker (L2), wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-VHH-L3-VH-L2-VI -L4-V_|',-L 1- V_a-COOH.

[00187] In one embodiment, the first peptide linker (LI) comprises the amino acid sequence of SEQ ID NO: 1.

[00188] In one embodiment, the second peptide linker (L2) comprises the amino acid sequence of SEQ ID NO: 3.

[00189] In one embodiment, the third peptide linker (L3) comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In another embodiment, the third peptide linker (L3) comprises the amino acid sequence of SEQ ID NO: 2. In another embodiment, the third peptide linker (L3) comprises the amino acid sequence of SEQ ID NO: 4.

[00190] In one embodiment, the fourth peptide linker (L4) comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In another embodiment, the fourth peptide linker (L4) comprises the amino acid sequence of SEQ ID NO: 2. In another embodiment, the fourth peptide linker (L4) comprises the amino acid sequence of SEQ ID NO: 4.

[00191] In one embodiment, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, wherein the sTCR comprises the amino acid sequence of SEQ ID NO: 13; b) a second domain (A) comprising a VHH domain which binds to albumin, wherein the VHH domain comprises the amino acid sequence of SEQ ID NO: 12; and c) a third domain (C) comprising a single chain variable fragment (scFv) which binds to human CD3, wherein the scFv comprises the amino acid sequence of SEQ ID NO: 17, wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-(A)-L3-(C)-L4-(T)-COOH. [00192] In one embodiment, a trispecific molecule that binds to (i) human CD3, (ii) human serum albumin, and (iii) a complex of the peptide Survivin, wherein the complex comprises the amino acid sequence of LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, comprising the amino acid sequence of SEQ ID NO: 22.

[00193] In one embodiment, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, wherein the sTCR comprises:

1 a sTCR variable beta region (Vp) comprising SEQ ID NO: 37 (CDR1), SEQ ID NO: 38 (CDR2) and SEQ ID NO: 39 (CDR3), and n. a sTCR variable alpha region (V_a) comprising SEQ ID NO: 49 (CDR1), SEQ ID NO: 35 (CDR2) and SEQ ID NO: 36 (CDR3), wherein the V and V_a regions of the sTCR are connected via a first peptide linker (LI); b) a second domain (A) comprising a VHH domain which binds to albumin, wherein the VHH domain comprises SEQ ID NO: 46 (CDR1), SEQ ID NO: 47 (CDR2) and SEQ ID NO: 48 (CDR3); and c) a third domain (C) comprising a single chain variable fragment (scFv) which binds to human CD3, wherein the scFv comprises i. a heavy chain variable region (V_H) comprising SEQ ID NO: 40 (CDR1), SEQ ID NO: 41 (CDR2) and SEQ ID NO: 42 (CDR3), and ii. a light chain variable region (V_L) comprising SEQ ID NO: 43 (CDR1), SEQ ID NO: 44 (CDR2) and SEQ ID NO: 45 (CDR3), wherein V_H and VL are connected via a second peptide linker (L2), wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-V_HH-L3-V_H-L2-V_I -L4-V_|',-L 1- V_a-COOH.

[00194] In one embodiment, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, wherein the sTCR comprises: i. a sTCR variable beta region ( V _[,) comprising the amino acid sequence of SEQ ID NO: 5, and ii. a sTCR variable alpha region (V_a) comprising the amino acid sequence of SEQ ID NO: 9, wherein the V p, and V_a regions of the sTCR are connected via a first peptide linker (LI); b) a second domain (A) comprising a VHH domain which binds to albumin, wherein the VHH domain comprises the amino acid sequence of SEQ ID NO: 12; and c) a third domain (C) comprising a single chain variable fragment (scFv) which binds to human CD3, wherein the scFv comprises i. a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 10, and ii. a light chain variable region (V_L) comprising the amino acid sequence of SEQ ID NO: 11, wherein V_H and V_L are connected via a second peptide linker (L2), wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-V_HH-L3-V_H-L2-V_L-L4-V_[;-L 1- V_a-COOH.

[00195] In one embodiment, the first peptide linker (LI) comprises the amino acid sequence of SEQ ID NO: 1.

[00196] In one embodiment, the second peptide linker (L2) comprises the amino acid sequence of SEQ ID NO: 3.

[00197] In one embodiment, the third peptide linker (L3) comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In another embodiment, the third peptide linker (L3) comprises the amino acid sequence of SEQ ID NO: 2. In another embodiment, the third peptide linker (L3) comprises the amino acid sequence of SEQ ID NO: 4. [00198] In one embodiment, the fourth peptide linker (L4) comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In another embodiment, the fourth peptide linker (L4) comprises the amino acid sequence of SEQ ID NO: 2. In another embodiment, the fourth peptide linker (L4) comprises the amino acid sequence of SEQ ID NO: 4.

[00199] In one embodiment, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, wherein the sTCR comprises the amino acid sequence of SEQ ID NO: 14; b) a second domain (A) comprising a VHH domain which binds to albumin, wherein the VHH domain comprises the amino acid sequence of SEQ ID NO: 12; and c) a third domain (C) comprising a single chain variable fragment (scFv) which binds to human CD3, wherein the scFv comprises the amino acid sequence of SEQ ID NO: 17, wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-(A)-L3-(C)-L4-(T)-COOH.

[00200] In one embodiment, a trispecific molecule that binds to (i) human CD3, (ii) human serum albumin, and (iii) a complex of the peptide Survivin, wherein the complex comprises the amino acid sequence of LTLGEFLKL and the HLA-A2 molecule, comprising the amino acid sequence of SEQ ID NO: 28.

[00201] In one embodiment, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, wherein the sTCR comprises: i. a sTCR variable beta region (Vp) comprising SEQ ID NO: 37 (CDR1), SEQ ID NO: 38 (CDR2) and SEQ ID NO: 39 (CDR3), and ii. a sTCR variable alpha region (V_a) comprising SEQ ID NO: 34 (CDR1), SEQ ID NO: 35 (CDR2) and SEQ ID NO: 36 (CDR3), wherein the V and V_a regions of the sTCR are connected via a first peptide linker (LI); b) a second domain (A) comprising a VHH domain which binds to albumin, wherein the VHH domain comprises SEQ ID NO: 46 (CDR1), SEQ ID NO: 47 (CDR2) and SEQ ID NO:

48 (CDR3); and c) a third domain (C) comprising a single chain variable fragment (scFv) which binds to human CD3, wherein the scFv comprises i. a heavy chain variable region (V_H) comprising SEQ ID NO: 40 (CDR1), SEQ ID NO: 41 (CDR2) and SEQ ID NO: 42 (CDR3), and ii. a light chain variable region (V_L) comprising SEQ ID NO: 43 (CDR1), SEQ ID NO: 44 (CDR2) and SEQ ID NO: 45 (CDR3), wherein V_H and V_L are connected via a second peptide linker (L2), wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-VHH-L3-VH-L2-VI -L4-V_|',-L 1- V_a-COOH.

[00202] In one embodiment, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL and the HLA-A2 molecule, wherein the sTCR comprises: i. a sTCR variable beta region ( V _[,) comprising the amino acid sequence of SEQ ID NO: 5, and ii. a sTCR variable alpha region (V_a) comprising the amino acid sequence of SEQ ID NO: 8, wherein the V _f. and V_a regions of the sTCR are connected via a first peptide linker (LI); b) a second domain (A) comprising a VHH domain which binds to albumin, wherein the VHH domain comprises the amino acid sequence of SEQ ID NO: 12; and c) a third domain (C) comprising a single chain variable fragment (scFv) which binds to human CD3, wherein the scFv comprises i. a heavy chain variable region (V_H) comprising the amino acid sequence of SEQ ID NO: 10, and ii. a light chain variable region (V_L) comprising the amino acid sequence of SEQ ID NO: 11, wherein V_H and V_L are connected via a second peptide linker (L2), wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-VHH-L3-VH-L2-VI -L4-V_|',-L 1- V_a-COOH.

[00203] In one embodiment, the first peptide linker (LI) comprises the amino acid sequence of SEQ ID NO: 1. [00204] In one embodiment, the second peptide linker (L2) comprises the amino acid sequence of SEQ ID NO: 3.

[00205] In one embodiment, the third peptide linker (L3) comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In another embodiment, the third peptide linker (L3) comprises the amino acid sequence of SEQ ID NO: 2. In another embodiment, the third peptide linker (L3) comprises the amino acid sequence of SEQ ID NO: 4.

[00206] In one embodiment, the fourth peptide linker (L4) comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In another embodiment, the fourth peptide linker (L4) comprises the amino acid sequence of SEQ ID NO: 2. In another embodiment, the fourth peptide linker (L4) comprises the amino acid sequence of SEQ ID NO: 4.

[00207] In one embodiment, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, wherein the sTCR comprises the amino acid sequence of SEQ ID NO: 13; b) a second domain (A) comprising a VHH domain which binds to albumin, wherein the VHH domain comprises the amino acid sequence of SEQ ID NO: 12; and c) a third domain (C) comprising a single chain variable fragment (scFv) which binds to human CD3, wherein the scFv comprises the amino acid sequence of SEQ ID NO: 17, wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-(A)-L3-(C)-L4-(T)-COOH.

[00208] In one embodiment, a trispecific molecule that binds to (i) human CD3, (ii) human serum albumin, and (iii) a complex of the peptide Survivin, wherein the complex comprises the amino acid sequence of LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, comprising the amino acid sequence of SEQ ID NO: 33.

[00209] In one embodiment, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, wherein the sTCR comprises: i. a sTCR variable beta region (Vp) comprising SEQ ID NO: 37 (CDR1), SEQ ID NO: 38 (CDR2) and SEQ ID NO: 39 (CDR3), and ii. a sTCR variable alpha region (V_a) comprising SEQ ID NO: 34 (CDR1), SEQ ID NO: 35 (CDR2) and SEQ ID NO: 36 (CDR3), wherein the Vp and V_a regions of the sTCR are connected via a first peptide linker (LI); b) a second domain (A) comprising a VHH domain which binds to albumin, wherein the VHH domain comprises SEQ ID NO: 46 (CDR1), SEQ ID NO: 47 (CDR2) and SEQ ID NO:

48 (CDR3); and c) a third domain (C) comprising a single chain variable fragment (scFv) which binds to human CD3, wherein the scFv comprises i. a heavy chain variable region (VH) comprising SEQ ID NO: 40 (CDR1), SEQ ID NO: 41 (CDR2) and SEQ ID NO: 42 (CDR3), and ii. a light chain variable region (V_L) comprising SEQ ID NO: 43 (CDR1), SEQ ID NO: 44 (CDR2) and SEQ ID NO: 45 (CDR3), wherein V_H and V_L are connected via a second peptide linker (L2), wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-V_HH-L3-V_H-L2-V_I -L4-V_|',-L 1- V_a-COOH.

[00210] In one embodiment, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, wherein the sTCR comprises: i. a sTCR variable beta region ( V _[,) comprising the amino acid sequence of SEQ ID NO: 6, and ii. a sTCR variable alpha region (V_a) comprising the amino acid sequence of SEQ ID NO: 8, wherein the V p, and V_a regions of the sTCR are connected via a first peptide linker (LI); b) a second domain (A) comprising a VHH domain which binds to albumin, wherein the VHH domain comprises the amino acid sequence of SEQ ID NO: 12; and c) a third domain (C) comprising a single chain variable fragment (scFv) which binds to human CD3, wherein the scFv comprises i. a heavy chain variable region (V_H) comprising the amino acid sequence of SEQ ID NO: 10, and ii. a light chain variable region (V_L) comprising the amino acid sequence of SEQ ID NO: 11, wherein V_H and V_L are connected via a second peptide linker (L2), wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-V_HH-L3-V_H-L2-V_I -L4-V_(;-L 1- V_a-COOH.

[00211] In one embodiment, the first peptide linker (LI) comprises the amino acid sequence of SEQ ID NO: 1.

[00212] In one embodiment, the second peptide linker (L2) comprises the amino acid sequence of SEQ ID NO: 3.

[00213] In one embodiment, the third peptide linker (L3) comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In another embodiment, the third peptide linker (L3) comprises the amino acid sequence of SEQ ID NO: 2. In another embodiment, the third peptide linker (L3) comprises the amino acid sequence of SEQ ID NO: 4.

[00214] In one embodiment, the fourth peptide linker (L4) comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In another embodiment, the fourth peptide linker (L4) comprises the amino acid sequence of SEQ ID NO: 2. In another embodiment, the fourth peptide linker (L4) comprises the amino acid sequence of SEQ ID NO: 4.

[00215] In one embodiment, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, wherein the sTCR comprises the amino acid sequence of SEQ ID NO: 15; b) a second domain (A) comprising a VHH domain which binds to albumin, wherein the VHH domain comprises the amino acid sequence of SEQ ID NO: 12; and c) a third domain (C) comprising a single chain variable fragment (scFv) which binds to human CD3, wherein the scFv comprises the amino acid sequence of SEQ ID NO: 17, wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-(A)-L3-(C)-L4-(T)-COOH.

[00216] In one embodiment, a trispecific molecule that binds to (i) human CD3, (ii) human serum albumin, and (iii) a complex of the peptide Survivin, wherein the complex comprises the amino acid sequence of LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, comprising the amino acid sequence of SEQ ID NO: 30.

[00217] In one embodiment, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, wherein the sTCR comprises: i. a sTCR variable beta region (Vp) comprising SEQ ID NO: 37 (CDR1), SEQ ID NO: 38 (CDR2) and SEQ ID NO: 39 (CDR3), and ii. a sTCR variable alpha region (V_a) comprising SEQ ID NO: 34 (CDR1), SEQ ID NO: 35 (CDR2) and SEQ ID NO: 36 (CDR3), wherein the V p, and V_a regions of the sTCR are connected via a first peptide linker (LI); b) a second domain (A) comprising a VHH domain which binds to albumin, wherein the VHH domain comprises SEQ ID NO: 46 (CDR1), SEQ ID NO: 47 (CDR2) and SEQ ID NO:

48 (CDR3); and c) a third domain (C) comprising a single chain variable fragment (scFv) which binds to human CD3, wherein the scFv comprises i. a heavy chain variable region (VH) comprising SEQ ID NO: 40 (CDR1), SEQ ID NO: 41 (CDR2) and SEQ ID NO: 42 (CDR3), and ii. a light chain variable region (VL) comprising SEQ ID NO: 43 (CDR1), SEQ ID NO: 44 (CDR2) and SEQ ID NO: 45 (CDR3), wherein V_H and VL are connected via a second peptide linker (L2), wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-VHH-L3-VH-L2-VI -L4-V_|',-L 1- V_a-COOH.

[00218] In one embodiment, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, wherein the sTCR comprises: i. a sTCR variable beta region (VQ comprising the amino acid sequence of SEQ ID NO: 7, and ii. a sTCR variable alpha region (V_a) comprising the amino acid sequence of SEQ ID NO: 8, wherein the V _f. and V_a regions of the sTCR are connected via a first peptide linker (LI); b) a second domain (A) comprising a VHH domain which binds to albumin, wherein the VHH domain comprises the amino acid sequence of SEQ ID NO: 12; and c) a third domain (C) comprising a single chain variable fragment (scFv) which binds to human CD3, wherein the scFv comprises i. a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 10, and ii. a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 11, wherein VH and VL are connected via a second peptide linker (L2), wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-VHH-L3-VH-L2-VI -L4-V_|',-L 1- V_a-COOH.

[00219] In one embodiment, the first peptide linker (LI) comprises the amino acid sequence of SEQ ID NO: 1.

[00220] In one embodiment, the second peptide linker (L2) comprises the amino acid sequence of SEQ ID NO: 3.

[00221] In one embodiment, the third peptide linker (L3) comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In another embodiment, the third peptide linker (L3) comprises the amino acid sequence of SEQ ID NO: 2. In another embodiment, the third peptide linker (L3) comprises the amino acid sequence of SEQ ID NO: 4.

[00222] In one embodiment, the fourth peptide linker (L4) comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In another embodiment, the fourth peptide linker (L4) comprises the amino acid sequence of SEQ ID NO: 2. In another embodiment, the fourth peptide linker (L4) comprises the amino acid sequence of SEQ ID NO: 4.

[00223] In one embodiment, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, wherein the sTCR comprises the amino acid sequence of SEQ ID NO: 16; b) a second domain (A) comprising a VHH domain which binds to albumin, wherein the VHH domain comprises the amino acid sequence of SEQ ID NO: 12; and c) a third domain (C) comprising a single chain variable fragment (scFv) which binds to human CD3, wherein the scFv comprises the amino acid sequence of SEQ ID NO: 17, wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-(A)-L3-(C)-L4-(T)-COOH. [00224] In one embodiment, a trispecific molecule that binds to (i) human CD3, (ii) human serum albumin, and (iii) a complex of the peptide Survivin, wherein the complex comprises the amino acid sequence of LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, comprising the amino acid sequence of SEQ ID NO: 31.

[00225] In some embodiments, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, wherein the sTCR comprises: i. a sTCR variable beta region (Vp), and ii. a sTCR variable alpha region (V_a), wherein the V_[i and V_a regions of the sTCR are connected via a first peptide linker (LI); b) a second domain (A) comprising a V_HH domain which binds to albumin; and c) a third domain (C) comprising a single chain variable fragment (scFv) which binds to human CD3, wherein the scFv comprises a heavy chain variable region (V_H) and a light chain variable region (V_L), wherein V_H and V_L are connected via a second peptide linker (L2), wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-(A)-L3-(T)-L4-(C)-COOH.

[00226] In one embodiment, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, wherein the sTCR comprises: i. a sTCR variable beta region (Vp), and ii. a sTCR variable alpha region (V_a), wherein the V_[i and V_a regions of the sTCR are connected via a first peptide linker (LI); b) a second domain (A) comprising a V_HH domain which binds to albumin; and c) a third domain (C) comprising a single chain variable fragment (scFv) which binds to human CD3, wherein the scFv comprises a heavy chain variable region (V_H) and a light chain variable region (V_L), wherein V_H and V_L are connected via a second peptide linker (L2), wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-V_HH-L3-V_|',-L 1-V„-L4-V_H-L2- V_L-COOH.

[00227] In one embodiment, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, wherein the sTCR comprises: i. a sTCR variable beta region (Vp) comprising SEQ ID NO: 37 (CDR1), SEQ ID NO: 38 (CDR2) and SEQ ID NO: 39 (CDR3), and ii. a sTCR variable alpha region (V_a) comprising SEQ ID NO: 34 (CDR1), SEQ ID NO: 35 (CDR2) and SEQ ID NO: 36 (CDR3), wherein the V p, and V_a regions of the sTCR are connected via a first peptide linker (LI); b) a second domain (A) comprising a VHH domain which binds to albumin, wherein the VHH domain comprises SEQ ID NO: 46 (CDR1), SEQ ID NO: 47 (CDR2) and SEQ ID NO: 48 (CDR3); and c) a third domain (C) comprising a single chain variable fragment (scFv) which binds to human CD3, wherein the scFv comprises i. a heavy chain variable region (VH) comprising SEQ ID NO: 40 (CDR1), SEQ ID NO: 41 (CDR2) and SEQ ID NO: 42 (CDR3), and ii. a light chain variable region (V_L) comprising SEQ ID NO: 43 (CDR1), SEQ ID NO: 44 (CDR2) and SEQ ID NO: 45 (CDR3), wherein V_H and V_L are connected via a second peptide linker (L2), wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-V_HH-L3-V_|',-L 1-V„-L4-V_H-L2- V_L-COOH.

[00228] In one embodiment, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, wherein the sTCR comprises: i. a sTCR variable beta region ( V _[,) comprising the amino acid sequence of SEQ ID NO: 5, and ii. a sTCR variable alpha region (V_a) comprising the amino acid sequence of SEQ ID NO: 8, wherein the V p, and V_a regions of the sTCR are connected via a first peptide linker (LI); b) a second domain (A) comprising a VHH domain which binds to albumin, wherein the VHH domain comprises the amino acid sequence of SEQ ID NO: 12; and c) a third domain (C) comprising a single chain variable fragment (scFv) which binds to human CD3, wherein the scFv comprises i. a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 10, and ii. a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 11, wherein VH and VL are connected via a second peptide linker (L2), wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-VHH-L3-V_[;-L 1-V„-L4-VH-L2- VL-COOH.

[00229] In one embodiment, the first peptide linker (LI) comprises the amino acid sequence of SEQ ID NO: 1.

[00230] In one embodiment, the second peptide linker (L2) comprises the amino acid sequence of SEQ ID NO: 3.

[00231] In one embodiment, the third peptide linker (L3) comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In another embodiment, the third peptide linker (L3) comprises the amino acid sequence of SEQ ID NO: 2. In another embodiment, the third peptide linker (L3) comprises the amino acid sequence of SEQ ID NO: 4.

[00232] In one embodiment, the fourth peptide linker (L4) comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In another embodiment, the fourth peptide linker (L4) comprises the amino acid sequence of SEQ ID NO: 2. In another embodiment, the fourth peptide linker (L4) comprises the amino acid sequence of SEQ ID NO: 4.

[00233] In one embodiment, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, wherein the sTCR comprises the amino acid sequence of SEQ ID NO: 13; b) a second domain (A) comprising a VHH domain which binds to albumin, wherein the VHH domain comprises the amino acid sequence of SEQ ID NO: 12; and c) a third domain (C) comprising a single chain variable fragment (scFv) which binds to human CD3, wherein the scFv comprises the amino acid sequence of SEQ ID NO: 17, wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-(A)-L3-(T)-L4-(C)-COOH. [00234] In one embodiment, a trispecific molecule that binds to (i) human CD3, (ii) human serum albumin, and (iii) a complex of the peptide Survivin, wherein the complex comprises the amino acid sequence of LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, comprising the amino acid sequence of SEQ ID NO: 20.

[00235] In one embodiment, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, wherein the sTCR comprises: i. a sTCR variable beta region (Vp) comprising SEQ ID NO: 37 (CDR1), SEQ ID NO: 38 (CDR2) and SEQ ID NO: 39 (CDR3), and ii. a sTCR variable alpha region (V_a) comprising SEQ ID NO: 49 (CDR1), SEQ ID NO: 35 (CDR2) and SEQ ID NO: 36 (CDR3), wherein the V and V_a regions of the sTCR are connected via a first peptide linker (LI); b) a second domain (A) comprising a VHH domain which binds to albumin, wherein the VHH domain comprises SEQ ID NO: 46 (CDR1), SEQ ID NO: 47 (CDR2) and SEQ ID NO: 48 (CDR3); and c) a third domain (C) comprising a single chain variable fragment (scFv) which binds to human CD3, wherein the scFv comprises i. a heavy chain variable region (V_H) comprising SEQ ID NO: 40 (CDR1), SEQ ID NO: 41 (CDR2) and SEQ ID NO: 42 (CDR3), and ii. a light chain variable region (V_L) comprising SEQ ID NO: 43 (CDR1), SEQ ID NO: 44 (CDR2) and SEQ ID NO: 45 (CDR3), wherein V_H and V_L are connected via a second peptide linker (L2), wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-V_HH-L3-V_[;-L 1-V„-L4-V_H-L2-

V_L-COOH. [00236] In one embodiment, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, wherein the sTCR comprises: i. a sTCR variable beta region ( V p.) comprising the amino acid sequence of SEQ ID NO: 5, and ii. a sTCR variable alpha region (V_a) comprising the amino acid sequence of SEQ ID NO: 9, wherein the V p, and V_a regions of the sTCR are connected via a first peptide linker (LI); b) a second domain (A) comprising a VHH domain which binds to albumin, wherein the VHH domain comprises the amino acid sequence of SEQ ID NO: 12; and c) a third domain (C) comprising a single chain variable fragment (scFv) which binds to human CD3, wherein the scFv comprises i. a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 10, and ii. a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 11, wherein VH and VL are connected via a second peptide linker (L2), wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-VHH-L3-V_[I-L 1-V„-L4-VH-L2- VL-COOH.

[00237] In one embodiment, the first peptide linker (LI) comprises the amino acid sequence of SEQ ID NO: 1.

[00238] In one embodiment, the second peptide linker (L2) comprises the amino acid sequence of SEQ ID NO: 3.

[00239] In one embodiment, the third peptide linker (L3) comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In another embodiment, the third peptide linker (L3) comprises the amino acid sequence of SEQ ID NO: 2. In another embodiment, the third peptide linker (L3) comprises the amino acid sequence of SEQ ID NO: 4.

[00240] In one embodiment, the fourth peptide linker (L4) comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In another embodiment, the fourth peptide linker (L4) comprises the amino acid sequence of SEQ ID NO: 2. In another embodiment, the fourth peptide linker (L4) comprises the amino acid sequence of SEQ ID NO: 4. [00241] In one embodiment, a trispecific molecule comprises: a) a first domain (T) comprising 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 LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, wherein the sTCR comprises the amino acid sequence of SEQ ID NO: 14; b) a second domain (A) comprising a VHH domain which binds to albumin, wherein the VHH domain comprises the amino acid sequence of SEQ ID NO: 12; and c) a third domain (C) comprising a single chain variable fragment (scFv) which binds to human CD3, wherein the scFv comprises the amino acid sequence of SEQ ID NO: 17, wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-(A)-L3-(T)-L4-(C)-COOH. [00242] In one embodiment, a trispecific molecule that binds to (i) human CD3, (ii) human serum albumin, and (iii) a complex of the peptide Survivin, wherein the complex comprises the amino acid sequence of LTLGEFLKL (SEQ ID NO: 51) and the HLA-A2 molecule, comprising the amino acid sequence of SEQ ID NO: 26.

[00243] In another aspect, the present disclosure pertains to a pharmaceutical composition comprising a trispecific molecule of the present invention.

[00244] In another aspect, the present disclosure pertains to a method of treating a colorectal or prostate cancer, comprising administering to a patient in need thereof, a trispecific molecule of the present invention, or a pharmaceutical composition thereof.

[00245] In another aspect, the present disclosure pertains to nucleic acid molecules encoding the trispecific molecules of the present invention as well as polynucleotides encoding the amino acid sequences listed in Table 1.

[00246] In another aspect, the present disclosure pertains to vectors comprising nucleic acid molecules encoding the trispecific molecules of the present invention as well as vectors comprising polynucleotides encoding the amino acid sequences listed in Table 1.

[00247] In another aspect, the present disclosure pertains to host cells capable of producing the trispecific molecules of the present invention.

EXAMPLES

[00248] The following Examples are provided for purposes of illustration, and not limitation.

[00249] TCR-CD3 Trispecific Molecule Generation [00250] Trispecific molecules were generated. The polypeptide sequence of each component of the trispecific molecules is listed in Table 1. CDRs within such polypeptides are underlined and their sequences are separately identified.

Table 1

[00251] Example 1

[00252] Two soluble T cell receptor (sTCR) sequences targeting SURVIVIN, la5EP-wt and la5EP- Y29Ga (SEQ ID NOS 13 and 14), were cloned into TriTAC expression constructs. Each TriTAC contained a target domain (T), in this case a sTCR targeting SURVIVIN, a CD3 binding domain (C) in the form of an anti-CD3-scFv (SEQ ID NO 17), and an albumin binding domain (A) in the form of an anti-albumin single domain antibody (SEQ ID NO 12). For each TriTAC, the three domains could be arranged in six different configurations (Fig 1): T:A:C, T:C:A, A:T:C, C:T:A, A:C:T, and C:A:T. Thus, for each of the two sTCR sequences, six expression constructs were created (SEQ ID NOS 18 to 29).

Each construct contained a signal domain for secreted expression from mammalian cells. Connecting each domain were GGGGSGGGS amino acid linkers (SEQ ID NO. 2). The C-terminus of each construct contained a HHHHHH amino acid sequence (SEQ ID NO. 50). In preferred embodiments, the C- terminus HHHHHH amino acid sequence is not present in the final trispecific molecule product.

[00253] The SURVIVIN-targeting TriTAC constructs were transfected into Expi293 cells or CHO-EBNA cells (Life Technologies) for secreted protein expression. These SURVIVIN-targeting TriTAC proteins were engineered with a protein A binding site, and the amount of TriTAC protein in the conditioned media from the transfected Expi293 or CHO-EBNA cells was quantitated using by using an Octet instrument with protein A tips using a TriTAC protein of similar molecular weight to the SURVIVIN- targeting TriTAC proteins as a standard.

[00254] The conditioned media were tested in a T-cell dependent cellular cytotoxicity (TDCC) assay to confirm the SURVIVIN-targeting TriTACs direct T cells to kill cancer cell that have MHC presenting SURVIVIN peptide (Nazarian AA, Archibeque IL, Nguyen YH, Wang P, Sinclair AM, Powers DA.

2015. J Biomol Screen. 20:519-27). In this assay, luciferase labelled HCT116 cells were combined with purified human T cells from two different healthy human donors and a titration of SURVIVIN-targeting TriTAC proteins. The T cell donors were healthy anonymous subjects and were referred to as Donor 02 (D02) and Donor 35 (D35). The titration was based on the quantitation described in the previous paragraph. If a SURVIVIN-targeting TriTAC protein directs T cells to kill the HCT116 cells, then the viability of the HCT116 cells, determined by running a luciferase assay at 96 hours after starting the experiment, should decrease. Because the TriTACs molecules contain an anti -albumin domain, an additional assay was run in parallel in the presence of 15 mg/mL human serum albumin (HSA) to assess the activity of the SURVIVIN-targeting TriTACs when bound to HSA. The viability results from the TDCC assay versus the concentration of TriTACs added were plotted in Fig. 2 (in the graphs, plotted are the mean viability data ± standard error of the mean, n=2). The EC50 values for cell killing are listed in Table 2. The graphs show that at the highest concentrations of TriTACs tested, either with the la5EP-wt or the la5EP-Y29Ga sequence and in the absence or presence of HSA, that the C:T:A configuration had the fewest HCT116 cells remaining alive at the end of the assay. In addition, the C:T:A configuration had most potent killing in the absence or presence of HSA (Table 2). In the presence of HSA, the A:C:T configuration had the second highest amount of HCT116 cell killing and the second highest potency. A negative control TriTAC in the T:A:C configuration targeting GFP did not direct killing of the HCT116 cells except for slight activity at the highest concentration tested in the presence of HSA.

Table 2 Activity of SURVIVIN-targeting TriTACs in HCT116 Cells.

[00255] Example 2

[00256] To confirm that results with the HCT116 cells could be extended to additional cell lines, the SURVIVIN-targeting TriTACs (SEQ ID NOS 18 to 29) present in conditioned media were tested in TDCC assays with T2 cells and PC3 cells. Because T2 cells do not endogenously present SURVIVIN peptide on MHC, SURVIVIN peptide was included in the co-cultures of T2 cells and T cells. The PC3 cells were engineered to express HEAa2/b2M (abbreviated as R6'3-a2/b2M). Otherwise, the assays were performed identically to the HCT116 assay (Example 1) except that viability of the T2 or R03-a2/b2M cells was measured at 48 hours, and the R03-a2/b2M were only tested with T cells from Donor 35. The T2 cell viability versus the concentration of TriTAC added was plotted in Fig. 3, and the EC50 values for cell killing are listed in Table 3. The R03-a2/b2M cell viability versus the concentration of TriTAC added is plotted in Fig. 4, and the EC50 values for cell killing are listed in Table 4. The results with the T2 and R6'3-s.2/b2M cells differ from the results with HCT116 cells in two ways. First, unlike with the HCT116 cells, all of the T2 and RE3-s.2/b2M cells were killed in the killing assay. Second, there was more potent killing of the T2 and RE3-s.2/b2M cells compared to the HCT116 cells as evident by the lower EC50 values with the T2 cells (compare results in Tables 2, 3, and 4). As observed with the HCT116 cells, TriTACs in the C:T:A and A:C:T configuration generally had the more potent directed cell killing activity with T cells from both donors and in the absence or presence of albumin than other configurations. Table 3 Activity of SURVIVIN-targeting TriTACs with T2 Cells with SURVIVIN peptide.

Table 4 Activity of SURVIVIN-targeting TriTACs with R0'3-s.2/b2M.

[00257] Example 3

[00258] To assess if the configuration of the TriTACs affected binding to human serum albumin (HSA), measurements were made using an Octet instrument. The instrument tips contained streptavidin, and first they were loaded with biotinylated albumin. Subsequently, the tips were incubated with conditioned medium containing 50 nM of each SURVIVIN-targeting TriTAC. Because the K_D measurements were made using a single 50 nM concentration of TriTAC protein, the results provided a rank ordering potency but did not provide definitive K_D values. These measured relative affinities are listed in Table 5. All of the sequences bound to HSA, and the KD values ranged from 1.5 to 12.5 nM, with the KD values being the smallest in the configurations with the albumin binding domain on the N-terminus (A:T:C and A:C:T).

[00259] Example 4

[00260] To test the impact of linker selections on the activity of the SURVIVIN-targeting TriTACs, different linkers were tested. Starting with TriTACs in the C:T:A and the A:C:T configurations and containing the la5EP-wt sequence, different linker sequences were inserted between the binding domains. These linker sequences were GGGGS (G4S) (SEQ ID NO. 4) and GGGGSGGGS (G4SG3S) (SEQ ID NO. 2). For two configurations, different linker combinations were tested. For the C:T:A configuration, SEQ ID NO: 21 (aCD3:: la5EP-wt::aALB) was compared to SEQ ID NO: 32 (aCD3:la5EP-wt::aALB-2). For the A:C:T configurations, SEQ ID NO: 22 (aALB::aCD3::la5EP-wt) was compared to SEQ ID NO: 33 (aALB::aCD3: la5EP-wt-3). These molecules were tested in a TDCC assay with R€3-s.2/b2M cells performed as described in Example 2 using two different T cell donors. These molecules were also tested in a TDCC assay with HCT116 cells performed as described in Example 1 using T cells from one donor. As shown in Table 6, altering the length of the linkers had minimal or no effect on the activities of TriTACs molecules such as the ability of directing T cells.

Table 6 TDCC Assays on TriTACs with different linkers

[00261] Example 5

[00262] Four SURVIVIN-targeting TriTACs (SEQ ID NOS 21, 22, 32, and 33) were selected for larger scale expression and protein purification. Plasmid DNAs were transfected into Expi293 or CHO-EBNA cells (Life Technologies) and were purified using Protein A affinity chromatography and mixed mode ion exchange chromatography. For example, and without being bound by any particular methodology, 1 mg of DNA per liter of cell culture was transfected into CHO-EBNA cells at a density of 5 x 10⁶ cells/mL using Polyethylenimine Max (PEI Max , Polysciences Inc) at a PEEDNA ratio of 6: 1. CHO-EBNA cells were cultivated in chemically defined medium in suspension at 5% CO2 at 37 °C, in baffled shaking flasks (140 RPM). Chemically defined feeds were added 1, 4, 6, and 8 days post-transfection. On day 14 post-transfection, transfected cell cultures were cleared by centrifugation followed by filtration through 0.2 pm PES filter (Millipore). The cell-free harvest was loaded on a MabSelect™ column (Cytiva) equilibrated with 20 mM Acetate, 22 mM Tris, pH 7.2. The column was washed with 20 mM Sodium Acetate, 22 mM Tris, 500 mM Sodium Chloride, pH 7.2 and bound protein was eluted with 50 mM Acetic Acid, pH 3.0. The pH of Protein A eluate was lowered to 3.5 using 0.5 M phosphoric acid and held at this pH for 30 to 60 minutes at room temperature. The pH was then increased to 6.5 using 1.0 M Tris. Neutralized protein was further purified by Phenyl Sepharose™ 6 Fast Flow (high sub) column (Cytiva) equilibrated and run with 20 mM Acetate, 22 mM Tris, pH 6.5. Fractions containing protein were pooled and concentration was measured by absorbance at 280 nm, and samples were analyzed by capillary electrophoresis (CE)-SDS, and mass spectrometry. The final material was stored in aliquots at - 80 °C. A representative SDS-PAGE of the purified proteins is provided in Figure 5 and demonstrates the purity of the proteins. The proteins were also analyzed by size exclusion chromatography (SEC) to evaluate their purity under non-denaturing conditions. Analysis of the SEC data (Table 7) indicated that the proteins purified by the method described here had 94.4% to 97.3% monomer SURIVIN-TriTAC protein. In addition, in order to mitigate deamidation and possible isomerization, aALB: :aCD3: : la5EP- wt-1 (SEQ ID NO: 30) was made by inserting Ala at the C-terminus of Vp variable chain of the sTCR of aALB::aCD3::la5EP-wt (SEQ ID NO: 22) ; and aALB : : aCD3 : : 1 a5 EP-wt-2 (SEQ ID NO: 31) was also made by replacing Asn at the C-terminus of aALB::aCD3::la5EP-wt (SEQ ID NO: 22) with Asp.

Table 7 Purity of purified SURVIVIN-targeting TriTACs by size exclusion chromatography

[00263] The affinities of SURVIVIN-targeting TriTACs were measured for binding to CD3s and HSA protein in vitro. Using an Octet instrument with streptavidin tips loaded with biotinylated-CD3s or biotinylated-albumin proteins, affinity measurements were made by incubating these tips in solutions containing 9, 27, or 81 nM solutions of purified TriTAC proteins. For the CD3s binding measurements, the TriTAC solutions also contained 15 mg/mL HSA. The results of these affinity measurements are listed in Table 8. The TriTACs bound to similar affinities to CD3s, with the K_D values ranging from 2.7 to 4.6 nM. The KD values for the TriTACs in the A:C:T configuration were smaller than in the C:T:A configuration (2.5 to 2.7 nM versus 13 to 15 nM). Table 8 Binding affinities of purified SURVIVIN-targeting TriTACs to human CD3s and HSA proteins

[00264] SURVIVIN-targeting TriTACs were tested in TDCC assays with R€3-s.2/b2M. T2, and HCT116 cells performed as described in Examples 1 and 2 except the T cells from anonymous donor 47 were used, and instead of running the assay in the presence or absence of HSA, the assay was run with bovine serum albumin (BSA) versus HSA. The anti-albumin domain in the SURVIVIN-targeting TriTACs does not measurably bind to BSA (data not shown). The results of these TDCC assays are in plotted in Figure 6 and the EC50 values for directed T cell killing are listed in Table 9. T cell directed killing was observed with all three cell lines. HSA reduced the potency of killing, and the killing was less potent with the HCT116 cells versus the RE3-s.2/b2M and T2 cells.

Table 9 TDCC activity of purified SURVIVIN-targeting TriTACs with PC-a2^2M, T2, and HCT116 cells

[00265] As shown, both the C:T:A construct and A:C:T construct exhibited similar in vitro activities, and both constructs contained a His-tag at the C-terminus. The C:T:A construct was not chosen for further development because it was hypothesized that removal of the His-tag may negatively impact the efficacy of that construct due to the potential for immunogenicity from the C-terminus of the albumin binding domain. However, this characteristic is not shared with the A:C:T construct as the His-tag can be removed from the construct without negative impact, i.e., the C-terminus of the albumin binding domain is not exposed in such a construct. Therefore, the A:C:T construct was selected for further development.

[00266] Example 6 [00267] SURVIVIN-targeting TriTACs were tested in functional assays using various acute myeloid leukemia (AML) cell lines. Briefly, cell lines OCI-AML2 (ACC-99), OCI-AML3 (ACC-582), and OCI- Lyl9 (ACC-528) were purchased from Leibniz-Institute DSMZ (German Collection of Microorganisms and Cell Cultures GmbH) and were cultured in a-MEM supplemented with 20% FBS and incubated at 37 °C and 5% CO2 for use as target cells. The target cells were stained with CellVue™ Burgundy (Invitrogen) prior to co-culture with T cells (e.g., effector 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 mM 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.

[00268] Effector T cells for use in the functional assays were prepared as follows. Effector T cells were isolated from donor PBMC stocks by negative selection using a T cell isolation kit (Miltenyi) on FS 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% CO2 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 10⁷ per 50 mL tube in 10 mL PBS. CellTrace™ stock solution was prepared immediately prior to use by adding the 20 pL 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% C02 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.

[00269] Functional analysis of SURVIVIN-targeting TriTACs comprised redirected T cell cytotoxicity and activation assays. The target cells, labeled with CellVue™ Burgundy, were seeded at 20,000 cells per well into a round bottom 96-well plate (BD) in 50 pi volume per well. The CellTrace™ Violet-labeled effector T cells were added to appropriate wells (in duplicate) at 200,000 cells per well in 50 pi volume, for approximate Effector T-cell / Target ratio (E:T) of 10: 1. Serially diluted TriTAC was added to appropriate wells in a 50 pi volume, and titrated in a 3-fold dilution across 9 wells (in duplicate). The mixed cultures were placed at 37 °C and 5% C02 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 mΐ/ml FACS buffer and 25 pi 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 mΐ/ml and 15 mΐ/ml, respectively, and added to wells at 25 mΐ/well for 15 minutes at room temperature in the dark. At the end of the incubation, 75 mΐ 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 EC50 values calculated with the aid of GraphPad 5.0 Software.

[00270] As shown in Figure 7, the A:C:T construct aALB::aCD3:: la5EP-wt-l (SEQ ID NO: 30) was able to induce potent redirected killing by CD3⁺ T cells against the HLA-A2, Survivin-positive AML cell lines OCI-AML2 and OCI-AML3 across 4 healthy CD3⁺ T cell donors, while no activity was observed with a negative control (irrelevant TriTAC). However, such cell death was not observed in the HLA-A2 negative, Survivin-positive AML cell line OCI-Lyl9 due to the lack of HLA-A2 expression by this cell line. (See Figure 7, top row.)

[00271] Likewise, aALB::aCD3:: la5EP-wt-l induced potent activation of CD3⁺ T cells against HLA-A2, Survivin-positive AML cell lines OCI-AML2 and OCI-AML3 across 4 healthy CD3⁺ T cell donors (no activity observed with irrelevant TriTAC), but only minimal activation was observed against the HLA-A2 negative, Survivin-positive AML cell line OCI-Lyl9, as measured by CD69 expression. (See Figure 7, bottom row.)

[00272] Example 7

[00273] Tumor growth inhibition of PC3M A2/B2M following treatment with 848-1 with or without donor-derived (RV228) CD3⁺ T cells. Briefly, the prostate cancer cell line PC3M was transduced to express the HLA-A2 and B2M genes. By engineering PC3M to express HLA-A2, it potentially becomes susceptible to targeting by aALB::aCD3:: la5EP-wt-l. The cells were grown by tissue culture and then transferred to immunodeficient C.B-17 SCID mice. Cells, both tumor and T cells, were co-injected in a O.lmL Matrigel suspension, at a 1: 1 ratio, thus ensuring tumor cells and T cells remain in close proximity and do not diffuse throughout the animal from the sight of injection (the right hindflank). As illustrated in Figure 8, the administration of SURVIVIN-targeting TriTACs in the presence of CD3⁺ T cells inhibited tumor growth in mice.

[00274] 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.

[00275] 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).

Claims

WE CLAIM:

1. A trispecific molecule that binds to human Survivin, CD3 and albumin, wherein the trispecific molecule comprises: a) a first domain (T) comprising a sTCR comprising: i. a V_p comprising SEQ ID NO: 37 (CDR1), SEQ ID NO: 38 (CDR2) and SEQ ID NO: 39 (CDR3), and ii. a V_a comprising SEQ ID NO: 34 (CDR1), SEQ ID NO: 35 (CDR2) and SEQ ID NO: 36 (CDR3), wherein the V_p and V_a regions of the sTCR are connected via a first peptide linker (LI); b) a second domain (A) comprising a VHH comprising SEQ ID NO: 46 (CDR1), SEQ ID NO: 47 (CDR2) and SEQ ID NO: 48 (CDR3); and c) a third domain (C) comprising a scFv comprising: i. a V_H comprising SEQ ID NO: 40 (CDR1), SEQ ID NO: 41 (CDR2) and SEQ ID NO: 42 (CDR3), and ii. a V_L comprising SEQ ID NO: 43 (CDR1), SEQ ID NO: 44 (CDR2) and SEQ ID NO: 45 (CDR3), wherein V_H and V_L are connected via a second peptide linker (L2), wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-V_HH-L3-V_H-L2-V_L-L4-V_P-L1- V_a-COOH.

2. A trispecific molecule that binds to human Survivin, CD3 and albumin, wherein the trispecific molecule comprises: a) a first domain (T) comprising a sTCR comprising: i. aVp comprising the amino acid sequence of SEQ ID NO: 6, and ii. a V_a comprising the amino acid sequence of SEQ ID NO: 8, wherein the V_[i and V_a regions of the sTCR are connected via a first peptide linker (LI); b) a second domain (A) comprising a V_HH comprising the amino acid sequence of SEQ ID NO: 12; and c) a third domain (C) comprising a scFv comprising: i. a V_H comprising the amino acid sequence of SEQ ID NO: 10, and ii. a V_L comprising the amino acid sequence of SEQ ID NO: 11, wherein V_H and V_L are connected via a second peptide linker (L2), wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order 1¾N-V_HH-L3-V_H-L2-V_L-L4-VP-L1- V_a-COOH.

3. The trispecific molecule of claim 1 or 2, wherein the LI comprises the amino acid sequence of SEQ ID NO: 1.

4. The trispecific molecule of claim 1 or 2, wherein the L2 comprises the amino acid sequence of SEQ ID NO: 3.

5. The trispecific molecule of claim 1 or 2, wherein the L3 comprises the amino acid sequence of SEQ ID NO: 2.

6. The trispecific molecule of claim 1 or 2, wherein the L4 comprises the amino acid sequence of SEQ ID NO: 2.

7. A trispecific molecule that binds to human Survivin, CD3 and albumin, wherein the trispecific molecule comprises: a) a first domain (T) comprising a sTCR comprising the amino acid sequence of SEQ ID NO: 15; b) a second domain (A) comprising a V_HH domain comprising the amino acid sequence of SEQ ID NO: 12; and c) a third domain (C) comprising a scFv comprising the amino acid sequence of SEQ ID NO: 17, wherein the domains of T, A and C are connected via a third linker (L3) and a fourth linker (L4) to form the trispecific molecule in the order H₂N-(A)-L3-(C)-L4-(T)-COOH.

8. The trispecific molecule of claim 7, wherein the L3 comprises the amino acid sequence of SEQ

ID NO: 4.

9. The trispecific molecule of claim 7, wherein the L4 comprises the amino acid sequence of SEQ ID NO: 4.

10. A trispecific molecule that binds to human Survivin, CD3 and albumin, wherein the trispecific molecule comprises the amino acid sequence of SEQ ID NO: 30.