Bispecific molecules

Abstract

The present invention provides bispecific molecules comprising a soluble TCR and an immune cell engaging domain, half-life extended bispecific molecules comprising such bispecific molecules and a half-life extending domain, methods of producing such bispecific molecules and half-life extended bispecific molecules, and uses thereof.

Description

[0001] BISPECIFIC MOLECULES

[0002] Field of the invention

[0003] The present invention relates generally to bispecific molecules comprising a soluble TCR and an immune cell engaging domain, half-life extended bispecific molecules comprising such bispecific molecules and a half-life extending domain, methods of producing such bispecific molecules and halflife extended bispecific molecules, and uses thereof.

[0004] Background to the invention

[0005] Bispecific molecules that redirect the T cell response against diseased cells are revolutionising the treatment of certain diseases including cancer. The vast majority of bispecific molecules that are currently in clinical trials, or that have received regulatory approval, are designed to target cell surface antigens. However, many diseases are caused by aberrant intracellular proteins and such antigens are not targetable by traditional bispecific antibodies. Bispecific molecules based on T cell receptors are instead able to target intracellular antigens which are presented as a short peptide fragment in complex with a cell surface expressed MHC molecule. In 2022 the first TCR bispecific molecule was approved for the treatment of metastatic uveal melanoma.

[0006] The design and construction of soluble TCR bispecific molecules that are suitable for therapeutic use is challenging and many factors need to be considered. Desirable features of such molecules include supraphysiological affinity to target pMHC antigen, which may be present at very low levels, potent and specific T cell activation at picomolar concentrations of reagent, and a suitable in vivo half-life to maximise efficacy of drug and minimise dosing frequency. TCR bispecific molecules based on the ImmTAC™ platform address many of these challenges. ImmTAC™ molecules are engineered for high affinity target recognition and generate a potent and specific T cell response against antigen positive cells even at low antigen density. However, the smaller size of ImmTAC™ molecules (~75 kDa) and lack of FcRn receptor binding leads to a relatively short in vivo half-life, on the order of several hours.

[0007] Increasing the molecule size and inclusion of half-life extending (HLE) domains, such as IgG Fc and other domains that bind to FcRn, are known strategies in the art to improve the retention time of antibody based bispecific molecules in vivo. However, larger molecules, and especially those T cell engagers based on TCRs, also risk interfering with optimal immune synapse formation by, for example, increasing the intermembrane distance between pMHC on the target cell and the receptor on the immune cell resulting in loss of potency. This risk may be particularly acute when targeting low density pMHC antigens, frequently associated with disease. There is also a risk that larger molecules could hinder tumour penetration of the drug in vivo. The format of the TCR bispecific molecule is hence critical to ensure optimal integration all the desirable properties. The inventors of the present application have unexpectedly identified formats of TCR bispecific molecules that retain, and in some cases further improve, the beneficial features of ImmTAC™ molecules even when the molecule size is decreased (e.g. to less than 60 kDa) and when a HLE domain is attached. Such molecules are suitable for use in the development of therapeutic reagents for treating various diseases.

[0008] Summary of the invention

[0009] The inventors tested over 20 different formats (i.e., orientations and positions of each domain in the polypeptide) for a bispecific molecule comprising a soluble TCR and an immune cell engaging domain. The inventors compared small (e.g., less than 60 kDa in size) formats lacking TCR constant domains with the original molecule (comprising the same domains and same sequences but additionally including the TCR constant domains, i.e. the format of an ImmTAC™). In doing so, they found that many small formats exhibited a loss of potency in vitro compared with the original molecule. However, the inventors surprisingly identified small formats which have increased potency compared with the original molecule. The inventors also tested different formats for a bispecific molecule comprising a soluble TCR, an immune cell engaging domain and a half-life extending (HLE) domain. It has previously been shown that attaching a HLE domain to a molecule in the format of an ImmTAC™ reduces their potency. However, the inventors surprisingly identified small formats which have comparable or increased potency compared with the original molecule in the format of an ImmTAC™.

[0010] In a first aspect, the invention provides a bispecific molecule comprising a soluble TCR and an immune cell engaging domain, wherein said soluble TCR binds to a peptide-major histocompatibility complex (pMHC) and comprises a TCR alpha chain variable domain (Va) and a TCR beta chain variable domain (Vp); wherein said immune cell engaging domain comprises an antibody light chain variable domain (VL), and an antibody heavy chain variable domain (VH); wherein said bispecific molecule is not greater than 60 kDa in size; and wherein said VH is linked to the N-terminus of the Vp.

[0011] In certain embodiments, the soluble TCR binds to the pMHC with a KD of less than 10 nM, less than 1 nM, less than 900 pM, less than 800 pM, less than 700 pM, less than 600 pM, less than 500 pM, less than 400 pM, less than 300 pM, less than 200 pM, or less than 100 pM.

[0012] In certain embodiments, the Va is linked to the VL. Thus, the invention also provides a bispecific molecule comprising a soluble TCR and an immune cell engaging domain, wherein said soluble TCR binds to a peptide-major histocompatibility complex (pMHC) and comprises a TCR alpha chain variable domain (Va) and a TCR beta chain variable domain (Vp); wherein said immune cell engaging domain comprises an antibody light chain variable domain (VL), and an antibody heavy chain variable domain (VH); wherein said bispecific molecule is not greater than 60 kDa in size; wherein said VH is linked to the N-terminus of the Vp; and wherein said Va is linked to said VL.

[0013] In certain embodiments, the bispecific molecule is a single chain molecule. The single chain bispecific molecule may have a format selected from the group consisting of:

[0014] (i) VL-VH-Vp-Va;

[0015] (ii) VH-Vp-Va-VL; and

[0016] (iii) Va-VL-VH-Vp.

[0017] In certain embodiments, the bispecific molecule is a dual chain molecule. The dual chain bispecific molecule may comprise:

[0018] (i) a first chain comprising VH-Vp; and

[0019] (ii) a second chain comprising Va-VL.

[0020] In certain embodiments, the Vp is linked to the VL via a disulphide bond.

[0021] In certain embodiments, two or more of the Va, Vp, VH and VL are linked via a linker and / or an IgG hinge sequence.

[0022] In certain embodiments, the bispecific molecule comprises a soluble TCR which has the property of binding to a ALWGPDAAA (SEQ ID NO: 45) HLA-A*02 complex. In certain embodiments:

[0023] (a) the Va comprises a CDR1 , a CDR2 and a CDR3 comprising the following sequences: CDR1 - DKHSQG (SEQ ID NO: 28), optionally with one, two or three mutations therein, CDR2 - IYSQGD (SEQ ID NO: 80), optionally with one, two or three mutations therein, CDR3 - AVRGNEKLT (SEQ ID NO: 30), optionally with one, two or three mutations therein, and / or

[0024] (b) the Vp comprises a CDR1 , a CDR2 and a CDR3 comprising the following sequences: CDR1 - LQHSY (SEQ ID NO: 81), optionally with one, two or three mutations therein, CDR2 - SVGVGF (SEQ ID NO: 33), optionally with one, two or three mutations therein, CDR3 - ASAYMTGELF (SEQ ID NO: 34), optionally with one, two or three mutations therein; optionally wherein the soluble TCR comprises a Va comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or is 100% identical to SEQ ID NO: 27 and a Vp comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or is 100% identical to SEQ ID NO: 31.

[0025] In certain embodiments, the immune cell engaging domain of the bispecific molecule binds to a T cell surface antigen. In certain embodiments, the T cell surface antigen is CD3. In certain embodiments, the immune cell engaging domain comprises:

[0026] (a) a VL comprising a CDR1 , a CDR2 and a CDR3 comprising the following sequences:

[0027] CDR1 - QDIRNY (SEQ ID NO: 36), CDR2 - YTS (SEQ ID NO: 37),

[0028] CDR3 - QQGNTLPWT (SEQ ID NO: 38); and

[0029] (b) a VH comprising a CDR1 , a CDR2 and a CDR3 comprising the following sequences: CDR1 - GYSFTGYA (SEQ ID NO: 40),

[0030] CDR2 - INPYKGVS (SEQ ID NO: 41),

[0031] CDR3 - ARSGYYGDSDWYFDV (SEQ ID NO: 42); optionally wherein the immune cell engaging domain comprises a VL comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or is 100% identical to SEQ ID NO: 35 and a VH comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or is 100% identical to the sequence of SEQ ID NO: 39.

[0032] Bispecific molecules of the invention may comprise the amino acid sequence provided in any one of SEQ ID Nos: 1-7.

[0033] In certain embodiments, the bispecific molecule comprises a soluble TOR which has the property of binding to a SLLQHLIGL (SEQ ID NO: 111) HLA-A*02 complex. In certain embodiments:

[0034] (a) the Va comprises a CDR1 , a CDR2 and a CDR3 comprising the following sequences: CDR1 - TISGTDY (SEQ ID NO: 92), optionally with one, two or three mutations therein, CDR2 - GLTSN (SEQ ID NO: 93), optionally with one, two or three mutations therein, CDR3 - CILILGHSRLGNYIATF (SEQ ID NO: 94), optionally with one, two or three mutations therein, and / or

[0035] (b) the Vp comprises a CDR1 , a CDR2 and a CDR3 comprising the following sequences: CDR1 - LNHDA (SEQ ID NO: 96), optionally with one, two or three mutations therein, CDR2 - SQIMGD (SEQ ID NO: 97), optionally with one, two or three mutations therein, CDR3 - CASSWWTGGASPIRF (SEQ ID NO: 98), optionally with one, two or three mutations therein; optionally wherein the soluble TOR comprises a Va comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or is 100% identical to SEQ ID NO: 91 and a Vp comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or is 100% identical to SEQ ID NO: 95.

[0036] In certain embodiments, the immune cell engaging domain of the bispecific molecule binds to a T cell surface antigen. In certain embodiments, the T cell surface antigen is CD3. In certain embodiments, the immune cell engaging domain comprises:

[0037] (a) a VL comprising a CDR1 , a CDR2 and a CDR3 comprising the following sequences:

[0038] CDR1 - QGIRKY (SEQ ID NO: 100),

[0039] CDR2 - AAS (SEQ ID NO: 101),

[0040] CDR3 - QQGNTLPWT (SEQ ID NO: 102); and / or

[0041] (b) a VH comprising a CDR1 , a CDR2 and a CDR3 comprising the following sequences: CDR1 - GYRFTGYL (SEQ ID NO: 104),

[0042] CDR2 - INPYKGST (SEQ ID NO: 105),

[0043] CDR3 - ARSGYYGDSDWYFDL (SEQ ID NO: 106); optionally wherein the immune cell engaging domain comprises a VL comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or is 100% identical to SEQ ID NO: 99 and a VH comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or is 100% identical to the sequence of SEQ ID NO: 103. Bispecific molecules of the invention may further comprise a half-life extending domain (HLE). Thus, in another aspect, the invention provides a half-life extended bispecific molecule comprising the bispecific molecule of the first aspect and a HLE. The half-life extended bispecific molecule thus comprises:

[0044] (a) a bispecific molecule comprising:

[0045] (i) a soluble TOR which binds to a pMHC and which comprises a TOR alpha chain variable domain (Va) and a TOR beta chain variable domain (Vp); and

[0046] (ii) an immune cell engaging domain comprising an antibody light chain variable domain (VL), and an antibody heavy chain variable domain (VH); wherein said bispecific molecule is not greater than 60 kDa in size; wherein said VH is linked to the N-terminus of the Vp; and

[0047] (b) a half-life extending domain (HLE).

[0048] In certain embodiments, the half-life extended bispecific molecule is a single chain molecule. In this regard, the half-life extended bispecific molecule may have a format selected from the group consisting of:

[0049] (i) A / -VL-VH-Vp-Va-HLE-C;

[0050] (ii) A / -HLE-VL-VH-Vp-Va-C;

[0051] (iii) A / -Va-VL-VH-Vp-HLE-C;

[0052] (iv) A / -HLE-Va-VL-VH-Vp-C;

[0053] (v) A / -VH-Vp-Va-VL-HLE-C;

[0054] (vi) A / -HLE-VH-Vp-Va-VL-C;

[0055] (vii) A / -Va-VL-HLE-VH-Vp-C; and

[0056] (viii) A / -VH-Vp-HLE-Va-VL-C.

[0057] In certain embodiments, the half-life extended bispecific molecule is a dual chain molecule. In this regard, the half-life extended bispecific molecule may comprise or consist of:

[0058] (i) a first chain having the format A / -VH-Vp-C and a second chain having the format N- Va-VL-HLE-C;

[0059] (ii) a first chain having the format A / -VH-Vp-HLE-C and a second chain having the format A / -Va-VL-C;

[0060] (iii) a first chain having the format A / -VH-Vp-C and a second chain having the format N- HLE-Va-VL-C; or

[0061] (iv) a first chain having the format A / -HLE-VH-Vp-C and a second chain having the format M-Va-VL-C. In certain embodiments, the HLE is selected from the group consisting of (i) monomeric Fc; (ii) monomeric Fc CH2; (iii) monomeric Fc CH3; (iv) HSA Dill; (v) HSA Dll IA; (vi) HSA DI II b; (vii) an FcRn binding peptide; (vii) a scFv that binds to FcRn; and (ix) a VHH that binds to FcRn. In certain embodiments, the HLE is monomeric Fc. In certain embodiments, the monomeric Fc has the sequence of SEQ ID NO: 43. In certain embodiments, the HLE is HSA Dill. In certain embodiments, the HSA Dill has the sequence of SEQ ID NO: 44.

[0062] In certain embodiments, the HLE is an IgG-Fc, and wherein said IgG-Fc comprises or consists of a first domain (FC1) and a second domain (FC2).

[0063] In certain embodiments, the half-life extended bispecific molecule is a triple chain molecule comprising or consisting of a first chain having (i) a first chain having the format A / -Va-FC1-C, a second chain having the format A / -VL-FC2-C, and a third chain having the format A / -VH-Vp-C.

[0064] In certain embodiments, the half-life extended bispecific molecule is a dual chain molecule comprising or consisting of:

[0065] (i) a first chain having the format A / -VH-Vp-Va-FC1-C and a second chain having the format A / -VL-FC2-C;

[0066] (ii) a first chain having the format A / -Va-VH-Vp-FC1-C and a second chain having the format A / -VL-FC2-C; or

[0067] (iii) a first chain having the format A / -VH-Vp-VL-FC1-C and a second chain having the format A / -Va-FC2-C.

[0068] The FC1 may comprise or consist of the sequence of SEQ ID NO: 107 and the FC2 may comprise or consist of the sequence of SEQ ID NO: 108, or the FC1 may comprise or consist of the sequence of SEQ ID NO: 108 and the FC2 may comprise or consist of the sequence of SEQ ID NO: 107

[0069] In certain embodiments, the HLE of a bispecific molecule of the invention is linked to the VH, VL, Va or Vp via a linker and / or an IgG hinge sequence.

[0070] The one or more linkers linking the various domains of the bispecific molecule and / or half-life extended bispecific molecule of the invention may have the sequence of any one of SEQ ID NOs: 46, 48-61 and / or the amino acid sequence GG. The one or more IgG hinge sequences linking the various domains of the bispecific molecule and / or half-life extended bispecific molecule of the invention may have the sequence of any one of SEQ ID NOs: 47, 62-68 or 109-110. Alternatively, any of the linkers and / or hinge sequences described herein may be used.

[0071] Half-life extended bispecific molecules of the invention may comprise the amino acid sequences provided in any one of SEQ ID Nos: 8-26, 78-79 or 82-90. In another aspect, the invention provides a nucleic acid encoding the bispecific molecule or half-life extended bispecific molecule. The invention also provides an expression vector comprising the nucleic acid of this aspect. In addition, the invention provides a cell harbouring the nucleic acid or the expression vector of this aspect. The invention also provides a cell harbouring a first expression vector comprising a nucleic acid encoding a first chain of a dual chain bispecific molecule or a dual chain half-life extended bispecific molecule of the invention, and a second expression vector comprising a nucleic acid encoding a second chain of the dual chain bispecific molecule or the dual chain half-life extended bispecific. The invention also provides a cell harbouring a first expression vector comprising a nucleic acid encoding a first chain of a triple chain half-life extended bispecific molecule, a second expression vector comprising a nucleic acid encoding a second chain of the triple chain half-life extended bispecific molecule, and a third expression vector comprising a nucleic acid encoding a third chain of the triple chain half-life extended bispecific molecule. In certain embodiments, the cell is a mammalian cell expressing one or more chains of a bispecific molecule of the invention. The mammalian cell may be a CHO cell.

[0072] In a further aspect, the invention provides a pharmaceutical composition comprising the bispecific molecule, the half-life extended bispecific molecule, the nucleic acid, the expression vector, and / or the cell of the any of the above aspects, together with one or more pharmaceutically acceptable carriers or excipients.

[0073] The bispecific molecule, the half-life extended bispecific molecule, the nucleic acid, the expression vector, the cell and / or the pharmaceutical composition of any of the above aspects may be used in the treatment of diseases such as cancer, infectious diseases and autoimmune diseases. Thus, in a further aspect, there is provided a method of treating a disease comprising administering the half-life extended bispecific molecule, the nucleic acid, the expression vector, the host cell and / or the pharmaceutical composition to a subject in need thereof. Also provided is the bispecific molecule, the half-life extended bispecific molecule, the nucleic acid, the expression vector, the cell and / or the pharmaceutical composition for use in a method of treatment, the method comprising administering the half-life extended bispecific molecule, the nucleic acid, the expression vector, the host cell and / or the pharmaceutical composition to a subject in need thereof. In certain embodiments, the disease to be treated is cancer, an infectious disease and / or an autoimmune disease.

[0074] The bispecific molecule, the half-life extended bispecific molecule, the nucleic acid, the expression vector, the cell and / or the pharmaceutical composition of any of the above aspects may be used to target a tumor; and / or redirect T cells to a tumor. Thus, in a further aspect, also provided is a method of targeting a tumor and / or redirecting T cells to a tumor comprising administering the bispecific molecule, the nucleic acid, the expression vector, the host cell and / or the pharmaceutical composition to a subject in need thereof. Also provided, in a further aspect, is a method of producing the bispecific molecule or half-life extended bispecific molecule, comprising maintaining the cell described above under optimal conditions for expression of the bispecific molecule or half-life extended bispecific molecule and isolating the bispecific molecule or half-life extended bispecific molecule.

[0075] Description of the invention

[0076] Bispecific molecules

[0077] As used herein, the term “bispecific molecule” generally refers to a molecule capable of binding to two target antigens. The bispecific molecules comprise a soluble TCR which binds to a peptide-major histocompatibility complex (pMHC), comprising a TCR alpha chain variable domain (Va) and a TCR beta chain variable domain (Vp); and an immune cell engaging domain comprising an antibody light chain variable domain (VL), and an antibody heavy chain variable domain (VH). The bispecific molecule of the invention is not greater than 60 kDa in size; and the VH is linked to the N-terminus of the Vp.

[0078] The bispecific molecule comprises two antigen-binding sites. Specifically, the Va and Vp dimerise to form the antigen-binding site of the soluble TCR, and the VH and VL dimerise to form the antigenbinding site of the immune cell engaging domain. The soluble TCR of the bispecific molecule thus comprises the binding site that specifically binds to a pMHC complex displayed on the surface of an antigen presenting cell, and the immune cell engaging domain of the bispecific molecule thus comprises the binding site that specifically binds to an antigen such as a T cell surface antigen. Such molecules may adopt a number of different formats as discussed herein.

[0079] Preferably, the soluble TCR and the immune cell engaging domain of the bispecific molecule lack TCR and antibody constant domains, respectively. As will be understood by the skilled person, the inclusion of such domains may result in the bispecific molecule being greater than 60 kDa in size. As mentioned, the bispecific molecules of the invention are not greater than 60 kDa in size.

[0080] Half-life extended bispecific molecules of the invention comprise a bispecific molecule of the invention and a half-life extending domain (HLE). Depending on the size of the HLE domain, half-life extended bispecific molecules of the invention may be less than 60 kDa in size or greater than 60 kDa in size.

[0081] The bispecific molecule may comprise one or more polypeptide chain(s). As used herein, the term “polypeptide chain” refers to a polymer (i.e., a chain) of amino acids, typically twenty or more amino acids, linked by peptide bonds and having an N- and C- terminus. As is known in the art, a protein may comprise multiple polypeptide chains assembled together by non-covalent or covalent interactions. The bispecific molecule of the invention may comprise one polypeptide chain (i.e., a single chain molecule). The bispecific molecule of the invention may comprise two polypeptide chains (i.e., a dual chain molecule). The bispecific molecule of the invention may comprise more than two polypeptide chains, e.g., three polypeptide chains (i.e., a triple chain molecule).

[0082] The bispecific molecule is soluble. Thus, the bispecific molecule may not comprise any transmembrane regions, such as TCR transmembrane regions. Such bispecific molecules may be used as soluble therapeutic or diagnostic agents.

[0083] Soluble TCRs

[0084] A “soluble TCR”, as used herein, is a protein domain capable of binding to a peptide-major histocompatibility (pMHC) complex. In this context, the term “soluble” refers to a TCR that does not comprise any transmembrane domain. The terms “MHC” and human leukocyte antigen “HLA” as used herein are used interchangeably. The soluble TCR of the bispecific molecule of the invention preferably lacks the TCRa and TCRp constant domains.

[0085] The TCR sequences defined herein are described with reference to IMGT nomenclature which is widely known and accessible to those working in the TCR field. For example, see: LeFranc and LeFranc, (2001). “T cell Receptor Factsbook”, Academic Press; Lefranc, (2011), Cold Spring Harb Protoc 2011 (6): 595-603; Lefranc, (2001), Curr Protoc Immunol Appendix 1 : Appendix 100; and Lefranc, (2003), Leukemia 17(1): 260-266. Briefly, TCRs consist of two disulfide linked chains. Each chain (alpha and beta) is generally regarded as having two extracellular regions, namely a variable and a constant region. A short joining region connects the variable and constant regions and is typically considered part of the alpha variable region (i.e., the alpha chain variable domain “Va”). Additionally, the beta chain usually contains a short diversity region next to the joining region, which is also typically considered part of the beta variable region (i.e., the beta chain variable domain “Vp”). The variable region of each chain of a typical TCR is located N-terminally and comprises three Complementarity Determining Regions (CDRs) embedded in a framework sequence (the framework regions being the variable domain residues other than the CDR residues). The CDRs comprise the recognition site for peptide-MHC binding.

[0086] The amino acid sequence of the Va and Vp may correspond to those found in nature, or they may contain one or more mutations relative to a natural protein. Such mutations may be made to increase the affinity of the soluble TCR for a given antigen. Additionally or alternatively, mutations may be incorporated to improve stability and manufacturability. The Va and Vp sequences may be derived from human sequences.

[0087] In certain embodiments, the Va comprises the CDRs of DKHSQG (SEQ ID NO: 28), IYSQGD (SEQ ID NO: 80) and AVRGNEKLT (SEQ ID NO: 30) as CDR1 , CDR2 and CDR3 respectively. In certain embodiments, the Va comprises the CDRs of DKHSQG (SEQ ID NO: 28), IYSNGD (SEQ ID NO: 29) and AVRGNEKLT (SEQ ID NO: 30) as CDR1 , CDR2 and CDR3 respectively.

[0088] In certain embodiments, the Vp comprises the CDRs of LQHSY (SEQ ID NO: 81), SVGVGF (SEQ ID NO: 33) and ASAYMTGELF (SEQ ID NO: 34) as CDR1 , CDR2 and CDR3 respectively. In certain embodiments, the Vp comprises the CDRs of MNHSY (SEQ ID NO: 32), SVGVGF (SEQ ID NO: 33) and ASAYMTGELF (SEQ ID NO: 34) as CDR1 , CDR2 and CDR3 respectively

[0089] Alternatively, the Va and Vp CDR sequences may each optionally have one, two or three amino acid substitutions relative to the sequences recited above. The substitutions may be conservative substitutions and preferably do not reduce (i.e., weaken) the binding affinity of the soluble TCR.

[0090] The Va may comprise CDRs that are at least 90%, at least 95%, at least 98%, or at least 99% identical to the sequences of DKHSQG (SEQ ID NO: 28), IYSQGD (SEQ ID NO: 80) and AVRGNEKLT (SEQ ID NO: 30) as CDR1 , CDR2 and CDR3 respectively and / or the Vp may comprise CDRs that are at least 90%, at least 95%, at least 98%, or at least 99% identical to the sequences of LQHSY (SEQ ID NO: 81), SVGVGF (SEQ ID NO: 33) and ASAYMTGELF (SEQ ID NO: 34) as CDR1 , CDR2 and CDR3 respectively.

[0091] The Va may comprise CDRs that correspond to the sequences of DKHSQG (SEQ ID NO: 28), IYSQGD (SEQ ID NO: 80) and AVRGNEKLT (SEQ ID NO: 30) as CDR1 , CDR2 and CDR3 respectively, and comprise framework regions (FRs) that are at least 90%, at least 95%, at least 98%, or at least 99% identical to the sequences of amino acid residues 1 to 26 (FR1), 33 to 49 (FR2), 56 to 89 (FR3) and 99 to 113 (FR4) of SEQ ID NO: 27; and / or the Vp may comprise CDRs that correspond to the sequences of LQHSY (SEQ ID NO: 81), SVGVGF (SEQ ID NO: 33) and ASAYMTGELF (SEQ ID NO: 34), and comprise FRs that are at least 90%, at least 95%, at least 98%, or at least 99% identical to the sequences of amino acid residues 1 to 26 (FR1), 32 to 48 (FR2), 55 to 91 (FR3) and 102 to 116 (FR4) of SEQ ID NO: 31.

[0092] The Va may be at least 80% identical to the sequence of SEQ ID NO: 27 and the Vp may be at least 80% identical to the sequence of SEQ ID NO: 31 . The Va may be at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 27 and the Vp may be at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 31 .

[0093] The Va may comprise or consist of the amino acid sequence provided in SEQ ID NO: 27 and the Vp may comprise or consist of the amino acid sequence provided in SEQ ID NO: 31.

[0094] In certain embodiments, the Va comprises the CDRs of TISGTDY (SEQ ID NO: 92), GLTSN (SEQ ID NO: 93) and CILILGHSRLGNYIATF (SEQ ID NO: 94) as CDR1 , CDR2 and CDR3 respectively. In certain embodiments, the Vp comprises the CDRs of LNHDA (SEQ ID NO: 96), SQIMGD (SEQ ID NO: 97) and CASSWWTGGASPIRF (SEQ ID NO: 98) as CDR1 , CDR2 and CDR3 respectively.

[0095] Alternatively, the Va and Vp CDR sequences may each optionally have one, two or three amino acid substitutions relative to the sequences recited above. The substitutions may be conservative substitutions and preferably do not reduce (i.e., weaken) the binding affinity of the soluble TOR.

[0096] The Va may comprise CDRs that are at least 90%, at least 95%, at least 98%, or at least 99% identical to the sequences of TISGTDY (SEQ ID NO: 92), GLTSN (SEQ ID NO: 93) and CILILGHSRLGNYIATF (SEQ ID NO: 94) as CDR1 , CDR2 and CDR3 respectively and / or the Vp may comprise CDRs that are at least 90%, at least 95%, at least 98%, or at least 99% identical to the sequences of LNHDA (SEQ ID NO: 96), SQIMGD (SEQ ID NO: 97) and CASSWWTGGASPIRF (SEQ ID NO: 98) as CDR1 , CDR2 and CDR3 respectively.

[0097] The Va may comprise CDRs that correspond to the sequences of TISGTDY (SEQ ID NO: 92), GLTSN (SEQ ID NO: 93) and CILILGHSRLGNYIATF (SEQ ID NO: 94) as CDR1 , CDR2 and CDR3 respectively, and comprise framework regions (FRs) that are at least 90%, at least 95%, at least 98%, or at least 99% identical to the sequences of amino acid residues 1 to 26 (FR1), 34 to 50 (FR2), 56 to 88 (FR3) and 106 to 115 (FR4) of SEQ ID NO: 91 ; and / or the Vp may comprise CDRs that correspond to the sequences of LNHDA (SEQ ID NO: 96), SQIMGD (SEQ ID NO: 97) and CASSWWTGGASPIRF (SEQ ID NO: 98), and comprise FRs that are at least 90%, at least 95%, at least 98%, or at least 99% identical to the sequences of amino acid residues 1 to 26 (FR1), 32 to 48 (FR2), 55 to 90 (FR3) and 106 to 114 (FR4) of SEQ ID NO: 95.

[0098] The Va may be at least 80% identical to the sequence of SEQ ID NO: 91 and the Vp may be at least 80% identical to the sequence of SEQ ID NO: 95. The Va may be at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 91 and the Vp may be at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 95.

[0099] The Va may comprise or consist of the amino acid sequence provided in SEQ ID NO: 91 and the Vp may comprise or consist of the amino acid sequence provided in SEQ ID NO: 95.

[0100] The skilled person would appreciate that the format of the bispecific molecule of the invention could equally be applied to TCR sequences other than those recited above. For example, other suitable TOR chain amino acid sequences are provided in WO2011001152, WO2017109496, WO2017175006, WO2018234319, WO2023156663, WO2024146951 , WO2024146936, and WO2024223842, and, for example, in U.S. Patent Nos. 8,519,100, 11 ,639,374, 11 ,505,590, and 11 ,427,624, the contents of each of which are herein incorporated by reference. As is well-known in the art, protein molecules may be subject to post-translational modifications. Glycosylation is one such modification, which comprises the covalent attachment of oligosaccharide moieties to defined amino acids in a TCR or antibody chain. For example, asparagine residues, or serine / threonine residues are well-known locations for oligosaccharide attachment. The glycosylation status of a particular protein depends on a number of factors, including protein sequence, protein conformation and the availability of certain enzymes. Furthermore, glycosylation status (i.e. oligosaccharide type, covalent linkage and total number of attachments) can influence protein function. Therefore, when producing recombinant proteins, controlling glycosylation is often desirable. Controlled glycosylation has been used to improve antibody based therapeutics.. Glycosylation may be controlled, by using particular cell lines for example (including but not limited to mammalian cell lines such as Chinese hamster ovary (CHO) cells or human embryonic kidney (HEK) cells), or by chemical modification. Such modifications may be desirable, since glycosylation can improve pharmacokinetics, reduce immunogenicity and more closely mimic a native human protein. Alternatively, glycosylation can lead to a lack of consistency in manufacturing which is not desirable for a therapeutic molecule. Residues at high risk of glycosylation, such as asparagine, may be substituted with an alternative amino acid, such as glutamine.

[0101] The soluble TCR may not be fully aglycosylated, i.e., the pMHC may retain one or more glycosylation site(s) from its native sequence. For example, the soluble TCR may be glycosylated at a single glycosylation site (i.e., the pMHC binding domain may contain only one glycosylation site). The single glycosylation site may be in the Va or Vp. Bispecific molecules with single glycosylated sites may have better manufacturability (e.g., protein production yield, resistance to thermal stress and aggregation), as compared to other glycosylated and / or aglycosylated variants, in addition to retaining affinity for peptide-MHC binding and potency of target cell killing.

[0102] The soluble TCR binds to MHC in complex with a peptide antigen. The peptide antigen may be a disease associated antigen. For example, the peptide antigen may be a peptide derived from HIV as described in WO2023156663. The soluble TCR may bind to a tumour associated antigen peptide in complex with an MHC. For example, the peptide antigen may be a peptide derived from GP100, NYESO, MAGEA4, PRAME, PRAMEA24 or PIWIL as described in WO2011001152, WO2017109496, WO2017175006, WO2018234319, WO2024146951 and WO2024146936. The disease associated antigen may be associated with an autoimmune disease, such as type 1 diabetes. For example, the peptide antigen may be PPI as described in WO2024223842. For example, the pMHC binding domain may bind to a ALWGPDAAA (SEQ ID NO: 45) HLA-A*02 complex. Preferably, the pMHC binding domain binds to a ALWGPDAAA (SEQ ID NO: 45) HLA-A*02 complex. For example, the MHC binding domain may bind to a KLVELEHTL (SEQ ID NO: 112) HLA-A*02 complex. For example, the MHC binding domain may bind to a SLLQHLIGL (SEQ ID NO: 111) HLA-A*02 complex.

[0103] Bispecific molecules described herein may comprise a soluble TCR which binds to the pMHC with high affinity (e.g. KD of less than 10 nM) and may therefore be advantageous for binding antigen presenting cells (APC) presenting a pMHC at less than 100 copies per cell, such as about 5 copies per cell to about 50 copies per cell (i.e. , low copy number targets). A bispecific molecule of the present invention may alternatively comprise a soluble TCR which binds to a pMHC at above 100 copies on the surface of an APC (i.e., high copy number targets). Thus, the bispecific molecule may be used for both high copy number targets (e.g. above 100 copies per cell) and low copy number targets (e.g. below 100 copies per cell, such as about 5 copies per cell to about 50 copies per cell).

[0104] Immune cell engaging domains

[0105] A bispecific molecule described herein comprises an immune cell engaging domain. An “immune cell engaging domain”, as used herein, is a protein domain that is capable of binding to a target on an immune cell and / or modifying an immune response, for example promoting or suppressing an immune response such as T cell activation. The immune cell engaging domain of the bispecific molecule of the invention comprises an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH). The VL and VH may both be in the same polypeptide chain or they may be in separate polypeptide chains. In either case, the VL and the VH associate together to form an antigen binding moiety that binds to an antigen. Thus, the immune cell engaging domain may comprise an antibody, or antigen-binding fragment thereof. One or more disulphide bonds may be incorporated between the VH and VL domains to e.g., enhance stability of the immune cell engaging domain. Suitable positions of such bonds are known in the art. The immune cell engaging domain of the bispecific molecule of the invention preferably lacks antibody constant domains.

[0106] The term “antibody” as used herein is meant to include conventional / native antibodies and engineered antibodies, in particular functional antibody fragments, single chain antibodies, and bispecific or multispecific antibodies. “Native” or “conventional”, in this context, refers to an antibody that has the same type of domains and domain arrangements as an antibody found in nature and comprises antibody-derived CDR and FR sequences. In a native / conventional four-chain, e.g. human, antibody, two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. The variable domains of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen. Conventional antibody binding sites are made up of residues that are primarily from the CDRs or hypervariable regions.

[0107] “Engineered” antibody formats include functional antibody fragments, single chain antibodies, single domain antibodies, and chimeric, humanized, bispecific or multispecific antibodies. A “functional antibody fragment” or “antigen-binding fragment” (used interchangeably herein) refers to a portion of a full-length antibody, or a protein that resembles a portion of a full-length antibody, that retains the ability to bind to its target antigen, in particular the antigen binding region or variable region of the full- length antibody. As used herein, the term "complementarity determining regions” (CDRs; i.e., CDR1 , CDR2, and CDR3) refers to the amino acid residues of an antibody variable region which define the antigen specificity of an antibody. "Framework regions" (FRs) are those variable domain residues other than the CDR residues. Each variable domain typically has three CDR regions identified as CDR1 , CDR2 and CDR3. The amino acid positions assigned to CDRs and FRs can be defined according to Kabat, Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991 or other numbering systems, e.g., the canonical numbering system of Chothia; the IMGT numbering system; or the AHO numbering system. For example, according to the numbering system of Kabat, VH framework regions (FRs) and CDRs are positioned as follows: residues 1-30 (FR1 ), SI- 35 (CDR1), 36-49 (FR2), 50-65 (CDR2), 66-94 (FR3), 95-102 (CDR3) and 103- 113 (FR4). According to the numbering system of Kabat, VL FRS and CDRs are positioned as follows: residues 1-23 (FR1), 24-34 (CDR1), 35-49 (FR2), 50-56 (CDR2), 57-88 (FR3), 89-97 (CDR3) and 98-107 (FR4). The present disclosure is not limited to FRs and CDRs as defined by the Kabat numbering system, but includes all numbering systems, including those discussed above.

[0108] As used herein, “variable domain" refers to the portions of the light and / or heavy chains of an antibody as defined herein that is capable of specifically binding to an antigen and includes amino acid sequences of complementarity determining regions (CDRs); i.e., CDR1 , CDR2, and CDR3, and framework regions (FRs). Exemplary variable domains comprise three or four FRs (e.g., FR1 , FR2, FR3 and optionally FR4) together with three CDRs, i.e., in the format FR1-CDR1-FR2-CDR2-FR3- CD3-FR4.

[0109] The VL and VH of the immune cell engaging domain in the bispecific molecules of the invention each have three CDRs, designated CDR1-L, CDR2-L, CDR3-L and CDR1-H, CDR2-H, CDR3-H, respectively. The antigen-binding site of the immune cell engaging domain, therefore, comprises six CDRs, comprising the CDR set from each of the VH and VL.

[0110] The immune cell engaging domain comprises an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH) which dimerise to form an antibody variable fragment (“Fv”). Thus, the immune cell engaging domain may comprise an Fv. As used herein, the term “Fv” shall be taken to mean any protein, whether comprised of multiple polypeptides or a single polypeptide, in which a VL and a VH associate and form a complex having an antigen binding site, i.e., capable of binding to an antigen. The VH and the VL which form the antigen binding site can be in a single polypeptide chain or in different polypeptide chains. Furthermore, the VH and VL may be linked by a disulphide bond and thus the Fv may be a dsFv. The term “Fv” shall be understood to encompass fragments directly derived from an antibody as well as proteins corresponding to such a fragment produced using recombinant means.

[0111] The antigen of the immune cell engaging domain may be located on an immune cell. In the context of the present invention, “immune cell” may refer to, for example, a T cell or a B cell. In particular, the antigen of the antigen-binding moiety may be a T cell surface antigen. The antigen may be selected from the group consisting of CD2, CD3 (such as the CD3y, CD36 and CD3e chains), CD4, CD5, CD7, CD8, CD10, CD11 b, CD11c, CD14, CD16, CD18, CD22, CD25, CD28, CD32a, CD32b, CD33, CD41 , CD41 b, CD42a, CD42b, CD44, CD45RA, CD49, CD55, CD56, CD61 , CD64, CD68, CD90, CD94, CD95, CD117, CD123, CD125, CD134, CD137, CD152, CD163, CD193, CD203c, CD235a, CD278, CD279, CD287, Nkp46, NKG2D, GITR, FCERI, TCRa / p, TCRy / 5, HLA-DR and 4-1 BB, or combinations thereof. “Combinations thereof’ refers to complexes of two or more of said antigens, e.g. a TCRa / p CD3 complex. Preferably, the antigen is CD3.

[0112] The immune cell engaging domain may be a single-chain variable fragment (scFv). “Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain. The scFv polypeptide may further comprise a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.

[0113] The immune cell engaging domain may be a T cell engaging immune effector domain. A “T cell engaging immune effector domain”, as used herein, is a protein domain that is capable of binding to a target on a T cell to promote an immune response (e.g., promote T cell recruitment and / or activation).

[0114] The T cell engaging immune effector domain may bind to a protein expressed on a cell surface of a T cell to promote activation of the T cell. For example, the T cell engaging immune effector domain may be a CD3 effector domain. The T cell engaging immune effector domain may bind to, for example specifically bind to, CD3 (i.e., the T cell engaging immune effector domain may be a CD3-binding protein).

[0115] CD3 effectors include but are not limited to anti-CD3 antibodies or antibody fragments, in particular an anti-CD3 scFv or Fv or antibody-like scaffolds. The immune cell engaging domain may be a T cell engaging immune effector domain which may be an anti-CD3 scFv or Fv. Further immune effectors include but are not limited to antibodies, including fragments, derivatives and variants thereof, that bind to antigens on T cells. Such antigens include CD28, 4-1 bb (CD137) or CD16 or any molecules that exert an effect at the immune synapse. A particularly preferred immune effector is an anti-CD3 antibody, or a functional fragment or variant of said anti-CD3 antibody, such as an anti-CD3 scFv or Fv. As used herein, the term “antibody” encompasses such fragments and variants. Examples of anti-CD3 antibodies include but are not limited to OKT3, UCHT-1 , BMA-031 and 12F6. Antibody fragments and variants / analogues which are suitable for use in the compositions and methods described herein include Fv fragments, scFv fragments and dsFv (disulfide linked Fv fragments).

[0116] Suitable immune cell engaging domains for binding to CD3 include binding domains derived from the CD3-specific, humanized antibody hUCHT 1 . In particular VH and VL domains derived from the UCHT1 variants UCHT1-V17, UCHT1-V17opt, UCHT1-V21 or UCHT1-V23 may be used. Alternatively, VH and VL domains derived from the antibody BMA031 , which targets the TCRa / p CD3 complex, and humanized versions thereof may be used, in particular VH and VL domains derived from BMA031 variants BMA031 (V36) or BMA031 (V10). Suitable BMA031 antibody variant sequences are described in WO 2022 / 233957. As another alternative, VH and VL domains derived from the CD3-specific antibody H2C (described in EP 2155783) may be used.

[0117] Suitable VH and VL domains include those of the exemplary bispecific molecules described herein. In this regard, the immune cell engaging domain may comprise:

[0118] (a) a VL comprising a CDR1 , a CDR2 and a CDR3 comprising the following sequences:

[0119] CDR1 - QDIRNY (SEQ ID NO: 36),

[0120] CDR2 - YTS (SEQ ID NO: 37),

[0121] CDR3 - QQGNTLPWT (SEQ ID NO: 38); and

[0122] (b) a VH comprising a CDR1 , a CDR2 and a CDR3 comprising the following sequences:

[0123] CDR1 - GYSFTGYA (SEQ ID NO: 40),

[0124] CDR2 - INPYKGVS (SEQ ID NO: 41),

[0125] CDR3 - ARSGYYGDSDWYFDV (SEQ ID NO: 42).

[0126] The immune cell engaging domain may comprise a VL comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 35 and a VH comprising an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 39. For example, the immune cell engaging domain may comprise a VL comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 35 and a VH comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 39.

[0127] The immune cell engaging domain may comprise or consist of a VL comprising the amino acid sequence provided in SEQ ID NO: 35 and a VH comprising the amino acid sequence provided in SEQ ID NO: 39.

[0128] Another suitable immune cell engaging domain described herein may comprise:

[0129] (a) a VL comprising a CDR1 , a CDR2 and a CDR3 comprising the following sequences:

[0130] CDR1 - QGIRKY (SEQ ID NO: 100),

[0131] CDR2 - AAS (SEQ ID NO: 101),

[0132] CDR3 - QQGNTLPWT (SEQ ID NO: 102); and

[0133] (b) a VH comprising a CDR1 , a CDR2 and a CDR3 comprising the following sequences:

[0134] CDR1 - GYRFTGYL (SEQ ID NO: 104),

[0135] CDR2 - INPYKGST (SEQ ID NO: 105),

[0136] CDR3 - ARSGYYGDSDWYFDL (SEQ ID NO: 106).

[0137] The immune cell engaging domain may comprise a VL comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 99 and a VH comprising an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 103. For example, the immune cell engaging domain may comprise a VL comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 99 and a VH comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 103.

[0138] The immune cell engaging domain may comprise or consist of a VL comprising the amino acid sequence provided in SEQ ID NO: 99 and a VH comprising the amino acid sequence provided in SEQ ID NO: 103.

[0139] Other suitable VH and VL domains include those derived from the anti-CD3 scFvs described in, for example, WO 2020 / 157210. In this regard, the immune cell engaging domain may comprise:

[0140] (a) a VL comprising a CDR1 , a CDR2 and a CDR3 comprising the following sequences:

[0141] CDR1 - QDIRNY (SEQ ID NO: 71),

[0142] CDR2 - YTS (SEQ ID NO: 72),

[0143] CDR3 - QQGNTLPWT (SEQ ID NO: 73); and

[0144] (b) a VH comprising a CDR1 , a CDR2 and a CDR3 comprising the following sequences: CDR1 - GYSFTGYT (SEQ ID NO: 75),

[0145] CDR2 - INPYKGVS (SEQ ID NO: 76),

[0146] CDR3 - ARSGYYGDSDWYFDV (SEQ ID NO: 77).

[0147] The immune cell engaging domain may comprise a VL comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 70 and a VH comprising an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 74. For example, the immune cell engaging domain may comprise a VL comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 70 and a VH comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 74.

[0148] The immune cell engaging domain may comprise or consist of a VL comprising the amino acid sequence provided in SEQ ID NO: 70 and a VH comprising the amino acid sequence provided in SEQ ID NO: 74.

[0149] The VL and VH CDR sequences above may each optionally have one, two, three, or four amino acid substitutions relative to the sequences recited above. The substitutions may be conservative substitutions and preferably do not reduce (i.e., weaken) the binding affinity of the immune cell engaging domain.

[0150] Alternatively, the immune cell engaging domain may be an immune suppressor. As used herein, the term “immune suppressor” refers to any molecule, e.g., a protein, that is capable of inhibiting an immune response, such as inhibiting T cell activation. The immune suppressor may bind to a target (e.g. antigen). For example, the immune suppressor may be an immune checkpoint agonist, i.e., a molecule that induces immune checkpoint signalling (such as a PD-1 agonist). The immune suppressor may comprise an antigen-binding moiety that is capable of binding to an antigen. The antigen of the immune suppressor may be located on an immune cell, such as a T cell. The immune suppressor may comprise an antibody or antigen binding fragment thereof. For example, the immune suppressor may be an Fv, dsFv and / or scFv or similar sized antibody scaffold. Such immune suppressors are described below.

[0151] For example, the target of the immune suppressor may be an immune checkpoint molecule, such as PD-1 (Programmed Death 1 receptor), A2AR (Adenosine A2A receptor), A2BR (Adenosine A2B receptor), B7-H3 (B7 Homolog 3, also called CD276) B7-H4 (B7 Homolog 4, also called VTCN1), BTLA (B and T Lymphocyte Attenuator, also called CD272), CTLA-4 (Cytotoxic T-Lymphocyte- Associated protein 4, also called CD152), IDO (Indoleamine 2,3-dioxygenase), CD200 Receptor, KIR (Killer-cell Immunoglobulin-like Receptor), TIGIT (T cell Immunoreceptor with Ig and ITIM domains), LAG3 (Lymphocyte Activation Gene-3), NOX2 (nicotinamide adenine dinucleotide phosphate NADPH oxidase isoform 2), TIM-3 (T-cell Immunoglobulin domain and Mucin domain 3), VISTA (V-domain Ig suppressor of T cell activation), SIGLEC7 (Sialic acid-binding immunoglobulin-type lectin 7, also called CD328), and SIGLEC9 (Sialic acid-binding immunoglobulin-type lectin 9, also called CD329).

[0152] In this regard, the immune suppressor may be an agonist of one or more of the above immune checkpoint molecules. Thus, the immune suppressor may be an immune checkpoint agonist (i.e., to inhibit immune activation). Suitable immune checkpoint agonists, including native ligands and antibodies, are reviewed in Paluch et al Front Immunol, 2018, 9:2306, for example.

[0153] The immune suppressor may comprise an agonist antibody that binds to, and may stimulate signalling of, an immune checkpoint molecule. For example, the immune suppressor may be, or comprise, a PD-1 agonist antibody. Such PD-1 agonists preferably do not compete with PD-L1 for binding to PD- 1. The PD-1 agonist may be a Fv, dsFv and / or scFv fragment. Examples of such antibodies are provided in WO2011110621 (US 9102728), WO2010029434 (US9181342) and WO2018024237 (US11274153), the contents of which are herein incorporated by reference. Thus, the antigen of the immune suppressor may be PD-1 and the antigen binding moiety of the immune suppressor may be a PD-1 agonist.

[0154] As used herein, the term “PD-1 agonist” refers to any molecule that is capable of binding to PD-1 and activating PD-1 signalling, including e.g., the PD-1 ligand, PD-L1 , and PD-1 agonist antibodies. Activation of the PD-1 pathway down-regulates immune activity, promoting peripheral immune tolerance and preventing autoimmunity.

[0155] Half-life extending (HLE) domains The bispecific molecules described herein may comprise a half-life extending domain (HLE). A “halflife extending domain”, as used herein, refers to a protein domain for extending the half-life of the binding protein, relative to a binding protein lacking the half-life extending domain.

[0156] The term “half-life”, as used herein, refers to a pharmacokinetic property of a binding molecule that is a measure of the mean survival time of binding molecules following their administration. Half-life can be expressed as the time required to eliminate 50 percent of a known quantity of a bispecific molecule from the patient's body (or other mammal) or a specific compartment thereof, for example, as measured in serum, i.e., circulating half-life, or in other tissues.

[0157] An increase in half-life allows for the reduction in amount of bispecific molecule given to a patient as well as reducing the frequency of administration. An increase in half-life can be beneficial, for example, for treatment of cancer, infectious disease or an autoimmune disease or condition.

[0158] The half-life extending domain may comprise an immunoglobulin Fc domain. The term “Fc domain”, as used herein, refers to a dimer of two Fc regions, or a single Fc region. As used herein, the term “Fc region” is used to refer to a region of a single polypeptide chain comprising at least a CH2 region and a CH3 region sequence. The Fc regions may comprise all or part of a hinge sequence. The hinge sequence may correspond substantially or partially to a hinge region from an IgG, for example lgG1 , lgG2, lgG3 or lgG4. The hinge sequence may be a hybrid hinge sequence, for example an IgG 112 hybrid hinge sequence.

[0159] The immunoglobulin Fc domain may be any antibody Fc domain. The Fc domain is the tail region of an antibody that interacts with cell surface Fc receptors and some proteins of the complement system. The Fc domain comprises two polypeptide chains (i.e., two Fc “regions”) both having two or three heavy chain constant domains (termed CH2, CH3 and CH4), and optionally a hinge region. The two Fc region chains may be linked by one or more disulphide bonds within the hinge region. Alternatively, the Fc domain may comprise a single polypeptide chain (i.e. one Fc “region”). An Fc domain comprising a single polypeptide chain may be termed “monomeric” or “mFc”. Preferably the Fc domain is monomeric Fc. Fc domains from immunoglobulin subclasses IgG 1 , lgG2 and lgG4 bind to and undergo FcRn mediated recycling, affording a long circulatory half-life (3 - 4 weeks), thus extending the half-life of the bispecific molecule described herein. The interaction of IgG with FcRn has been localized in the Fc region covering parts of the CH2 and CH3 domains. Preferred immunoglobulin Fc domains for use in the present invention include, but are not limited to Fc domains from IgG 1 or lgG4. For example, the IgG 1 Fc domain. Preferably, the Fc domain retains pH dependent binding to FcRn.

[0160] Bispecific molecules with increased half-lives may also be generated by modifying amino acid residues identified as involved in the interaction between the Fc and the FcRn receptor. Bispecific molecules comprising Fc regions that comprise one or more modifications which promote binding to FcRn may have an increased half-life of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 80%, about 85%, about 90%, about 95%, about 100%, about 125%, about 150% or more as compared to a bispecific molecule comprising a native Fc region. Bispecific molecules comprising Fc regions that comprise one or more modifications which promote binding to FcRn may have an increased half-life of about 2 fold, about 3 fold, about 4 fold, about 5 fold, about 10 fold, about 20 fold, about 50 fold or more, or is between 2 fold and 10 fold, or between 5 fold and 25 fold, or between 15 fold and 50 fold, as compared to bispecific molecules comprising a native Fc region.

[0161] The Fc regions may comprise mutations relative to a wild-type or unmodified Fc sequence. Mutations include substitutions, insertions and deletions. Such mutations may be made for the purpose of introducing desirable therapeutic properties such as to enhance dimerisation of the Fc regions and / or enhance binding to FcRn and / or to attenuate an effector function of the Fc domain. Additionally or alternatively, mutations may be made for manufacturing reasons, for example to remove or replace amino acids that may be subject to post-translational modifications such as glycosylation, as described herein. The immunoglobulin Fc may be fused to the other domains (i.e. , TCR or immune cell engaging domain, if present) in the molecule of the invention via a linker, and / or a hinge sequence as described herein. Alternatively no linker may be used.

[0162] The Fc regions may comprise mutations made to facilitate hetero-dimerisation, for example, knobs into holes (KiH) mutations maybe engineered into the CH3 domain. Thus, the half-life extending domain may comprise one or more amino acid substitutions which facilitate dimerisation of the FC1 region and the FC2 region. Such substitutions include “Knob-in-hole” substitutions. In this case, one chain (i.e. one of the FC1 or FC2 regions) is engineered to contain a bulky protruding residue (i.e. the knob), such as Y, and the other chain (i.e., the other of the FC1 and FC2 regions) is engineered to contain a complementary pocket (i.e. the hole). For example, a knob may be constructed by replacing a small amino acid side chain with a larger side chain. A hole may be constructed by replacing a large amino acid side chain with a smaller side chain. Without wishing to be bound to theory, this is thought to stabilize a hetero-dimer of the FC1 and FC2 regions by favouring formation of the hetero-dimer over other species, for example homomultimers of FC1 and FC2, thereby enhancing the stability and manufacturability of the bispecific molecule of the invention. Suitable positions and substitutions for KiH mutations, and other mutations for facilitating dimerisation of Fc regions, are known in the art. For example, the FC1 or FC2 region may comprise the amino acid substitution T366W, according to the EU numbering scheme, and the other of the FC1 and FC2 regions may comprise one or more amino acid substitutions selected from the group consisting of T366S, L368A and Y407V, according to the EU numbering scheme. Suitable sequences of FC1 and FC2 regions are provided in SEQ ID NO: 107 and 108, respectively. In this regard, one of the FC1 or FC2 regions may comprise or consist of SEQ ID NO: 107, and the other of the FC1 or FC2 regions may comprise or consist of SEQ ID NO: 108. The Fc domain may also comprise one or more mutations that attenuate an effector function of the Fc domain. Exemplary effector functions include, without limitation, complement-dependent cytotoxicity (CDC) and / or antibody-dependent cellular cytotoxicity (ADCC). The modification to attenuate effector function may be a modification that alters the glycosylation pattern of the Fc domain, e.g., a modification that results in an aglycosylated Fc domain. Alternatively, the modification to attenuate effector function may be a modification that does not alter the glycosylation pattern of the Fc domain. The modification to attenuate effector function may reduce or eliminate binding to human effector cells, binding to one or more Fc receptors, and / or binding to cells expressing an Fc receptor. For example, the half-life extending domain may comprise one or more amino acid substitutions selected from the group consisting of S228P, E233P, L234A, L235A, L235E, L235P, G236R, G237A, P238S, F241A, V264A D265A, H268A, D270A, N297A, N297G, N297Q, E318A, K322A, L328R, P329G, P329A, A330S, A330L, P331 A and P331 S, according to the EU numbering scheme. Particular modifications include a N297G or N297A substitution in the Fc region of human lgG1 (EU numbering). Other suitable modifications include L234A, L235A and P329G substitutions in the Fc region of human lgG1 (EU numbering), that result in attenuated effector function. The Fc regions in the bispecific molecule of the invention may comprise a substitution at residue N297, numbering according to EU index. For example, the substitution may be an N297G or N297A substitution. Other suitable mutations (e.g., at residue N297) are known to those skilled in the art. For a monomeric Fc (mFc), mutations may be made to introduce one or more non-native disulphide bonds as discussed further below.

[0163] Fc variants having reduced effector function refers to Fc variants that reduce effector function (e.g., CDC, ADCC, and / or binding to FcR, etc. activities) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or more as compared to the effector function achieved by a wildtype Fc region (e.g., an Fc region not having a mutation to reduce effector function, although it may have other mutations). The Fc variants having reduced effector function may be Fc variants that eliminate all detectable effector function as compared to a wild-type Fc region. Assays for measuring effector function are known in the art and described below.

[0164] In vitro and / or in vivo cytotoxicity assays can be conducted to confirm the reduction / depletion of CDC and / or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the Fc region or fusion protein lacks FcyR binding (hence likely lacking ADCC activity), but retains FcRn binding ability. The primary cells for mediating ADCC, NK cells, express FcyRII I only, whereas monocytes express FcyRI, FcyRII and FcyRII I .

[0165] Substitutions may be introduced into the FC1 and FC2 regions that abrogate or reduce binding to Fey receptors and / or increase binding to FcRn, and / or prevent Fab arm exchange, and / or remove protease sites. In this regard, the half-life extending domain may also comprise one or more amino acid substitutions which prevent or reduce binding to activating receptors. The half-life extending domain may comprise one or more amino acid substitutions which prevent or reduce binding to FcyR. The Fc region may be a monomeric Fc (mFc), comprising only one Fc region. The mFc may have one or two heavy chain constant domains (termed CH2, CH3). Preferably the mFc has two heavy chain constant regions, for example CH2 and CH3. Alternatively, the mFc may have one heavy chain constant region, for example CH3. The mFc may comprise one or more mutations relative to a native constant domain sequence. Mutations may be made for the purpose of stabilisation, for example by introducing one or more non-native disulphide bonds. Examples of mFc sequences are known in the art, for example those described in WO2013138643, Ying 2012 (Ying, et al. JBC 2012, 287 (23) 19399-19408), Gong 2009 (Gong, et al. JBC 2009, 284 (21) 14203-14210) and Gong 2011 (Gong, et al. JBC 2011 , 286 (31) 27288-27293) and Ying 2013 (Ying, et al. 2013, 288 (35) 25154-25164). Preferably, the mFc domain contains stabilising mutations. Preferably the mFc retains pH dependent binding to FcRn. Alternatively, the mFc may include mutations to increase pH dependent binding to FcRn. A suitable mFc sequence is provided in SEQ ID NO: 43.

[0166] The half-life extending domain may alternatively comprise albumin, or an albumin-binding domain such as the Dill domain of albumin. Preferably the albumin binding domain is the Dill domain of albumin. The albumin binding domain may contain mutations, for example to enhance pH dependent binding to FcRn, for example as described in Andersen 2014 (Andersen, et al. JBC 2014, 289 (19) 13492-13502). A suitable sequence for Dill is provided in SEQ ID NO: 44. As is known in the art, albumin has a long circulatory half-life of 19 days, due in part to its size, being above the renal threshold, and by its specific interaction and recycling via FcRn. Attachment to albumin is a well- known strategy to improve the circulatory half-life of a therapeutic molecule in vivo. Albumin may be attached non-covalently, through the use of a specific albumin binding domain, or covalently, by conjugation or direct genetic fusion.

[0167] The half-life extending domains may alternatively comprise small peptide domains which mimic the interaction of IgG or albumin with FcRn, for example those described in Kelly 2022 (Kelly and Sirk ACS Chem. Biol. 2022, 17 (2) 404-413) or Sockolosky 2012 (Sockolosky, et al. PNAS 2012, 109 (40) 16095-16100) or any other suitable peptide domain known in the art.

[0168] Format and linkers

[0169] As used herein, the term “format” refers to the position and orientation of each domain, and the number of polypeptide chains, in the bispecific molecule of the invention. A schematic diagram of the formats of exemplary bispecific molecules is provided in Figures 1 , 4 and 9. The soluble TCR in the exemplary bispecific molecule shown in Figures 1 , 4 and 9 comprises TCR alpha and beta variable domains (i.e. , Va and Vp). The immune cell engaging domain in the exemplary bispecific molecule shown in Figures 1 , 4 and 9 comprises antibody light and heavy variable domains (i.e., VL and VH). The soluble TCR and the immune cell engaging domain of such molecules are capable of binding to a pMHC complex and an immune cell, respectively. In this regard, the soluble TCR and the immune cell engaging domain may be capable of simultaneously binding to a pMHC complex and an immune cell, respectively. The exemplary bispecific molecules shown in Figures 4 and 9 comprise a half-life extending (HLE) domain. In this regard, the HLE may be capable of extending the half-life of the bispecific molecule, relative to a bispecific molecule lacking the HLE (i.e. , a single chain HLE). The term "linker" as used herein refers to one or more amino acid residues inserted between domains, or a domain and an agent, to provide sufficient mobility for the domains or elements, for example the domains of the bispecific molecules described herein, to fold correctly to form the antigen binding sites, such that the bispecific molecule is capable of binding to its target pMHC complex and an immune cell. A linker may be inserted at the transition between variable domains or between variable domains and other domains, respectively, at the amino acid sequence level. The transition between domains can be identified because the approximate size of antibody domains as well as TOR domains is well understood by those skilled in the art. The precise location of a domain transition can be determined by locating peptide stretches that do not form secondary structural elements such as beta-sheets or alpha-helices as demonstrated by experimental data or as can be assumed by techniques of modelling or secondary structure prediction.

[0170] Two or more of the VL, VH, Va and / or Vp may be linked to each other via linkers and / or IgG hinge sequences. If the HLE is present, two or more of the VL, VH, Va, Vp and HLE may be linked to each other via linkers and / or IgG hinge sequences. Linker sequences may be flexible, in that they are made up primarily of amino acids such as glycine, alanine and serine, which do not have bulky side chains likely to restrict flexibility. Such linkers include “glycine-serine” linkers, which refer to linkers that comprise only, or predominantly, glycine and serine residues for example (GGGGS)n. Alternatively, linkers with greater rigidity may be desirable. Examples of more rigid linkers include alpha helix-forming linkers with the sequence of (EAAAK)n. Usable or optimum lengths of linker sequences may be easily determined. Often the linker sequence will be less than about 30, such as less than 25, less than 20, less than 10, or from 2-10 amino acids in length. The linker may be 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids in length. Examples of suitable linkers that may be used in bispecific molecules are known in the art and include those described in WO2010 / 133828 and Chen et al Adv Drug Deliv Rev.

[0171] 2013;65(10):1357-1369. For example, the linker or linkers present in the bispecific molecule of the invention may have a sequence selected from the group of GG, GGGGS (SEQ ID NO: 48), GGGSG (SEQ ID NO: 49), GGSGG (SEQ ID NO: 53), GSGGG (SEQ ID NO: 54), GSGGGP (SEQ ID NO: 55), GGEPS (SEQ ID NO: 56), GGEGGGP (SEQ ID NO: 57), GGEGGGSEGGGS (SEQ ID NO: 58), GGGSGGGG (SEQ ID NO: 50), GGSGGGGSGGGGSGGGGST (SEQ ID NO: 51), GGGGSGGGGSGGGGSGGGGSGGGS (SEQ ID NO: 52), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 46), GGGGSGGGGSGGGGS (SEQ ID NO: 59), EAAAK (SEQ ID NO: 60) and EAAAKEAAAKEAAAK (SEQ ID NO: 61).

[0172] Suitable IgG hinge sequences are known in the art and include the exemplary lgG1 / 2 hybrid hinge sequence EPKSSDKTHTCPPCPAPPVAGP (SEQ ID NO: 47) / EPKSSDKTHTCPPCPAPPVAG (SEQ ID NO: 109). Other suitable IgG hinge sequences include EPKSCDKTHTCPPCPAPELLGG (SEQ ID NO: 62), ERKCCVECPPCPAPPVAG (SEQ ID NO: 63), EPKSCDTPPPCPRCPAPELLGG (SEQ ID NO: 64), ESKYGPPCPSCPAPEFLGG (SEQ ID NO: 65), EPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 66), DKTHTCPPCPAPELLGG (SEQ ID NO: 67), DKTHTCPPCPAPPVAGP (SEQ ID NO: 68) / DKTHTCPPCPAPPVAG (SEQ ID NO: 110). As will be known to those skilled in the art, alternative sequences may be used in place of IgG hinge sequences to facilitate disulphide bond formation.

[0173] Examples of such sequences are disclosed herein and include GGCPPCGG (SEQ ID No: 69) and the amino acid sequence GGC.

[0174] Exemplary lgG1 / 2 hybrid hinge sequences EPKSSDKTHTCPPCPAPPVAGP (SEQ ID NO: 47) and EPKSSDKTHTCPPCPAPPVAG (SEQ ID NO: 109), and exemplary hinge sequences DKTHTCPPCPAPPVAGP (SEQ ID NO: 68) and DKTHTCPPCPAPPVAG (SEQ ID NO: 110), differ from one another in that a C-terminal Proline residue is either present or absent. This Proline residue generally represents the boundary between the hinge and the start of the Fc domain.

[0175] In the format of the bispecific molecule of the invention, the VH of the immune cell engaging domain is linked to the N-terminus of the Vp of the soluble TCR. The inventors have identified that molecules in this format have the highest potency of more than 20 different formats tested.

[0176] The bispecific molecule of the invention may be in a single-chain format (i.e., a single chain molecule). In this context, “single-chain” is used to describe a bispecific molecule that is expressed as a single polypeptide chain which contains the soluble TCR, the immune cell engaging domain and, if present, the half-life extending domain. As such, the VL, VH, Vp, Va and, if present, the HLE domain, may be on a single polypeptide chain. The HLE domain may be linked to one of the VL, VH, Vp, Va, i.e., the HLE domain may be attached at either end of the single chain. The HLE domain may be linked to two of the VL, VH, Vp, Va, i.e., the HLE domain may be within the single chain rather than attached to either end.

[0177] The bispecific molecule of the invention may be in a dual-chain format (i.e., a dual chain molecule). In this context, “dual-chain” is used to describe a bispecific molecule that is expressed as two polypeptide chains which together contain the soluble TCR, the immune cell engaging domain and, if present, the HLE domain. As such, the VL, VH, Vp, Va and, if present, the HLE, may be on either the first or the second polypeptide chain. The HLE domain may be linked to one of the VL, VH, Vp, Va, i.e., the HLE domain may be attached at either end of the first or second polypeptide chain. The two polypeptide chains may be linked by a disulphide bond. The disulphide bond may form between IgG hinge sequences fused to domains on separate chains.

[0178] The bispecific molecule of the invention may be in a triple-chain format (i.e., a triple chain molecule). In this context, “triple-chain” is used to describe a bispecific molecule that is expressed as three polypeptide chains which together contain the soluble TCR, the immune cell engaging domain and the HLE domain. As such, the VL, VH, Vp, Va and, if present, the HLE, may be present on either the first, second, or the third polypeptide chain. Any two of the three polypeptide chains may be linked by one or more disulphide bonds. The disulphide bonds may form between IgG hinge sequences fused to domains on separate chains.

[0179] The single chain bispecific molecule of the invention may have the following format: / V-VL-VH-Vp-Va- C.

[0180] In certain formats, the VL of the immune cell engaging domain may be linked to the Va of the soluble TOR. In this regard, preferably, the single chain bispecific molecule of the invention may have any one of the following formats: (i) / V-Va-VL-VH-Vp-C or (ii) / V-VH-Vp-Va-VL-C. In this regard, preferably, the dual chain bispecific molecule of the invention may comprise or consist of a first chain having the format A / -VH-Vp-C and a second chain having the format / V-Va-VL-C. In certain formats of the dual chain bispecific molecule, the Vp of the first chain may be linked to the VL of the second chain via a disulphide bond formed between IgG hinge sequences fused to said Vp and VL.

[0181] As shown in Figures 2B-C, in certain formats the bispecific molecule of the invention may comprise a linker sequence “L1 ” selected from the group consisting of GGGGSGGGGSGGGGSGGGGSGGGS (SEQ ID NO: 52), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 46) GGGGSGGGGSGGGGS (SEQ ID NO: 59), and GGSGGGGSGGGGSGGGGST (SEQ ID NO: 51); a linker sequence “L2” selected from the group consisting of GGGSGGGG (SEQ ID NO: 50), GGGGS (SEQ ID NO: 48) and GGGSG (SEQ ID NO: 49); and / or an IgG hinge sequence “L3” selected from the group consisting of EPKSCDKTHTCPPCPAPELLGG (SEQ ID NO: 62), ERKCCVECPPCPAPPVAG (SEQ ID NO: 63), EPKSCDTPPPCPRCPAPELLGG (SEQ ID NO: 64), ESKYGPPCPSCPAPEFLGG (SEQ ID NO: 65), EPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 66), DKTHTCPPCPAPELLGG (SEQ ID NO: 67), DKTHTCPPCPAPPVAGP (SEQ ID NO: 68), GGCPPCGG (SEQ ID No: 69) and the amino acid sequence GGC.

[0182] In this regard, the single chain bispecific molecule of the invention may comprise, in the following order, from N-terminus to C-terminus: a) a VL domain; b) a linker sequence “L1 ”; c) a VH domain; d) a linker sequence “L2”; e) a Vp domain; f) a linker sequence “L1 ”; g) a Va domain.

[0183] The single chain bispecific molecule may comprise, in the following order, from N-terminus to C- terminus: a) a Va domain; b) a linker sequence “L2”; c) a VL domain; d) a linker sequence “L1 e) a VH domain; f) a linker sequence “L2”; g) a Vp domain.

[0184] The single chain bispecific molecule may comprise, in the following order, from N-terminus to C- terminus: a) a VH domain; b) a linker sequence “L2”; c) a Vp domain; d) a linker sequence “L1 ”; e) a Va domain; f) a linker sequence “L2”; g) a VL domain.

[0185] The dual chain bispecific molecule may comprise:

[0186] (i) a first chain comprising, in the following order, from N-terminus to C-terminus: a) a VH domain; b) a linker sequence “L2”; c) a Vp domain; and (ii) a second chain comprising, in the following order, from N-terminus to C-terminus: a) a Va domain; b) a linker sequence “L2”; c) a VL domain.

[0187] The dual chain bispecific molecule may comprise:

[0188] (i) a first chain comprising, in the following order, from N-terminus to C-terminus: a) a VH domain; b) a linker sequence “L2”; c) a Vp domain; d) a hinge sequence “L3”; and (ii) a second chain comprising, in the following order, from N-terminus to C-terminus: a) a Va domain; b) a linker sequence “L2”; c) a VL domain; d) a hinge sequence “L3”.

[0189] If present, the HLE may be linked to one or more of the VH, VL, Va and / or Vp domains by a linker and / or an IgG hinge sequence. In this regard, preferably, the single chain half-life extended bispecific molecule of the invention may have any one of the following formats:

[0190] (i) / V-VL-VH-Vp-Va-HLE-C,

[0191] (ii) / V-HLE-VL-VH-Vp-Va-C,

[0192] (iii) / V-Va-VL-VH-Vp-HLE-C,

[0193] (iv) / V-HLE-Va-VL-VH-Vp-C,

[0194] (v) / V-VH-Vp-Va-VL-HLE-C,

[0195] (vi) / V-HLE-VH-Vp-Va-VL-C,

[0196] (vii) / V-Va-VL-HLE-VH-Vp-C, or

[0197] (viii) / V-VH-Vp-HLE-Va-VL-C.

[0198] In this regard, preferably, the dual chain half-life extended bispecific molecule of the invention may comprise or consist of:

[0199] (i) a first chain having the format A / -VH-Vp-C and a second chain having the format N- Va-VL-HLE-C;

[0200] (ii) a first chain having the format A / -VH-Vp-HLE-C and a second chain having the format A / -Va-VL-C;

[0201] (iii) a first chain having the format A / -VH-Vp-C and a second chain having the format N- HLE-Va-VL-C; or

[0202] (iv) a first chain having the format A / -HLE-VH-Vp-C and a second chain having the format M-Va-VL-C.

[0203] In certain dual chain formats, the HLE may be linked to the entire immune cell engaging domain or the entire soluble TOR. That is, the HLE may be linked to both the VH and VL, or both the Va and Vp.

[0204] In certain dual chain or triple chain formats, the HLE domain may comprise two chains (e.g., a first chain ‘FC1 ’ and a second chain ‘FC2’ of an Fc dimer). The first and second chain of the HLE may be linked to one or more of the VH, VL, Va and / or Vp domains on different chains of the bispecific molecule. In certain formats, the bispecific molecule may comprise an IgG hinge sequence “L4” selected from the group consisting of EPKSSDKTHTCPPCPAPPVAG (SEQ ID NO: 109) and DKTHTCPPCPAPPVAG (SEQ ID NO: 110).

[0205] In this regard, a multi chain half-life extended bispecific molecule may comprise:

[0206] (i) a first chain comprising, in the following order, from N-terminus to C-terminus: a) a Va domain; b) a linker sequence “L4”; c) a first chain of a HLE; (ii) a second chain comprising, in the following order, from N-terminus to C-terminus: a) a VL domain; b) a linker sequence “L4”; c) a second chain of a HLE; and (iii) a third chain comprising, in the following order, from N-terminus to C-terminus: a) a VH domain; b) a linker sequence “L2”; c) a Vp domain.

[0207] A dual chain half-life extended bispecific molecule may comprise:

[0208] (i) a first chain comprising, in the following order, from N-terminus to C-terminus: a) a Va domain; b) a linker sequence “L2”; c) a VL domain; d) a linker sequence “L1 ”; e) a VH domain; f) a linker sequence “L4”; g) a first chain of a HLE; and (ii) a second chain comprising, in the following order, from N-terminus to C-terminus: a) a Vp domain; b) a linker sequence “L4”; c) a second chain of a HLE.

[0209] A dual chain half-life extended bispecific molecule may comprise:

[0210] (i) a first chain comprising, in the following order, from N-terminus to C-terminus: a) a VH domain; b) a linker sequence “L2”; c) a Vp domain; d) a linker sequence “L1 ”; e) a Va domain; f) a linker sequence “L4”; g) a first chain of a HLE; and (ii) a second chain comprising, in the following order, from N-terminus to C-terminus: a) a VL domain; b) a linker sequence “L4”; c) a second chain of a HLE. A dual chain half-life extended bispecific molecule may comprise:

[0211] (i) a first chain comprising, in the following order, from N-terminus to C-terminus: a) a Va domain; b) a linker sequence “L1 ”; c) a VH domain; d) a linker sequence “L1 ”; e) a VL domain; f) a linker sequence “L4”; g) a first chain of a HLE; and (ii) a second chain comprising, in the following order, from N-terminus to C-terminus: a) a Vp domain; b) a linker sequence “L4”; c) a second chain of a HLE.

[0212] A dual chain half-life extended bispecific molecule may comprise:

[0213] (i) a first chain comprising, in the following order, from N-terminus to C-terminus: a) a VH domain; b) a linker sequence “L1 ”; c) a Va domain; d) a linker sequence “L1 ”; e) a Vp domain; f) a linker sequence “L4”; g) a first chain of a HLE; and (ii) a second chain comprising, in the following order, from N-terminus to C-terminus: a) a VL domain; b) a linker sequence “L4”; c) a second chain of a HLE.

[0214] A dual chain half-life extended bispecific molecule may comprise:

[0215] (i) a first chain comprising, in the following order, from N-terminus to C-terminus: a) a Va domain; b) a linker sequence “L1 ”; c) a VH domain; d) a linker sequence “L2”; e) a Vp domain; f) a linker sequence “L4”; g) a first chain of a HLE; and (ii) a second chain comprising, in the following order, from N-terminus to C-terminus: a) a VL domain; b) a linker sequence “L4”; c) a second chain of a HLE.

[0216] A dual chain half-life extended bispecific molecule may comprise:

[0217] (i) a first chain comprising, in the following order, from N-terminus to C-terminus: a) a VH domain; b) a linker sequence “L1 c) a Va domain; d) a linker sequence “L2”; e) a VL domain; f) a linker sequence “L4”; g) a first chain of a HLE; and (ii) a second chain comprising, in the following order, from N-terminus to C-terminus: a) a Vp domain; b) a linker sequence “L4”; c) a second chain of a HLE.

[0218] A dual chain half-life extended bispecific molecule may comprise:

[0219] (i) a first chain comprising, in the following order, from N-terminus to C-terminus: a) a VH domain; b) a linker sequence “L2”; c) a Vp domain; d) a linker sequence “L1 ”; e) a VL domain; f) a linker sequence “L4”; g) a first chain of a HLE; and (ii) a second chain comprising, in the following order, from N-terminus to C-terminus: a) a Va domain; b) a linker sequence “L4”; c) a second chain of a HLE.

[0220] In this regard, the triple chain half-life extended bispecific molecule of the invention may comprise or consist of a first chain having (i) a first chain having the format A / -Va-FC1-C, a second chain having the format A / -VL-FC2-C, and a third chain having the format A / -VH-Vp-C. In this regard, the dual chain half-life extended bispecific molecule may comprise or consist of:

[0221] (i) a first chain having the format A / -VH-Vp-Va-FC1-C and a second chain having the format A / -VL-FC2-C;

[0222] (ii) a first chain having the format A / -Va-VH-Vp-FC1-C and a second chain having the format A / -VL-FC2-C; or

[0223] (iii) a first chain having the format A / -VH-Vp-VL-FC1-C and a second chain having the format A / -Va-FC2-C.

[0224] Preferred formats are the triple chain half-life extended bispecific molecule of the invention comprising or consisting of a first chain having (i) a first chain having the format A / -Va-FC1-C, a second chain having the format A / -VL-FC2-C, and a third chain having the format A / -VH-Vp-C; and the dual chain half-life extended bispecific molecule comprising or consisting of a first chain having the format A / -VH- Vp-Va-FC1-C and a second chain having the format A / -VL-FC2-C.

[0225] Complete amino acid sequences of exemplary chains of bispecific molecules are provided in SEQ ID NOs: 1-26, 78-79 and 82-90. Complete amino acid sequences of exemplary chains of bispecific molecules without a HLE domain are provided in SEQ ID Nos: 1-7. Complete amino acid sequences of exemplary chains of bispecific molecules with a HLE domain (half-life extended bispecific molecules) are provided in SEQ ID Nos: 8-26, 78-79 and 82-90.

[0226] A single chain bispecific molecule may consist of an amino acid sequence selected from the group consisting of: SEQ ID NOs: 1 (Molecule A), 6 (Molecule D1), 7 (Molecule D2).

[0227] A single chain half-life extended bispecific molecule may consist of an amino acid sequence selected from the group consisting of: SEQ ID NOs: 8 (Molecule A mFc v1), 9 (Molecule A mFc v2), 16 (Molecule C mFc v5), 17 (Molecule C mFc v6), 18 (Molecule D1 mFc v1), 19 (Molecule D1 mFc v2), 24 (Molecule C Dill v6), 25 (Molecule D1 Dill v1), 26 (Molecule D2 Dill v1).

[0228] A dual chain bispecific molecule may consist of a first chain having the amino acid sequence of SEQ ID NO: 2 and a second chain having the amino acid sequence of SEQ ID NO: 3 (Molecule B).

[0229] A dual chain bispecific molecule may consist of a first chain having the amino acid sequence of SEQ ID NO: 4 and a second chain having the amino acid sequence of SEQ ID NO: 5 (Molecule C).

[0230] A dual chain half-life extended bispecific molecule may consist of a first chain having the amino acid sequence of SEQ ID NO: 10 and a second chain having the amino acid sequence of SEQ ID NO: 11 (Molecule C mFc v1).

[0231] A dual chain half-life extended bispecific molecule may consist of a first chain having the amino acid sequence of SEQ ID NO: 12 and a second chain having the amino acid sequence of SEQ ID NO: 13 (Molecule C mFc v2). A dual chain half-life extended bispecific molecule may consist of a first chain having the amino acid sequence of SEQ ID NO: 14 and a second chain having the amino acid sequence of SEQ ID NO: 15 (Molecule C mFc v3).

[0232] A dual chain half-life extended bispecific molecule may consist of a first chain having the amino acid sequence of SEQ ID NO: 20 and a second chain having the amino acid sequence of SEQ ID NO: 21 (Molecule C Dill v1).

[0233] A dual chain half-life extended bispecific molecule may consist of a first chain having the amino acid sequence of SEQ ID NO: 22 and a second chain having the amino acid sequence of SEQ ID NO: 23 (Molecule C Dill v2).

[0234] A dual chain half-life extended bispecific molecule may consist of a first chain having the amino acid sequence of SEQ ID NO: 78 and a second chain having the amino acid sequence of SEQ ID NO: 79 (Molecule C mFc v4).

[0235] A dual chain half-life extended bispecific molecule may consist of a first chain having the amino acid sequence of SEQ ID NO: 85 and a second chain having the amino acid sequence of SEQ ID NO: 86 (Molecule Ev3).

[0236] A dual chain half-life extended bispecific molecule may consist of a first chain having the amino acid sequence of SEQ ID NO: 87 and a second chain having the amino acid sequence of SEQ ID NO: 88 (Molecule Ev6).

[0237] A dual chain half-life extended bispecific molecule may consist of a first chain having the amino acid sequence of SEQ ID NO: 89 and a second chain having the amino acid sequence of SEQ ID NO: 90 (Molecule Ev8).

[0238] A triple chain half-life extended bispecific molecule may consist of a first chain having the amino acid sequence of SEQ ID NO: 82, a second chain having the amino acid sequence of SEQ ID NO: 83, and a third chain having the amino acid sequence of SEQ ID NO: 84 (Molecule Ev1).

[0239] Amino acid sequences

[0240] Within the scope of the invention are phenotypically silent variants of any bispecific molecule disclosed herein. As used herein the term “phenotypically silent variants” is understood to refer to a variant which incorporates one or more further amino acid changes, including substitutions, insertions and deletions, in addition to those set out above, and which variant has a similar phenotype to the corresponding molecule without said change(s). For the purposes of this application, phenotype comprises binding affinity (KD and / or binding half-life) and specificity. The phenotype for a soluble bispecific molecule may include potency of immune activation and purification yield, in addition to binding affinity and specificity.

[0241] Phenotypically silent variants may contain one or more conservative substitutions and / or one or more tolerated substitutions. By tolerated substitutions it is meant those substitutions which do not fall under the definition of conservative as provided below but are nonetheless phenotypically silent. The skilled person is aware that various amino acids have similar properties and thus are ‘conservative’. One or more such amino acids of a protein, polypeptide or peptide can often be substituted by one or more other such amino acids without eliminating a desired activity of that protein, polypeptide or peptide.

[0242] Thus the amino acids glycine, alanine, valine, leucine and isoleucine can often be substituted for one another (amino acids having aliphatic side chains). Of these possible substitutions it is preferred that glycine and alanine are used to substitute for one another (since they have relatively short side chains) and that valine, leucine and isoleucine are used to substitute for one another (since they have larger aliphatic side chains which are hydrophobic). Other amino acids which can often be substituted for one another include: phenylalanine, tyrosine and tryptophan (amino acids having aromatic side chains); lysine, arginine and histidine (amino acids having basic side chains); aspartate and glutamate (amino acids having acidic side chains); asparagine and glutamine (amino acids having amide side chains); and cysteine and methionine (amino acids having sulphur containing side chains). It should be appreciated that amino acid substitutions within the scope of the present invention can be made using naturally occurring or non-naturally occurring amino acids. For example, it is contemplated herein that the methyl group on an alanine may be replaced with an ethyl group, and / or that minor changes may be made to the peptide backbone. Whether or not natural or synthetic amino acids are used, it is preferred that only L- amino acids are present.

[0243] Substitutions of this nature are often referred to as “conservative” or “semi-conservative” amino acid substitutions. The present invention therefore extends to use of a molecule comprising any of the amino acid sequences described above but with one or more conservative substitutions and or one or more tolerated substitutions in the sequence, such that the amino acid sequence of the molecule, or any domain or region thereof, has at least 90% identity, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity, to the sequences disclosed herein.

[0244] “Identity” as known in the art is the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. While there exist a number of methods to measure identity between two polypeptide or two polynucleotide sequences, methods commonly employed to determine identity are codified in computer programs. Preferred computer programs to determine identity between two sequences include, but are not limited to, GCG program package, BLASTP, BLASTN, and FASTA.

[0245] One can use a program such as the CLUSTAL program to compare amino acid sequences. This program compares amino acid sequences and finds the optimal alignment by inserting spaces in either sequence as appropriate. It is possible to calculate amino acid identity or similarity (identity plus conservation of amino acid type) for an optimal alignment. A program like BLASTx will align the longest stretch of similar sequences and assign a value to the fit. It is thus possible to obtain a comparison where several regions of similarity are found, each having a different score. Both types of identity analysis are contemplated in the present invention.

[0246] The percent identity of two amino acid sequences or of two nucleic acid sequences is determined by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced in the first sequence for best alignment with the sequence) and comparing the amino acid residues or nucleotides at corresponding positions. The “best alignment” is an alignment of two sequences which results in the highest percent identity. The percent identity is determined by the number of identical amino acid residues or nucleotides in the sequences being compared (i.e. , % identity = number of identical positions / total number of positions x 100).

[0247] The determination of percent identity between two sequences can be accomplished using a mathematical algorithm known to those of skill in the art. Determination of percent identity between two nucleotide sequences can be performed with the BLASTn program. Determination of percent identity between two protein sequences can be performed with the BLASTp program. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilised . Alternatively, PSI-Blast can be used to perform an iterated search which detects distant relationships between molecules (Id.). When utilising BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., BLASTp and BLASTp) can be used. See http: / / www.ncbi.nlm.nih.gov. Default general parameters may include for example, Word Size = 3, Expect Threshold = 10. Parameters may be selected to automatically adjust for short input sequences. Another example of a mathematical algorithm utilised for the comparison of sequences is the algorithm CABIOS . The ALIGN program (version 2.0) which is part of the CGC sequence alignment software package has incorporated such an algorithm. Other algorithms for sequence analysis known in the art include ADVANCE and ADAM, and FASTA. Within FASTA, ktup is a control option that sets the sensitivity and speed of the search. For the purposes of evaluating percent identity in the present disclosure, BLASTp with the default parameters is used as the comparison methodology. In addition, when the recited percent identity provides a non-whole number value for amino acids (i.e., a sequence of 25 amino acids having 90% sequence identity provides a value of “22.5”, the obtained value is rounded down to the next whole number, thus “22”). Accordingly, in the example provided, a sequence having 22 matches out of 25 amino acids is within 90% sequence identity. As will be obvious to those skilled in the art, it may be possible to truncate, or extend, the sequences provided at the C-terminus and / or N-terminus thereof, by 1 , 2, 3, 4, 5 or more residues, without substantially affecting the functional characteristics of the bispecific molecule. The sequences provided at the C-terminus and / or N-terminus thereof may be truncated or extended by 1 , 2, 3, 4 or 5 residues. All such variants are encompassed by the present invention.

[0248] Mutations, including conservative and tolerated substitutions, insertions and deletions, may be introduced into the sequences provided using any appropriate method including, but not limited to, those based on polymerase chain reaction (PCR), restriction enzyme-based cloning, or ligation independent cloning (LIC) procedures. These methods are detailed in many of the standard molecular biology texts. The protein sequences provided herein may be obtained from recombinant expression, solid state synthesis, or any other appropriate method known in the art.

[0249] Assessing binding characteristics and activity of bispecific molecules

[0250] Methods to determine binding affinity (inversely proportional to the equilibrium constant KD) and binding half-life (expressed as T14) are known to those skilled in the art. Binding affinity and binding half-life may be determined using Surface Plasmon Resonance (SPR) or Bio-Layer Interferometry (BLI), for example using a BIAcore instrument or Octet instrument, respectively. For example, binding affinity of a bispecific molecule for a peptide-MHC complex may be determined using SPR at 25°C, and / or at 37°C, wherein the peptide-MHC complex is immobilised on a solid support (e.g., a sensor chip) and is contacted with a solution comprising the bispecific molecule. Suitable experimental conditions and methods for determining binding parameters are described in the Examples (e.g., Example 1 and Example 2).

[0251] It will be appreciated by those skilled in the art that a higher affinity refers to a lower numerical value for KD and indicates stronger binding In other words, a doubling of affinity refers to halving the numerical value of the KD. T1 is calculated as In2 divided by the off-rate (koff). Therefore, doubling of TV2 results in a halving in koff. KD and koff values for TCRs are usually measured for soluble forms of the TCR, i.e. those forms which are truncated to remove cytoplasmic and transmembrane domain residues. To account for variation between independent measurements, and particularly for interactions with dissociation times in excess of 20 hours, the binding affinity and or binding half-life of a given protein may be measured several times, for example 3 or more times, using the same assay protocol, and an average of the results taken. To compare binding data between two samples (i.e. two different proteins and or two preparations of the same protein) it is preferable that measurements are made using the same assay conditions (e.g. temperature). Measurement methods described in relation to TCRs may also be applied to the bispecific molecules described herein.

[0252] Bispecific molecules of the invention, are able to generate potent activation or inhibition of a T cell response in vitro against antigen positive cells, in particular those cells presenting low levels of antigen typical of cancer cells (i.e. in the order of 5-100, for example 50, antigens per cell (Bossi et al., (2013) Oncoimmunol. 1 ;2 (11) :e26840; Purbhoo et a / ., (2006). J Immunol 176(12): 7308-7316.). The T cell response that is measured may be the release of T cell activation markers such as Interferon y or Granzyme B, or target cell killing, or other measure of T cell activation, such as T cell proliferation. A highly potent response may be one with EC50 or IC50 value in the nM - pM range, for example 500 nM or lower, preferably 1 nM or lower, or 500 pM or lower, or 100 pM or lower.

[0253] Molecules encompassed by the present invention may have an improved half-life. Methods for determining whether a protein has an improved half-life will be apparent to the skilled person. For example, the ability of a protein to bind to a neonatal Fc receptor (FcRn) in a pH dependent manner may be assessed. In this regard, increased binding affinity for FcRn increases the serum half-life of the protein.

[0254] The half-life of a protein disclosed herein can also be measured by in vivo pharmacokinetic studies, using an in vivo model, such as a mouse model. For example labelled protein may be injected intravenously into mice and plasma concentration periodically measured as a function of time, for example at 3 minutes to 72 hours after the injection. Alternatively, an unlabeled bispecific molecule of the invention can be injected and its plasma concentration periodically measured using an activity based ELISA assay. Experiments may be repeated at difference dose levels, for example two dose levels or three dose levels. Plasma clearance may be determined by non-compartmental analysis.

[0255] Nucleic acids, vectors and host cells

[0256] The present invention provides a nucleic acid encoding a bispecific molecule or a half-life extended bispecific molecule described herein. The nucleic acid may be DNA or RNA. For example, the nucleic acid may be cDNA. The nucleic acid may be mRNA. The nucleic acid may be non-naturally occurring and / or purified and / or engineered. The nucleic acid sequence may be codon optimised, in accordance with the expression system utilised. As is known to those skilled in the art, expression systems may include bacterial cells such as E. coli, or yeast cells, or mammalian cells, or insect cells, or they may be cell free expression systems.

[0257] The nucleic acid may encode domains of a bispecific molecule or a half-life extended bispecific molecule of the invention within a single open reading frame or within distinct open reading frames. For example, the VL, VH, Vp and Va and, if present, the HLE of a single chain bispecific molecule may be encoded within a single open reading frame or within distinct open reading frames. In a dual chain bispecific molecule, the two polypeptide chains may be encoded within a single open reading frame or within distinct open reading frames. In a triple chain bispecific molecule, the three polypeptide chains may be encoded within a single open reading frame or within distinct open reading frames. In this regard, the present invention also provides a nucleic acid encoding: (i) a single chain bispecific molecule as described herein, wherein the VL, VH, Vp and Va are encoded within a single open reading frame or within distinct open reading frames;

[0258] (ii) a dual chain bispecific molecule as described herein, wherein the first and second chains are encoded within a single open reading frame or within distinct open reading frames;

[0259] (iii) a single chain half-life extended bispecific molecule as described herein, wherein the VL, VH, Vp, Va and HLE are encoded within a single open reading frame or within distinct open reading frames;

[0260] (iv) a dual chain half-life extended bispecific molecule as described herein, wherein the HLE is linked to the first chain or the second chain, and wherein the first and second chains are encoded within a single open reading frame or within distinct open reading frames;

[0261] (v) a triple chain half-life extended bispecific molecule as described herein, wherein the HLE is linked to the first chain, the second chain and / or the third chain, and wherein the first, second and third chains are encoded within a single open reading frame or within distinct open reading frames.

[0262] The present invention also provides constructs in the form of plasmids, vectors, transcription or expression cassettes which comprise at least one nucleic acid as described above. The present invention also provides a recombinant host cell which comprises one or more constructs as above. As mentioned, a nucleic acid encoding a bispecific molecule or a half-life extended bispecific molecule of the invention forms an aspect of the present invention, as does an expression vector comprising said nucleic acid, as does a method of production of the bispecific molecule or a half-life extended bispecific molecule comprising expression from a nucleic acid encoding a bispecific molecule or a half-life extended bispecific molecule of the invention. Expression may conveniently be achieved by culturing recombinant host cells containing the nucleic acid under appropriate conditions. Following production by expression, a bispecific molecule or a half-life extended bispecific molecule may be isolated and / or purified using any suitable technique, then used as appropriate.

[0263] Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Vectors may be any suitable vectors known in the art, including plasmids or viral vectors (e.g. ‘phage, or phagemid), as appropriate. Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins.

[0264] The present invention also provides a host cell containing a nucleic acid or an expression vector as disclosed herein. As will be known to those skilled in the art, the polypeptide chains forming a molecule may be expressed by separate expression vectors. Accordingly, the two chains of a dual chain bispecific molecule described herein may be expressed by separate expression vectors. Thus, the present invention also provides a cell harbouring a first expression vector comprising a nucleic acid encoding a first chain of a dual chain bispecific molecule or a dual chain half-life extended bispecific molecule described herein, and a second expression vector comprising a nucleic acid encoding a second chain of a dual chain bispecific molecule or a dual chain half-life extended bispecific molecule described herein.

[0265] Further, the invention provides a method comprising introducing such nucleic acid into a host cell. The introduction may employ any available technique. For eukaryotic cells, suitable techniques may include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus, e.g. vaccinia or, for insect cells, baculovirus. For bacterial cells, suitable techniques may include calcium chloride transformation, electroporation and transfection using bacteriophage. The introduction may be followed by causing or allowing expression from the nucleic acid, e.g. by culturing host cells under conditions for expression of the gene.

[0266] Suitable host cells for cloning or expression of nucleic acids and / or vectors of the present invention are known in the art. Suitable host cells for protein expression include bacteria (for example, E. coli), fungi (for example, yeast), plant and animal (invertebrates and vertebrates) cells. Examples of invertebrate cells include insect cells. Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures can also be utilized as hosts. Vertebrate cells may also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (HEK cells, for example 293 or 293T cells); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells as described; monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells (as described; MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR- CHO cells); and myeloma cell lines such as Y0, NSO and Sp2 / 0. The host cell may be eukaryotic, e.g., a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NSO, Sp20 cell).

[0267] The nucleic acid of the invention may be integrated into the genome (e.g. chromosome) of the host cell. Integration may be promoted by inclusion of sequences which promote recombination with the genome, in accordance with standard techniques.

[0268] Methods of making bispecific molecules

[0269] Methods of producing a bispecific molecule or a half-life extended bispecific molecule of the invention may comprise recombinantly expressing the bispecific molecule or half-life extended bispecific molecule in a host cell and subsequently purifying the bispecific molecule or half-life extended bispecific molecule. The methods may comprise maintaining the host cell under optimal conditions for expression of a nucleic acid or expression vector encoding the bispecific molecule or half-life extended bispecific molecule and isolating the bispecific molecule or half-life extended bispecific molecule.

[0270] Methods of producing recombinant proteins are well known in the art. Nucleic acids encoding the protein can be cloned into expression constructs or vectors, which are then transfected into host cells, as described herein above. Molecular cloning techniques to achieve these ends are known in the art. A wide variety of cloning and in vitro amplification methods are suitable for the construction of recombinant nucleic acids. Methods of producing recombinant antibodies are also known in the art.

[0271] The nucleic acid may be inserted operably linked to a promoter in an expression construct or expression vector for further cloning (amplification of the DNA) or for expression in a cell-free system or in cells. As used herein, the term “promoter” is to be taken in its broadest context and includes the transcriptional regulatory sequences of a genomic gene, including the TATA box or initiator element, which is required for accurate transcription initiation, with or without additional regulatory elements (e.g., upstream activating sequences, transcription factor binding sites, enhancers and silencers) that alter expression of a nucleic acid, e.g., in response to a developmental and / or external stimulus, or in a tissue specific manner. In the present context, the term “promoter” is also used to describe a recombinant, synthetic or fusion nucleic acid, or derivative which confers, activates or enhances the expression of a nucleic acid to which it is operably linked. Exemplary promoters can contain additional copies of one or more specific regulatory elements to further enhance expression and / or alter the spatial expression and / or temporal expression of said nucleic acid. As used herein, the term “operably linked to" means positioning a promoter relative to a nucleic acid such that expression of the nucleic acid is controlled by the promoter.

[0272] Many vectors for expression in cells are commercially available. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, a sequence encoding a protein (e.g., derived from the information provided herein), an enhancer element, a promoter, and a transcription termination sequence. The skilled person will be aware of suitable sequences for expression of a protein. Exemplary signal sequences include prokaryotic secretion signals (e.g., pe1 B, alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II), yeast secretion signals (e.g., invertase leader, a factor leader, or acid phosphatase leader) or mammalian secretion signals (e.g., herpes simplex gD signal).

[0273] Exemplary promoters active in mammalian cells include cytomegalovirus immediate early promoter (CMV-IE), human elongation factor 1-a promoter (EF1), small nuclear RNA promoters (Ula and Ulb), a-myosin heavy chain promoter, Simian virus 40 promoter (SV40), Rous sarcoma virus promoter (RSV), Adenovirus major late promoter, p-actin promoter; hybrid regulatory element comprising a CMV enhancer / p-actin promoter or an immunoglobulin promoter or an active fragment thereof. Typical promoters suitable for expression in yeast cells such as for example a yeast cell selected from the group comprising Pichia pastoris, Saccharomyces cerevisiae and S. pombe, include, but are not limited to, the ADH1 promoter, the GAL1 promoter, the GALA promoter, the CUP1 promoter, the PH05 promoter, the nmt promoter, the RPR1 promoter, or the TEF1 promoter.

[0274] The host cells used to produce the protein may be cultured in a variety of media, depending on the cell type used. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPM1-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing mammalian cells. Media for culturing other cell types discussed herein are known in the art.

[0275] Methods for isolating a protein are known in the art. Where a protein is secreted into culture medium, supernatants from such expression systems can be first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants. Alternatively, or additionally, supernatants can be filtered and / or separated from cells expressing the protein, e.g., using continuous centrifugation.

[0276] The protein prepared from the cells can be purified using, for example, ion exchange, hydroxyapatite chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, affinity chromatography (e.g., protein A affinity chromatography or protein G chromatography), or any combination of the foregoing. These methods are known in the art.

[0277] The skilled person will also be aware that a protein can be modified to include a tag to facilitate purification or detection, e.g., a poly-histidine tag, a hexa-histidine tag, an influenza virus hemagglutinin (HA) tag, a Simian Virus 5 (V5) tag, a LLAG tag, or a glutathione S-transferase (GST) tag. The resulting protein is then purified using methods known in the art, such as, affinity purification. For example, a protein comprising a hexa-his tag is purified by contacting a sample comprising the protein with nickel-nitrilotriacetic acid (Ni-NTA) that specifically binds a hexa-his tag immobilized on a solid or semi-solid support, washing the sample to remove unbound protein, and subsequently eluting the bound protein. Alternatively, or in addition a ligand or antibody that binds to a tag is used in an affinity purification method.

[0278] Bispecific molecules or half-life extended bispecific molecules described herein may be amenable to high yield purification. Yield may be determined based on the amount of material retained during the purification process (i.e. the amount of correctly folded material obtained at the end of the purification process relative to the amount of solubilised material obtained prior to refolding), and / or yield may be based on the amount of correctly folded material obtained at the end of the purification process, relative to the original culture volume. High yield means greater than 1 %, or greater than 5%, or higher yield. High yield means greater than 1 mg / ml, or greater than 3 mg / ml, or greater than 5 mg / ml, or higher yield.

[0279] Pharmaceutical compositions and medical methods

[0280] For administration to patients, the bispecific molecules, half-life extended bispecific molecules, nucleic acids, expression vectors or cells of the invention may be provided as part of a pharmaceutical composition together with one or more pharmaceutically acceptable carriers or excipients. This pharmaceutical composition may be in any suitable form (e.g. depending upon the desired method of administering it to a patient). It may be provided in unit dosage form and will generally be provided in a sealed container and may be provided as part of a kit. Such a kit would normally (although not necessarily) include instructions for use. It may include a plurality of said unit dosage forms.

[0281] The pharmaceutical composition may be adapted for administration by any appropriate route, such as parenteral (including subcutaneous, intramuscular, intrathecal or intravenous), enteral (including oral or rectal), inhalation or intranasal routes. Such compositions may be prepared by any method known in the art of pharmacy, for example by mixing the active ingredient with the carrier(s) or excipient(s) under sterile conditions. Methods for preparing a protein into a suitable form for administration to a subject (e.g. a pharmaceutical composition) are known in the art.

[0282] The pharmaceutical compositions will commonly comprise a solution of a bispecific molecule or a half-life extended bispecific molecule of the invention (or the nucleic acid, cell, or vector of the invention) dissolved in a pharmaceutically acceptable carrier, for example an aqueous carrier. A variety of aqueous carriers can be used, e.g., buffered saline and the like. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of the bispecific molecules in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the patient's needs. Exemplary carriers include water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Nonaqueous vehicles such as mixed oils and ethyl oleate may also be used. Liposomes may also be used as carriers. The vehicles may contain minor amounts of additives that enhance isotonicity and chemical stability, e.g., buffers and preservatives.

[0283] Bispecific molecules or half-life extended bispecific molecules described herein may have an ideal safety profile for use as therapeutic agents. An ideal safety profile means that in addition to demonstrating good specificity, the bispecific molecules described herein may have passed further preclinical safety tests. Examples of such tests include whole blood assays to confirm minimal cytokine release in the presence of whole blood and thus low risk of causing a potential cytokine release syndrome in vivo, and alloreactivity tests to confirm low potential for recognition of alternative HLA types.

[0284] Dosages of the bispecific molecules or half-life extended bispecific molecules described herein can vary between wide limits, depending upon the disease or disorder to be treated, the age and condition of the individual to be treated, etc. A physician will ultimately determine appropriate dosages to be used.

[0285] Bispecific molecules, half-life extended bispecific molecules, pharmaceutical compositions, vectors, nucleic acids and cells of the invention may be provided in substantially pure form, for example, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% pure.

[0286] The bispecific molecule, half-life extended bispecific molecule, nucleic acid, vector, pharmaceutical composition and cell of the invention may be used for treating diseases such as cancer, particularly cancers which are associated with expression of a tumour-associated antigen. For example, the cancer may be associated with expression of GP100, PRAME, PIWIL1 , MAGEA4 or PRAME. For example, the cancer may be associated with expression of an antigen described in, for example, WO2011001152 (and US 2012-0225481 A1), WO2017109496 (and US 2019-0002523 A1), WG2017175006 (and US 2019- 0092834 A1) or WO2018234319 (and US 2021-0355188 A1), which are all incorporated herein by reference in their entirety.

[0287] Alternatively, the bispecific molecule, half-life extended bispecific molecule, nucleic acid, vector, pharmaceutical composition and cell of the invention may be used for treating an infectious disease. The infectious disease may be caused by a bacterial, viral, fungal or parasitic pathogen. Any infection with a pathogen which results in antigen-presenting cells presenting HI_A bound to a peptide from the pathogen may be suitable for treatment with the bispecific molecule of the invention. The bispecific molecule or half-life extended bispecific molecule of the invention is particularly well suited to infections where antigen-presenting cells present levels of pathogen peptide that are lower than optimal for the natural immune system to clear the infection without additional treatment. The infectious disease may be a chronic infection. Exemplary infectious diseases include Hepatitis B virus (HBV) infection and human immunodeficiency virus (HIV) infection. Thus, the peptide in the pHI_A may be a peptide from a HBV or HIV protein.

[0288] The bispecific molecule, half-life extended bispecific molecule, nucleic acid, vector, pharmaceutical composition and cell of the invention may alternatively be used in a method of treating an autoimmune disease, such as type 1 diabetes. For example, the type 1 diabetes may be associated with expression of PPI . For example, the type 1 diabetes may be associated with expression of an antigen described in, for example, WO2024223842, which is incorporated herein by reference in its entirety. Organ-specific immune suppression, rather than systemic immunosuppression, may be a beneficial route for treatment given the potential significant adverse events associated with systemic immunosuppression. In autoimmunity, there is also mounting evidence that PD-1 pathway impairment plays an important role in disease pathogenesis. PD-1 , PD-L1 and PD-L2 gene polymorphisms are associated with several autoimmune diseases. Abnormally low PD-L1 expression has been observed in samples from type 1 diabetes and Crohn’s disease patients. Activating PD-1 on autoreactive lymphocytes thus may serve as a mechanism to treat autoimmune diseases. Effective therapeutics for the treatment of autoimmune diseases include those having an advantageous risk profile (e.g., a high level of target and tissue specificity) and are capable of being administered with less frequency.

[0289] Also provided by the invention are:

[0290] • a bispecific molecule, nucleic acid, expression vector, pharmaceutical composition or cell of the invention for use in medicine, preferably for use in a method of treating cancer or a tumour or an autoimmune disease or an infectious disease;

[0291] • the use of a bispecific molecule, nucleic acid, expression vector, pharmaceutical composition or cell of the invention in the manufacture of a medicament for treating cancer or a tumour or an autoimmune disease or an infectious disease;

[0292] • a method of treating cancer or a tumour or an autoimmune disease or an infectious disease in a patient, comprising administering a bispecific molecule, nucleic acid, expression vector, pharmaceutical composition or cell of the invention to a subject in need thereof;

[0293] • a method of targeting a tumour and / or redirecting T cells to a tumour, comprising administering a bispecific molecule, nucleic acid, expression vector, pharmaceutical composition or cell of the invention to a subject in need thereof; and

[0294] • an injectable formulation for administering to a human subject comprising a bispecific molecule, nucleic acid, expression vector, pharmaceutical composition or cell of the invention.

[0295] The method of treatment may further include administering separately, in combination, or sequentially, an additional therapeutic agent, for example an anti-neoplastic agent. Example of such agents are known in the art and may include immune activating agents and / or T cell modulating agents.

[0296] Kits and articles of manufacture

[0297] In another aspect, a kit or an article of manufacture containing materials useful for the treatment and / or prevention of the diseases described above is provided.

[0298] The kit may comprise (a) a container comprising the bispecific molecule, nucleic acid, expression vector or cell of the invention, optionally in a pharmaceutically acceptable carrier or diluent; and (b) a package insert with instructions for treating a disease (e.g., cancer, immune disease or autoimmune disease) in a subject. The kit may further comprise (c) at least one further therapeutically active compound or drug. The package insert may be on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds or contains a composition that comprises the molecule, nucleic acid, vector or cell of the invention and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is the molecule, nucleic acid, vector or cell of the invention. The label or package insert indicates that the composition is used for treating a subject eligible for treatment, e.g., one having or predisposed to developing a disease described herein, with specific guidance regarding dosing amounts and intervals of the composition and any other medicament being provided. The kit may further comprise an additional container comprising a pharmaceutically acceptable diluent buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and / or dextrose solution. The kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0299] The kit optionally further comprises a container comprising a second medicament, wherein the molecule, nucleic acid, expression vector or cell of the invention is a first medicament, and which kit further comprises instructions on the package insert for treating the subject with the second medicament, in an effective amount.

[0300] Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. Features of each aspect of the invention are as for each of the other aspects mutatis mutandis. For example, any features disclosed herein in the context of a bispecific molecule of the invention, equally apply to the methods, and other aspects, of the invention. The documents referred to herein are incorporated by reference to the fullest extent permitted by law.

[0301] Description of the drawings

[0302] Figure 1 is a schematic showing a panel of TCR bispecific molecules. TCR alpha and beta variable domains and antibody light and heavy variable domains are indicated Va, Vp, VL and VH respectively.

[0303] Figure 2 includes schematics showing: A) panel of TCR bispecific molecules with improved function relative to ImmTAC™ (molecules A, B, C, D1 and D2). B) corresponding linear domain arrangement of molecules A, B, C, D1 and D2. The location of interdomain linkers / hinges is indicated with L (L1 , L2 and L3). C) panel of linker (L1 and L2) and IgG hinge or alternative (L3) options for interdomain linkers / hinges indicated in Figure 2B. Figure 3 shows T cell activation in the presence of molecules A-D and comprising a TCR that binds to a HLA restricted peptide from either PPI or CT83. The equivalent ImmTAC™ molecule is included for comparison.

[0304] Figure 4 is a schematic showing a panel of TCR bispecific molecules incorporating a half-life extending domain.

[0305] Figure 5 shows T cell activation in the presence of TCR bispecific molecules comprising a TCR that binds to an HLA restricted peptide from PPI, and monomeric Fc as a half-life extending domain. Equivalent ImmTAC™ molecule without HLE domain is included for comparison.

[0306] Figure 6 shows T cell activation in the presence of TCR bispecific molecules comprising a TCR that binds to an HLA restricted peptide from PPI, and HSA Dill as a half-life extending domain. Equivalent ImmTAC™ molecule without HLE domain is included for comparison.

[0307] Figure 7 shows T cell activation in the presence of TCR bispecific molecules comprising a TCR that binds to an HLA restricted peptide from CT83, and monomeric Fc as a half-life extending domain. Equivalent ImmTAC™ molecule without HLE domain is included for comparison.

[0308] Figure 8 shows T cell activation in the presence of TCR bispecific molecules comprising a TCR that binds to an HLA restricted peptide from CT83, and HSA Dill as a half-life extending domain. Equivalent ImmTAC™ molecule without HLE domain is included for comparison.

[0309] Figure 9 is a schematic showing a panel of TCR bispecific molecules incorporating a half-life extending domain (molecules E v1 , E v2, E v3, E v4, E v5, E v6, E v7 and E v8). TCR alpha and beta variable domains and antibody light and heavy variable domains are indicated Va, Vp, VL and VH respectively.

[0310] Figure 10 shows T cell activation in the presence of TCR bispecific molecules comprising a TCR that binds to an HLA restricted peptide from PRAME, and dimeric Fc having “knob-in-hole” substitutions as a half-life extending domain.

[0311] Figure 11 shows T cell activation in the presence of TCR bispecific molecules comprising a TCR that binds to an HLA restricted peptide from PRAME, and dimeric Fc having “knob-in-hole” substitutions as a half-life extending domain.

[0312] Description of the sequences

[0313] Exemplary peptide antigen

[0314] Exemplary soluble TCR sequences

[0315] AKEVEQNSGPLSVPEGANASLNCTYSDKHSQGFFWYRQYSGKSPELIMSIYSNGDKQDGRFTAQLN KASQYVSLLIRDSQPSDSATYLCAVRGNEKLTFGTGTRLTIIPNIQN (SEQ ID NO: 27)

[0316] SEQ ID NO: 27 is the amino acid sequence of an exemplary TCR alpha chain variable domain (Va) of a soluble TCR which binds to a peptide having the sequence of ALWGPDAAA (SEQ ID NO: 45) in complex with HLA-A*02. The CDRs (CDR1 , CDR2 and CDR3) within the Va are in bold and underlined and are designated SEQ ID NO: 28, 29 and 30.

[0317] NAGVTQTPKFRILKIGQSMTLQCTQDMNHSYMYWYRQDPGMGLKLIYYSVGVGFTDKGEVPNGYNV TRSTTEDFPLRLESAAPSQTSVYFCASAYMTGELFFGEGSRLTVLEDLKN (SEQ ID NO: 31)

[0318] SEQ ID NO: 31 is the amino acid sequence of an exemplary TCR beta chain variable domain (Vp) of a soluble TCR which binds to a peptide having the sequence of ALWGPDAAA (SEQ ID NO: 45) in complex with HLA-A*02. The CDRs (CDR1 , CDR2 and CDR3) within Vp are in bold and underlined and are designated SEQ ID NO: 32, 33 and 34.

[0319] Exemplary immune cell engaging domain sequences

[0320] U28:

[0321] AIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGS GTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIK (SEQ ID NO: 35)

[0322] SEQ ID NO: 35 is the amino acid sequence of an exemplary antibody light chain variable domain (VL) of an immune cell engaging domain referred to herein as “U0” which binds to CD3. The CDRs (CDR1 , CDR2 and CDR3) within the VL are in bold and underlined and are designated SEQ ID NO: 36, 37 and 38.

[0323] EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYAMNWVRQAPGKGLEWVALINPYKGVSTYNQKFK DRFTFSVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSS (SEQ ID NO: 39) SEQ ID NO: 39 is the amino acid sequence of an exemplary antibody heavy chain variable domain (VH) of an immune cell engaging domain referred to herein as “UO” which binds to CD3. The CDRs (CDR1 , CDR2 and CDR3) within the VH are in bold and underlined and are designated SEQ ID NO: 40, 41 and 42.

[0324] UO:

[0325] AIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGS GTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIK (SEQ ID NO: 70)

[0326] SEQ ID NO: 70 is the amino acid sequence of an exemplary antibody light chain variable domain (VL) of an immune cell engaging domain referred to herein as “UO” which binds to CD3. The CDRs (CDR1 , CDR2 and CDR3) within the VL are in bold and underlined and are designated SEQ ID NO: 71 , 72 and 73.

[0327] EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFK DRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSS (SEQ ID NO: 74)

[0328] SEQ ID NO: 74 is the amino acid sequence of an exemplary antibody heavy chain variable domain (VH) of an immune cell engaging domain referred to herein as “UO” which binds to CD3. The CDRs (CDR1 , CDR2 and CDR3) within the VH are in bold and underlined and are designated SEQ ID NO: 75, 76 and 77.

[0329] U144:

[0330] AIQMTQSPSSLSASVGDRVTITCRASQGIRKYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGS GTDFTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIK (SEQ ID NO: 99)

[0331] SEQ ID NO: 99 is the amino acid sequence of an exemplary antibody light chain variable domain (VL) of an immune cell engaging domain referred to herein as “U144” which binds to CD3. The CDRs (CDR1 , CDR2 and CDR3) within the VL are in bold and underlined and are designated SEQ ID NO: 100, 101 and 102.

[0332] EVQLLESGGGLVQPGGSLRLSCAASGYRFTGYLMNWVRQAPGKGLEWVSAINPYKGSTYYADSVK GRFTFSRDNSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDLWGRGTLVTVSS (SEQ ID NO: 103)

[0333] SEQ ID NO: 103 is the amino acid sequence of an exemplary antibody heavy chain variable domain (VH) of an immune cell engaging domain referred to herein as “U144” which binds to CD3. The CDRs (CDR1 , CDR2 and CDR3) within the VH are in bold and underlined and are designated SEQ ID NO: 104, 105 and 106.

[0334] Exemplary single domain HLE sequences

[0335] PSVFCFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVV SVLTVLHQDWLNGKEYKCKVSNKALPAPIECTISKAKGQCREPQVYTSPPSRDELTKNQVSLRCHVK GFYPSDIAVEWESNGQPENNYKTTKPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHECLHNHY TQKSLSLSPGK (SEQ ID NO: 43)

[0336] SEQ ID NO: 43 is the amino acid sequence of an exemplary monomeric Fc (mFc) half-life extending domain (HLE).

[0337] EEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPC AEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTL SEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGPKLVAASQAAL GL (SEQ ID NO: 44)

[0338] SEQ ID NO: 44 is the amino acid sequence of an exemplary Dill domain of human serum albumin (Dill) half-life extending domain (HLE).

[0339] Exemplary multi domain HLE sequences

[0340] PSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK (SEQ ID NO: 107)

[0341] PSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK (SEQ ID NO: 108)

[0342] SEQ ID NO: 107 is the amino acid sequence of the first domain / chain (also referred to herein as FC1) of an exemplary Fc half-life extending domain (HLE). SEQ ID NO: 108 is the amino acid sequence of a second domain / chain (also referred to herein as FC2) of an exemplary Fc half-life extending domain (HLE). The Fc regions comprise “knob-in-hole” substitutions shown in bold. The first chain (SEQ ID NO: 107) comprises the substitutions conferring a “hole” and the second chain (SEQ ID NO: 108) comprises substitutions conferring a “knob”. Exemplary bispecific molecule sequences

[0343] Molecule A:

[0344] AIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGS GTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSGGGS EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYAMNWVRQAPGKGLEWVALINPYKGVSTYNQKF KDRFTFSVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSGGGGSN AGVTQTPKFRILKIGQSMTLQCTQDMNHSYMYWYRQDPGMGLKLIYYSVGVGFTDKGEVPNGYNVT RSTTEDFPLRLESAAPSQTSVYFCASAYMTGELFFGEGSRLTVLEDLKNGSSGGGGSGGGGSGGG GSTAKEVEQNSGPLSVPEGANASLNCTYSDKHSQGFFWYRQYSGKSPELIMSIYSNGDKQDGRFTA QLNKASQYVSLLIRDSQPSDSATYLCAVRGNEKLTFGTGTRLTIIPNIQN (SEQ ID NO: 1)

[0345] SEQ ID NO: 1 is the full amino acid sequence of an exemplary bispecific molecule referred to herein as “Molecule A”. Molecule A is a single chain bispecific molecule comprising (i) a soluble TOR, which comprises a TOR alpha chain variable domain (Va) and a TOR beta chain variable domain (Vp), and (ii) an immune cell engaging domain, which comprises an antibody light chain variable domain (VL), and an antibody heavy chain variable domain (VH). In Molecule A, the soluble TOR binds to a peptide having the sequence of ALWGPDAAA (SEQ ID NO: 45) in complex with HLA-A*02 and the immune cell engaging domain binds to CD3. The light chain variable domain (VL) is in italics and is designated SEQ ID NO: 35. The CDRs (CDR1 , CDR2 and CDR3) within the VL are underlined and in italics and are designated SEQ ID NO: 36, 37 and 38. The heavy chain variable domain (VH) is in bold and is designated SEQ ID NO: 39. The CDRs (CDR1 , CDR2 and CDR3) within the VH are underlined and in bold and are designated SEQ ID NO: 40, 41 and 42. The TOR beta chain variable domain (Vp) is double underlined and is designated SEQ ID NO: 31 . The CDRs (CDR1 , CDR2 and CDR3) within Vp are double underlined and in bold and are designated SEQ ID NO: 32, 33 and 34. The TCR alpha chain variable domain (Va) is underlined and is designated SEQ ID NO: 27. The CDRs (CDR1 , CDR2 and CDR3) within the Va are underlined and in bold and are designated SEQ ID NO: 28, 29 and 30. A glycine-serine linker with the sequence of SEQ ID NO: 52, linking the VL and VH, is shown in plain text. A glycine-serine linker with the sequence of SEQ ID NO: 48, linking the VH and Vp, is shown in plain text. A glycine-serine linker with the sequence of SEQ ID NO: 51 , linking the Vp and Va, is shown in plain text.

[0346] Molecule B:

[0347] EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYAMNWVRQAPGKGLEWVALINPYKGVSTYNQKF KDRFTFSVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSGGGSGG GGNAGVTQTPKFRILKIGQSMTLQCTQDMNHSYMYWYRQDPGMGLKLIYYSVGVGFTDKGEVPNG YNVTRSTTEDFPLRLESAAPSQTSVYFCASAYMTGELFFGEGSRLTVLEDLKN (SEQ ID NO: 2) SEQ ID NO: 2 is the full amino acid sequence of a first chain of an exemplary bispecific molecule referred to herein as “Molecule B”. Molecule B is a dual chain bispecific molecule comprising (i) a soluble TCR, which comprises a TCR alpha chain variable domain (Va) and a TCR beta chain variable domain (Vp), and (ii) an immune cell engaging domain, which comprises an antibody light chain variable domain (VL), and an antibody heavy chain variable domain (VH). In Molecule B, the soluble TCR binds to a peptide having the sequence of ALWGPDAAA (SEQ ID NO: 45) in complex with HLA-A*02 and the immune cell engaging domain binds to CD3. The heavy chain variable domain (VH) is in bold and is designated SEQ ID NO: 39. The CDRs (CDR1 , CDR2 and CDR3) within the VH are underlined and are designated SEQ ID NO: 40, 41 and 42. The TCR beta chain variable domain (Vp) is double underlined and is designated SEQ ID NO: 31 . The CDRs (CDR1 , CDR2 and CDR3) within Vp are double underlined and in bold and are designated SEQ ID NO: 32, 33 and 34. A glycineserine linker with the sequence of SEQ ID NO: 50, linking the VH and Vp, is shown in plain text.

[0348] AKEVEQNSGPLSVPEGANASLNCTYSDKHSQGFFWYRQYSGKSPELIMSIYSNGDKQDGRFTAQLN KASQYVSLLIRDSQPSDSATYLCAVRGNEKLTFGTGTRLTIIPNIQNG GG SG GG G AIQMTQSPSSLSA SVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPE DFATYYCQQGNTLPWTFGQGTKVEIK (SEQ ID NO: 3)

[0349] SEQ ID NO: 3 is the full amino acid sequence of a second chain of an exemplary bispecific molecule referred to herein as “Molecule B”. The TCR alpha chain variable domain (Va) is underlined and is designated SEQ ID NO: 27. The CDRs (CDR1 , CDR2 and CDR3) within the Va are underlined and in bold and are designated SEQ ID NO: 28, 29 and 30. The light chain variable domain (VL) is in italics and is designated SEQ ID NO: 35. The CDRs (CDR1 , CDR2 and CDR3) within the VL are underlined and are designated SEQ ID NO: 36, 37 and 38. A glycine-serine linker with the sequence of SEQ ID NO: 50, linking the Va and VL is shown in plain text.

[0350] Molecule C:

[0351] EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYAMNWVRQAPGKGLEWVALINPYKGVSTYNQKF KDRFTFSVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSGGGSGG GGNAGVTQTPKFRILKIGQSMTLQCTQDMNHSYMYWYRQDPGMGLKLIYYSVGVGFTDKGEVPNG YNVTRSTTEDFPLRLESAAPSQTSVYFCASAYMTGELFFGEGSRLTVLEDLKNEPKSSDKTHTCPPC PAPPVAGP (SEQ ID NO: 4)

[0352] SEQ ID NO: 4 is the full amino acid sequence of a first chain of an exemplary bispecific molecule referred to herein as “Molecule C”. Molecule C is a dual chain bispecific molecule comprising (i) a soluble TCR, which comprises a TCR alpha chain variable domain (Va) and a TCR beta chain variable domain (Vp), and (ii) an immune cell engaging domain, which comprises an antibody light chain variable domain (VL), and an antibody heavy chain variable domain (VH). In Molecule C, the soluble TCR binds to a peptide having the sequence of ALWGPDAAA (SEQ ID NO: 45) in complex with HLA-A*02 and the immune cell engaging domain binds to CD3. The heavy chain variable domain (VH) is in bold and is designated SEQ ID NO: 39. The CDRs (CDR1 , CDR2 and CDR3) within the VH are underlined and are designated SEQ ID NO: 40, 41 and 42. The TOR beta chain variable domain (Vp) is double underlined and is designated SEQ ID NO: 31 . The CDRs (CDR1 , CDR2 and CDR3) within Vp are double underlined and in bold and are designated SEQ ID NO: 32, 33 and 34. A glycineserine linker with the sequence of SEQ ID NO: 50, linking the VH and Vp, is shown in plain text. An lgG1 / 2 hybrid hinge sequence with the sequence of SEQ ID NO: 47, linked to the Vp, is shown in bold italics.

[0353] AKEVEQNSGPLSVPEGANASLNCTYSDKHSQGFFWYRQYSGKSPELIMSIYSNGDKQDGRFTAQLN KASQYVSLLIRDSQPSDSATYLCAVRGNEKLTFGTGTRLTIIPNIQNG GG SG GG G AIQMTQSPSSLSA SVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPE DFATYYCQQGNTLPWTFGQGTKVEIKEPKSSDKTHTCPPCPAPPVAGP (SEQ ID NO: 5)

[0354] SEQ ID NO: 5 is the full amino acid sequence of a second chain of an exemplary bispecific molecule referred to herein as “Molecule C”. The TOR alpha chain variable domain (Va) is underlined and is designated SEQ ID NO: 27. The CDRs (CDR1 , CDR2 and CDR3) within the Va are underlined and in bold and are designated SEQ ID NO: 28, 29 and 30. The light chain variable domain (VL) is in italics and is designated SEQ ID NO: 35. The CDRs (CDR1 , CDR2 and CDR3) within the VL are underlined and are designated SEQ ID NO: 36, 37 and 38. A glycine-serine linker with the sequence of SEQ ID NO: 50, linking the Va and VL is shown in plain text. An IgG 1 / 2 hybrid hinge sequence with the sequence of SEQ ID NO: 47, linked to the VL, is shown in bold italics.

[0355] Molecule D1 :

[0356] AKEVEQNSGPLSVPEGANASLNCTYSDKHSQGFFWYRQYSGKSPELIMSIYSNGDKQDGRFTAQLN KASQYVSLLIRDSQPSDSATYLCAVRGNEKLTFGTGTRLTIIPNIQNGGGGSA / QMTQSPSSLSA S VG DRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFA TYYCQQG / VTLPI / I / TPGQGTK' / E / KGGGGSGGGGSGGGGSGGGGSGGGSEVQLVESGGGLVQPGG SLRLSCAASGYSFTGYAMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTFSVDKSKNTAYL QMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSGGGSGNAGVTQTPKFRILKIGQSM TLQCTQDMNHSYMYWYRQDPGMGLKLIYYSVGVGFTDKGEVPNGYNVTRSTTEDFPLRLESAAPS QTSVYFCASAYMTGELFFGEGSRLTVLEDLKN (SEQ ID NO: 6)

[0357] SEQ ID NO: 6 is the full amino acid sequence of an exemplary bispecific molecule referred to herein as “Molecule D1 ”. Molecule D1 is a single chain bispecific molecule comprising (i) a soluble TCR, which comprises a TCR alpha chain variable domain (Va) and a TCR beta chain variable domain (Vp), and (ii) an immune cell engaging domain, which comprises an antibody light chain variable domain (VL), and an antibody heavy chain variable domain (VH). In Molecule D1 , the soluble TCR binds to a peptide having the sequence of ALWGPDAAA (SEQ ID NO: 45) in complex with HLA-A*02 and the immune cell engaging domain binds to CD3. The TCR alpha chain variable domain (Va) is underlined and is designated SEQ ID NO: 27. The CDRs (CDR1 , CDR2 and CDR3) within the Va are underlined and in bold and are designated SEQ ID NO: 28, 29 and 30. The light chain variable domain (VL) is in italics and is designated SEQ ID NO: 35. The CDRs (CDR1 , CDR2 and CDR3) within the VL are underlined and are designated SEQ ID NO: 36, 37 and 38. The heavy chain variable domain (VH) is in bold and is designated SEQ ID NO: 39. The CDRs (CDR1 , CDR2 and CDR3) within the VH are underlined and are designated SEQ ID NO: 40, 41 and 42. The TCR beta chain variable domain (Vp) is double underlined and is designated SEQ ID NO: 31. The CDRs (CDR1 , CDR2 and CDR3) within Vp are double underlined and in bold and are designated SEQ ID NO: 32, 33 and 34. A glycine-serine linker with the sequence of SEQ ID NO: 48, linking the Va and VL, is shown in plain text. A glycine-serine linker with the sequence of SEQ ID NO: 52, linking the VL and VH, is shown in plain text. A glycine-serine linker with the sequence of SEQ ID NO: 49, linking the VH and Vp, is shown in plain text.

[0358] Molecule D2:

[0359] EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYAMNWVRQAPGKGLEWVALINPYKGVSTYNQKF KDRFTFSVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSGGGGSN AGVTQTPKFRILKIGQSMTLQCTQDMNHSYMYWYRQDPGMGLKLIYYSVGVGFTDKGEVPNGYNVT RSTTEDFPLRLESAAPSQTSVYFCASAYMTGELFFGEGSRLTVLEDLKNGSSGGGGSGGGGSGGG GSTAKEVEQNSGPLSVPEGANASLNCTYSDKHSQGFFWYRQYSGKSPELIMSIYSNGDKQDGRFTA QLNKASQYVSLLIRDSQPSDSATYLCAVRGNEKLTFGTGTRLTIIPNIQNGGGSGA / QMTQSPSSLSA SVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPE DFATYYCQQGNTLPWTFGQGTKVEIK (SEQ ID NO: 7)

[0360] SEQ ID NO: 7 is the full amino acid sequence of an exemplary bispecific molecule referred to herein as “Molecule D2”. Molecule D2 is a single chain bispecific molecule comprising (i) a soluble TCR, which comprises a TCR alpha chain variable domain (Va) and a TCR beta chain variable domain (Vp), and (ii) an immune cell engaging domain, which comprises an antibody light chain variable domain (VL), and an antibody heavy chain variable domain (VH). In Molecule D2, the soluble TCR binds to a peptide having the sequence of ALWGPDAAA (SEQ ID NO: 45) in complex with HLA-A*02 and the immune cell engaging domain binds to CD3. The heavy chain variable domain (VH) is in bold and is designated SEQ ID NO: 39. The CDRs (CDR1 , CDR2 and CDR3) within the VH are underlined and are designated SEQ ID NO: 40, 41 and 42. The TCR beta chain variable domain (Vp) is double underlined and is designated SEQ ID NO: 31. The CDRs (CDR1 , CDR2 and CDR3) within Vp are double underlined and in bold and are designated SEQ ID NO: 32, 33 and 34. The TCR alpha chain variable domain (Va) is underlined and is designated SEQ ID NO: 27. The CDRs (CDR1 , CDR2 and CDR3) within the Va are underlined and in bold and are designated SEQ ID NO: 28, 29 and 30. The light chain variable domain (VL) is in italics and is designated SEQ ID NO: 35. The CDRs (CDR1 , CDR2 and CDR3) within the VL are underlined and are designated SEQ ID NO: 36, 37 and 38. A glycine-serine linker with the sequence of SEQ ID NO: 48, linking the VH and Vp, is shown in plain text. A glycine-serine linker with the sequence of SEQ ID NO: 51 , linking the Vp and Va, is shown in plain text. A glycine-serine linker with the sequence of SEQ ID NO: 49, linking the Va and VL, is shown in plain text.

[0361] Molecule A mFc v1 :

[0362] AIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGS GTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSGGGS EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYAMNWVRQAPGKGLEWVALINPYKGVSTYNQKF KDRFTFSVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSGGGGSN AGVTQTPKFRILKIGQSMTLQCTQDMNHSYMYWYRQDPGMGLKLIYYSVGVGFTDKGEVPNGYNVT RSTTEDFPLRLESAAPSQTSVYFCASAYMTGELFFGEGSRLTVLEDLKNGGSGGGGSGGGGSGGG GSTAKEVEQNSGPLSVPEGANASLNCTYSDKHSQGFFWYRQYSGKSPELIMSIYSNGDKQDGRFTA QLNKASQYVSLLIRDSQPSDSATYLCAVRGNEKLTFGTGTRLTIIPNIQNGGPSVFCFPPKPKDTLMIS

[0363] SEQ ID NO: 8 is the full amino acid sequence of an exemplary half-life extended bispecific molecule referred to herein as “Molecule A mFc v1 ”. Molecule A mFc v1 is a single chain half-life extended bispecific molecule comprising (i) a soluble TOR, which comprises a TOR alpha chain variable domain (Va) and a TOR beta chain variable domain (Vp), (ii) an immune cell engaging domain, which comprises an antibody light chain variable domain (VL), and an antibody heavy chain variable domain (VH), and (iii) an mFc half-life extending domain (HLE). In Molecule A mFc v1 , the soluble TOR binds to a peptide having the sequence of ALWGPDAAA (SEQ ID NO: 45) in complex with HLA-A*02 and the immune cell engaging domain binds to CD3. The light chain variable domain (VL) is in italics and is designated SEQ ID NO: 35. The CDRs (CDR1 , CDR2 and CDR3) within the VL are underlined and are designated SEQ ID NO: 36, 37 and 38. The heavy chain variable domain (VH) is in bold and is designated SEQ ID NO: 39. The CDRs (CDR1 , CDR2 and CDR3) within the VH are underlined and are designated SEQ ID NO: 40, 41 and 42. The TOR beta chain variable domain (Vp) is double underlined and is designated SEQ ID NO: 31. The CDRs (CDR1 , CDR2 and CDR3) within Vp are double underlined and in bold and are designated SEQ ID NO: 32, 33 and 34. The TCR alpha chain variable domain (Va) is underlined and is designated SEQ ID NO: 27. The CDRs (CDR1 , CDR2 and CDR3) within the Va are underlined and in bold and are designated SEQ ID NO: 28, 29 and 30. The mFc half-life extending (HLE) domain is dash-underlined and is designated SEQ ID NO: 43. A glycine-serine linker with the sequence of SEQ ID NO: 52, linking the VL and VH, is shown in plain text. A glycine-serine linker with the sequence of SEQ ID NO: 48, linking the VH and Vp, is shown in plain text. A glycine-serine linker with the sequence of SEQ ID NO: 51 , linking the Vp and Va, is shown in plain text. A glycine linker (GG), linking the Va and HLE, is shown in plain text.

[0364] Molecule A mFc v2: HYTQKSLSLSPGKGGGGSAIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIY YTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGGGGSGGG GSGGGGSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYAMNWVRQAPGKGLE WVALINPYKGVSTYNQKFKDRFTFSVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDV WGQGTLVTVSSGGGGSNAGVTQTPKFRILKIGQSMTLQCTQDMNHSYMYWYRQDPGMGLKLIYYS VGVGFTDKGEVPNGYNVTRSTTEDFPLRLESAAPSQTSVYFCASAYMTGELFFGEGSRLTVLEDLKN GGSGGGGSGGGGSGGGGSTAKEVEQNSGPLSVPEGANASLNCTYSDKHSQGFFWYRQYSGKSP ELIMSIYSNGDKQDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVRGNEKLTFGTGTRLTIIPNIQN (SEQ ID NO: 9)

[0365] SEQ ID NO: 9 is the full amino acid sequence of an exemplary half-life extended bispecific molecule referred to herein as “Molecule A mFc v2”. Molecule A mFc v2 is a single chain half-life extended bispecific molecule comprising (i) a soluble TOR, which comprises a TOR alpha chain variable domain (Va) and a TOR beta chain variable domain (Vp), (ii) an immune cell engaging domain, which comprises an antibody light chain variable domain (VL), and an antibody heavy chain variable domain (VH), and (iii) a mFc half-life extending domain (HLE). In Molecule A mFc v2, the soluble TOR binds to a peptide having the sequence of ALWGPDAAA (SEQ ID NO: 45) in complex with HLA-A*02 and the immune cell engaging domain binds to CD3. The mFc half-life extending (HLE) domain is dash- underlined and is designated SEQ ID NO: 43. The light chain variable domain (VL) is in italics and is designated SEQ ID NO: 35. The CDRs (CDR1 , CDR2 and CDR3) within the VL are underlined and are designated SEQ ID NO: 36, 37 and 38. The heavy chain variable domain (VH) is in bold and is designated SEQ ID NO: 39. The CDRs (CDR1 , CDR2 and CDR3) within the VH are underlined and are designated SEQ ID NO: 40, 41 and 42. The TOR beta chain variable domain (Vp) is double underlined and is designated SEQ ID NO: 31. The CDRs (CDR1 , CDR2 and CDR3) within Vp are double underlined and in bold and are designated SEQ ID NO: 32, 33 and 34. The TCR alpha chain variable domain (Va) is underlined and is designated SEQ ID NO: 27. The CDRs (CDR1 , CDR2 and CDR3) within the Va are underlined and in bold and are designated SEQ ID NO: 28, 29 and 30. A glycine-serine linker with the sequence of SEQ ID NO: 48, linking the HLE and VL, is shown in plain text. A glycine-serine linker with the sequence of SEQ ID NO: 52, linking the VL and VH, is shown in plain text. A glycine-serine linker with the sequence of SEQ ID NO: 48, linking the VH and Vp, is shown in plain text. A glycine-serine linker with the sequence of SEQ ID NO: 51 , linking the Vp and Va, is shown in plain text. Molecule C mFc v1 :

[0366] EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYAMNWVRQAPGKGLEWVALINPYKGVSTYNQKF KDRFTFSVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSGGGSGG

[0367] GGNAGVTQTPKFRILKIGQSMTLQCTQDMNHSYMYWYRQDPGMGLKLIYYSVGVGFTDKGEVPNG YNVTRSTTEDFPLRLESAAPSQTSVYFCASAYMTGELFFGEGSRLTVLEDLKNEPKSSDKTHTCPPC PAPPVAGP (SEQ ID NO: 10)

[0368] SEQ ID NO: 10 is the full amino acid sequence of a first chain of an exemplary half-life extended bispecific molecule referred to herein as “Molecule C mFc v1 ”. Molecule C mFc v1 is a dual chain half-life extended bispecific molecule comprising (i) a soluble TOR, which comprises a TOR alpha chain variable domain (Va) and a TOR beta chain variable domain (Vp), (ii) an immune cell engaging domain, which comprises an antibody light chain variable domain (VL), and an antibody heavy chain variable domain (VH), and (iii) a mFc half-life extending domain (HLE). In Molecule C mFc v1 , the soluble TOR binds to a peptide having the sequence of ALWGPDAAA (SEQ ID NO: 45) in complex with HLA-A*02 and the immune cell engaging domain binds to CD3. The heavy chain variable domain (VH) is in bold and is designated SEQ ID NO: 39. The CDRs (CDR1 , CDR2 and CDR3) within the VH are underlined and are designated SEQ ID NO: 40, 41 and 42. The TOR beta chain variable domain (Vp) is double underlined and is designated SEQ ID NO: 31 . The CDRs (CDR1 , CDR2 and CDR3) within Vp are double underlined and in bold and are designated SEQ ID NO: 32, 33 and 34. A glycineserine linker with the sequence of SEQ ID NO: 50, linking the VH and Vp, is shown in plain text. An lgG1 / 2 hybrid hinge with the sequence of SEQ ID NO: 47, linked to the Vp, is shown in bold italics.

[0369] AKEVEQNSGPLSVPEGANASLNCTYSDKHSQGFFWYRQYSGKSPELIMSIYSNGDKQDGRFTAQLN KASQYVSLLIRDSQPSDSATYLCAVRGNEKLTFGTGTRLTIIPNIQNG GG SG GG G AIQMTQSPSSLSA SVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPE

[0370] SEQ ID NO: 11 is the full amino acid sequence of a second chain of an exemplary half-life extended bispecific molecule referred to herein as “Molecule C mFc v1 ”. The TOR alpha chain variable domain (Va) is underlined and is designated SEQ ID NO: 27. The CDRs (CDR1 , CDR2 and CDR3) within the Va are underlined and in bold and are designated SEQ ID NO: 28, 29 and 30. The light chain variable domain (VL) is in italics and is designated SEQ ID NO: 35. The CDRs (CDR1 , CDR2 and CDR3) within the VL are underlined and are designated SEQ ID NO: 36, 37 and 38. The mFc half-life extending (HLE) domain is dash-underlined and is designated SEQ ID NO: 43. A glycine-serine linker with the sequence of SEQ ID NO: 50, linking the Va and VL, is shown in plain text. An IgG 1 / 2 hybrid hinge with the sequence of SEQ ID NO: 47, linked to the VL is shown in bold italics. A glycine linker (GG), linking the IgG hinge and HLE is shown in plain text.

[0371] Molecule C mFc v2:

[0372] EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYAMNWVRQAPGKGLEWVALINPYKGVSTYNQKF KDRFTFSVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSGGGSGG GGNAGVTQTPKFRILKIGQSMTLQCTQDMNHSYMYWYRQDPGMGLKLIYYSVGVGFTDKGEVPNG YNVTRSTTEDFPLRLESAAPSQTSVYFCASAYMTGELFFGEGSRLTVLEDLKNEPKSSDKTHTCPPC

[0373] SEQ ID NO: 12 is the full amino acid sequence of a first chain of an exemplary half-life extended bispecific molecule referred to herein as “Molecule C mFc v2”. Molecule C mFv v2 is a dual chain half-life extended bispecific molecule comprising (i) a soluble TOR, which comprises a TOR alpha chain variable domain (Va) and a TOR beta chain variable domain (Vp), (ii) an immune cell engaging domain, which comprises an antibody light chain variable domain (VL), and an antibody heavy chain variable domain (VH), and (iii) a mFc half-life extending domain. In Molecule C mFc v2, the soluble TOR binds to a peptide having the sequence of ALWGPDAAA (SEQ ID NO: 45) in complex with HLA- A*02 and the immune cell engaging domain binds to CD3. The heavy chain variable domain (VH) is in bold and is designated SEQ ID NO: 39. The CDRs (CDR1 , CDR2 and CDR3) within the VH are underlined and are designated SEQ ID NO: 40, 41 and 42. The TOR beta chain variable domain (Vp) is double underlined and is designated SEQ ID NO: 31 . The CDRs (CDR1 , CDR2 and CDR3) within Vp are double underlined and in bold and are designated SEQ ID NO: 32, 33 and 34. The mFC halflife extending (HLE) domain is dash-underlined and is designated SEQ ID NO: 43. A glycine-serine linker with the sequence of SEQ ID NO: 50, linking the VH and Vp, is shown in plain text. An IgG 112 hybrid hinge with the sequence of SEQ ID NO: 47, linked to the Vp, is shown in bold italics. A glycine linker (GG), linking the IgG 112 hybrid hinge to the HLE, is shown in plain text.

[0374] AKEVEQNSGPLSVPEGANASLNCTYSDKHSQGFFWYRQYSGKSPELIMSIYSNGDKQDGRFTAQLN KASQYVSLLIRDSQPSDSATYLCAVRGNEKLTFGTGTRLTIIPNIQNG GG SG GG G AIQMTQSPSSLSA SVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPE DFATYYCQQGNTLPWTFGQGTKVEIKEPKSSDKTHTCPPCPAPPVAGP (SEQ ID NO: 13)

[0375] SEQ ID NO: 13 is the full amino acid sequence of a second chain of an exemplary half-life extended bispecific molecule referred to herein as “Molecule C mFc v2”. The TOR alpha chain variable domain (Va) is underlined and is designated SEQ ID NO: 27. The CDRs (CDR1 , CDR2 and CDR3) within the Va are underlined and in bold and are designated SEQ ID NO: 28, 29 and 30. The light chain variable domain (VL) is in italics and is designated SEQ ID NO: 35. The CDRs (CDR1 , CDR2 and CDR3) within the VL are underlined and are designated SEQ ID NO: 36, 37 and 38. A glycine-serine linker with the sequence of SEQ ID NO: 50, linking the Va and VL, is shown in plain text. An IgG 1 / 2 hybrid hinge with the sequence of SEQ ID NO: 47, linked to the VL is shown in bold italics.

[0376] Molecule C mFc v3:

[0377] EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYAMNWVRQAPGKGLEWVALINPYKGVSTYNQKF KDRFTFSVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSGGGSGG GGNAGVTQTPKFRILKIGQSMTLQCTQDMNHSYMYWYRQDPGMGLKLIYYSVGVGFTDKGEVPNG YNVTRSTTEDFPLRLESAAPSQTSVYFCASAYMTGELFFGEGSRLTVLEDLKNEPKSSDKTHTCPPC PAPPVAGP (SEQ ID NO: 14)

[0378] SEQ ID NO: 14 is the full amino acid sequence of a first chain of an exemplary half-life extended bispecific molecule referred to herein as “Molecule C mFc v3”. Molecule C mFc v3 is a dual chain half-life extended bispecific molecule comprising (i) a soluble TOR, which comprises a TOR alpha chain variable domain (Va) and a TOR beta chain variable domain (Vp), (ii) an immune cell engaging domain, which comprises an antibody light chain variable domain (VL), and an antibody heavy chain variable domain (VH), and (iii) a mFc half-life extending domain (HLE). In Molecule C mFc v3, the soluble TOR binds to a peptide having the sequence of ALWGPDAAA (SEQ ID NO: 45) in complex with HLA-A*02 and the immune cell engaging domain binds to CD3. The heavy chain variable domain (VH) is in bold and is designated SEQ ID NO: 39. The CDRs (CDR1 , CDR2 and CDR3) within the VH are underlined and are designated SEQ ID NO: 40, 41 and 42. The TOR beta chain variable domain (Vp) is double underlined and is designated SEQ ID NO: 31 . The CDRs (CDR1 , CDR2 and CDR3) within Vp are double underlined and in bold and are designated SEQ ID NO: 32, 33 and 34. A glycineserine linker with the sequence of SEQ ID NO: 50, linking the VH and Vp, is shown in plain text. An lgG1 / 2 hybrid hinge with the sequence of SEQ ID NO: 47, linked to the Vp, is shown in bold italics. HYTQKSLSLSPGKGGGGSAKEVEQNSGPLSVPEGANASLNCTYSDKHSQGFFWYRQYSGKSPELI MSIYSNGDKQDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVRGNEKLTFGTGTRLTIIPNIQNGG GSGGGGAIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSR FSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKEPKSSDKTHTCPPCPAPPVAG P (SEQ ID NO: 15)

[0379] SEQ ID NO: 15 is the full amino acid sequence of a second chain of an exemplary half-life extended bispecific molecule referred to herein as “Molecule C mFc v3”. The mFC half-life extending (HLE) domain is dash-underlined and is designated SEQ ID NO: 43. The TCR alpha chain variable domain (Va) is underlined and is designated SEQ ID NO: 27. The CDRs (CDR1 , CDR2 and CDR3) within the Va are underlined and in bold and are designated SEQ ID NO: 28, 29 and 30. The light chain variable domain (VL) is in italics and is designated SEQ ID NO: 35. The CDRs (CDR1 , CDR2 and CDR3) within the VL are underlined and are designated SEQ ID NO: 36, 37 and 38. A glycine-serine linker with the sequence of SEQ ID NO: 48, linking the HLE and Va, is shown in plain text. A glycine-serine linker with the sequence of SEQ ID NO: 50, linking the Va and VL, is shown in plain text. An IgG 1 / 2 hybrid hinge with the sequence of SEQ ID NO: 47, linked to the VL is shown in bold italics.

[0380] Molecule C mFc v5:

[0381] AKEVEQNSGPLSVPEGANASLNCTYSDKHSQGFFWYRQYSGKSPELIMSIYSNGDKQDGRFTAQLN KASQYVSLLIRDSQPSDSATYLCAVRGNEKLTFGTGTRLTIIPNIQNG GG SG GG G AIQMTQSPSSLSA SVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPE

[0382] GSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYAMNWVRQAPGKGLEWVALINPYKGVSTYNQ KFKDRFTFSVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSGGGS

[0383] GGGGNAGVTQTPKFRILKIGQSMTLQCTQDMNHSYMYWYRQDPGMGLKLIYYSVGVGFTDKGEVP NGYNVTRSTTEDFPLRLESAAPSQTSVYFCASAYMTGELFFGEGSRLTVLEDLKNEPKSSDKTHTCP PCPAPPVAGP (SEQ ID NO: 16)

[0384] SEQ ID NO: 16 is the full amino acid sequence of an exemplary half-life extended bispecific molecule referred to herein as “Molecule C mFc v5”. Molecule C mFc v5 is a single chain half-life extended bispecific molecule comprising (i) a soluble TCR, which comprises a TCR alpha chain variable domain (Va) and a TCR beta chain variable domain (Vp), (ii) an immune cell engaging domain, which comprises an antibody light chain variable domain (VL), and an antibody heavy chain variable domain (VH), and (iii) a mFc half-life extending domain (HLE). In Molecule C mFc v5, the soluble TCR binds to a peptide having the sequence of ALWGPDAAA (SEQ ID NO: 45) in complex with HLA-A*02 and the immune cell engaging domain binds to CD3. The TCR alpha chain variable domain (Va) is underlined and is designated SEQ ID NO: 27. The CDRs (CDR1 , CDR2 and CDR3) within the Va are underlined and in bold and are designated SEQ ID NO: 28, 29 and 30. The light chain variable domain (VL) is in italics and is designated SEQ ID NO: 35. The CDRs (CDR1 , CDR2 and CDR3) within the VL are underlined and are designated SEQ ID NO: 36, 37 and 38. The mFC half-life extending (HLE) domain is dash-underlined and is designated SEQ ID NO: 43. The heavy chain variable domain (VH) is in bold and is designated SEQ ID NO: 39. The CDRs (CDR1 , CDR2 and CDR3) within the VH are underlined and are designated SEQ ID NO: 40, 41 and 42. The TCR beta chain variable domain (Vp) is double underlined and is designated SEQ ID NO: 31 . The CDRs (CDR1 , CDR2 and CDR3) within Vp are double underlined and in bold and are designated SEQ ID NO: 32, 33 and 34. A glycine-serine linker with the sequence of SEQ ID NO: 50, linking the Va and VL, is shown in plain text. An lgG1 / 2 hybrid hinge with the sequence of SEQ ID NO: 47, linked to the VL is shown in bold italics. A glycine linker (GG), linking the lgG1 / 2 hybrid hinge and HLE, is shown in plain text. A glycine-serine linker with the sequence of SEQ ID NO: 48, linking the HLE and VH, is shown in plain text. A glycine-serine linker with the sequence of SEQ ID NO: 50, linking the VH and Vp, is shown in plain text. An IgG 1 / 2 hybrid hinge with the sequence of SEQ ID NO: 47, linked to the Vp is shown in bold italics.

[0385] Molecule C mFc v6:

[0386] EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYAMNWVRQAPGKGLEWVALINPYKGVSTYNQKF KDRFTFSVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSGGGSGG GGNAGVTQTPKFRILKIGQSMTLQCTQDMNHSYMYWYRQDPGMGLKLIYYSVGVGFTDKGEVPNG YNVTRSTTEDFPLRLESAAPSQTSVYFCASAYMTGELFFGEGSRLTVLEDLKNEPKSSDKTHTCPPC VMHECLHNHYTQKSLSLSPGKGGGGSAKEVEQNSGPLSVPEGANASLNCTYSDKHSQGFFWYRQY SGKSPELIMSIYSNGDKQDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVRGNEKLTFGTGTRLTII PN\QNGGGSGGGGAIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRL ESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKEPKSSDKTHTCPPC PAPPVAGP (SEQ ID NO: 17)

[0387] SEQ ID NO: 17 is the full amino acid sequence of an exemplary half-life extended bispecific molecule referred to herein as “Molecule C mFc v6”. Molecule C mFc v6 is a single chain half-life extended bispecific molecule comprising (i) a soluble TOR, which comprises a TOR alpha chain variable domain (Va) and a TOR beta chain variable domain (Vp), (ii) an immune cell engaging domain, which comprises an antibody light chain variable domain (VL), and an antibody heavy chain variable domain (VH), and (iii) a mFc half-life extending domain (HLE). In Molecule C mFc v6, the soluble TOR binds to a peptide having the sequence of ALWGPDAAA (SEQ ID NO: 45) in complex with HLA-A*02 and the immune cell engaging domain binds to CD3. The heavy chain variable domain (VH) is in bold and is designated SEQ ID NO: 39. The CDRs (CDR1 , CDR2 and CDR3) within the VH are underlined and are designated SEQ ID NO: 40, 41 and 42. The TOR beta chain variable domain (Vp) is double underlined and is designated SEQ ID NO: 31. The CDRs (CDR1 , CDR2 and CDR3) within Vp are double underlined and in bold and are designated SEQ ID NO: 32, 33 and 34. The mFc half-life extending (HLE) domain is dash-underlined and is designated SEQ ID NO: 43. The TOR alpha chain variable domain (Va) is underlined and is designated SEQ ID NO: 27. The CDRs (CDR1 , CDR2 and CDR3) within the Va are underlined and in bold and are designated SEQ ID NO: 28, 29 and 30. The light chain variable domain (VL) is in italics and is designated SEQ ID NO: 35. The CDRs (CDR1 , CDR2 and CDR3) within the VL are underlined and are designated SEQ ID NO: 36, 37 and 38. A glycine-serine linker with the sequence of SEQ ID NO: 50, linking the VH and Vp, is shown in plain text. An lgG1 / 2 hybrid hinge with the sequence of SEQ ID NO: 47, linked to the Vp is shown in bold italics. A glycine linker (GG), linking the lgG1 / 2 hybrid hinge and HLE, is shown in plain text. A glycine-serine linker with the sequence of SEQ ID NO: 48, linking the HLE and Va, is shown in plain text. A glycine-serine linker with the sequence of SEQ ID NO: 50, linking the Va and VL, is shown in plain text. An IgG 1 / 2 hybrid hinge with the sequence of SEQ ID NO: 47, linked to the VL is shown in bold italics.

[0388] Molecule D1 mFc v1 :

[0389] AKEVEQNSGPLSVPEGANASLNCTYSDKHSQGFFWYRQYSGKSPELIMSIYSNGDKQDGRFTAQLN KASQYVSLLIRDSQPSDSATYLCAVRGNEKLTFGTGTRLTIIPNIQNGGGGSA / QMTQSPSSLSA S VG DRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFA TYYCQQG / VTLPI / I / TPGQGTK' / E / KGGGGSGGGGSGGGGSGGGGSGGGSEVQLVESGGGLVQPGG SLRLSCAASGYSFTGYAMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTFSVDKSKNTAYL QMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSGGGSGNAGVTQTPKFRILKIGQSM TLQCTQDMNHSYMYWYRQDPGMGLKLIYYSVGVGFTDKGEVPNGYNVTRSTTEDFPLRLESAAPS QTSVYFCASAYMTGELFFGEGSRLTVLEDLKNGGPSVFCFPPKPKDTLMISRTPEVTCVWDVSHED

[0390] SEQ ID NO: 18 is the full amino acid sequence of an exemplary half-life extended bispecific molecule referred to herein as “Molecule D1 mFc v1”. Molecule D1 mFc v1 is a single chain half-life extended bispecific molecule comprising (i) a soluble TOR, which comprises a TOR alpha chain variable domain (Va) and a TOR beta chain variable domain (Vp), (ii) an immune cell engaging domain, which comprises an antibody light chain variable domain (VL), and an antibody heavy chain variable domain (VH), and (iii) a mFc half-life extending domain (HLE). In Molecule D1 mFc v1 , the soluble TOR binds to a peptide having the sequence of ALWGPDAAA (SEQ ID NO: 45) in complex with HLA-A*02 and the immune cell engaging domain binds to CD3. The TOR alpha chain variable domain (Va) is underlined and is designated SEQ ID NO: 27. The CDRs (CDR1 , CDR2 and CDR3) within the Va are underlined and in bold and are designated SEQ ID NO: 28, 29 and 30. The light chain variable domain (VL) is in italics and is designated SEQ ID NO: 35. The CDRs (CDR1 , CDR2 and CDR3) within the VL are underlined and are designated SEQ ID NO: 36, 37 and 38. The heavy chain variable domain (VH) is in bold and is designated SEQ ID NO: 39. The CDRs (CDR1 , CDR2 and CDR3) within the VH are underlined and are designated SEQ ID NO: 40, 41 and 42. The TCR beta chain variable domain (Vp) is double underlined and is designated SEQ ID NO: 31. The CDRs (CDR1 , CDR2 and CDR3) within Vp are double underlined and in bold and are designated SEQ ID NO: 32, 33 and 34. The mFC half-life extending (HLE) domain is dash-underlined and is designated SEQ ID NO: 43. A glycine-serine linker with the sequence of SEQ ID NO: 48, linking the Va and VL, is shown in plain text. A glycine-serine linker with the sequence of SEQ ID NO: 52, linking the VL and VH, is shown in plain text. A glycine-serine linker with the sequence of SEQ ID NO: 49, linking the VH and Vp, is shown in plain text. A glycine linker (GG), linking the Vp and HLE, is shown in plain text.

[0391] Molecule D1 mFc v2: HYTQKSLSLSPGKGGGGSAKEVEQNSGPLSVPEGANASLNCTYSDKHSQGFFWYRQYSGKSPELI MSIYSNGDKQDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVRGNEKLTFGTGTRLTIIPNIQNGG GGSAIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSG SGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSG GGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYAMNWVRQAPGKGLEWVALINPYKGVSTYN QKFKDRFTFSVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSGGG SGNAGVTQTPKFRILKIGQSMTLQCTQDMNHSYMYWYRQDPGMGLKLIYYSVGVGFTDKGEVPNGY NVTRSTTEDFPLRLESAAPSQTSVYFCASAYMTGELFFGEGSRLTVLEDLKN (SEQ ID NO: 19)

[0392] SEQ ID NO: 19 is the full amino acid sequence of an exemplary half-life extended bispecific molecule referred to herein as “Molecule D1 mFc v2”. Molecule D1 mFc v2 is a single chain half-life extended bispecific molecule comprising (i) a soluble TOR, which comprises a TOR alpha chain variable domain (Va) and a TOR beta chain variable domain (Vp), (ii) an immune cell engaging domain, which comprises an antibody light chain variable domain (VL), and an antibody heavy chain variable domain (VH), and (iii) a mFc half-life extending domain (HLE). In Molecule D1 mFc v2, the soluble TOR binds to a peptide having the sequence of ALWGPDAAA (SEQ ID NO: 45) in complex with HLA-A*02 and the immune cell engaging domain binds to CD3. The mFc half-life extending (HLE) domain is dash- underlined and is designated SEQ ID NO: 43. The TOR alpha chain variable domain (Va) is underlined and is designated SEQ ID NO: 27. The CDRs (CDR1 , CDR2 and CDR3) within the Va are underlined and in bold and are designated SEQ ID NO: 28, 29 and 30. The light chain variable domain (VL) is in italics and is designated SEQ ID NO: 35. The CDRs (CDR1 , CDR2 and CDR3) within the VL are underlined and are designated SEQ ID NO: 36, 37 and 38. The heavy chain variable domain (VH) is in bold and is designated SEQ ID NO: 39. The CDRs (CDR1 , CDR2 and CDR3) within the VH are underlined and are designated SEQ ID NO: 40, 41 and 42. The TCR beta chain variable domain (Vp) is double underlined and is designated SEQ ID NO: 31. The CDRs (CDR1 , CDR2 and CDR3) within Vp are double underlined and in bold and are designated SEQ ID NO: 32, 33 and 34. A glycine-serine linker with the sequence of SEQ ID NO: 48, linking the HLE and Va, is shown in plain text. A glycine-serine linker with the sequence of SEQ ID NO: 48, linking the Va and VL, is shown in plain text. A glycine-serine linker with the sequence of SEQ ID NO: 52, linking the VL and VH, is shown in plain text. A glycine-serine linker with the sequence of SEQ ID NO: 49, linking the VH and Vp, is shown in plain text.

[0393] Molecule C Dill v1 :

[0394] EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYAMNWVRQAPGKGLEWVALINPYKGVSTYNQKF KDRFTFSVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSGGGSGG GGNAGVTQTPKFRILKIGQSMTLQCTQDMNHSYMYWYRQDPGMGLKLIYYSVGVGFTDKGEVPNG YNVTRSTTEDFPLRLESAAPSQTSVYFCASAYMTGELFFGEGSRLTVLEDLKNEPKSSDKTHTCPPC PAPPVAGP (SEQ ID NO: 20)

[0395] SEQ ID NO: 20 is the full amino acid sequence of a first chain of an exemplary half-life extended bispecific molecule referred to herein as “Molecule C Dill v1 ”. Molecule C Dill v1 is a dual chain halflife extended bispecific molecule comprising (i) a soluble TOR, which comprises a TOR alpha chain variable domain (Va) and a TOR beta chain variable domain (Vp), (ii) an immune cell engaging domain, which comprises an antibody light chain variable domain (VL), and an antibody heavy chain variable domain (VH), and (iii) a Dill half-life extending domain (HLE). In Molecule C Dill v1 , the soluble TOR binds to a peptide having the sequence of ALWGPDAAA (SEQ ID NO: 45) in complex with HLA-A*02 and the immune cell engaging domain binds to CD3. The heavy chain variable domain (VH) is in bold and is designated SEQ ID NO: 39. The CDRs (CDR1 , CDR2 and CDR3) within the VH are underlined and are designated SEQ ID NO: 40, 41 and 42. The TOR beta chain variable domain (Vp) is double underlined and is designated SEQ ID NO: 31 . The CDRs (CDR1 , CDR2 and CDR3) within Vp are double underlined and in bold and are designated SEQ ID NO: 32, 33 and 34. A glycineserine linker with the sequence of SEQ ID NO: 50, linking the VH and Vp, is shown in plain text. An lgG1 / 2 hybrid hinge with the sequence of SEQ ID NO: 47, linked to the Vp, is shown in bold italics.

[0396] AKEVEQNSGPLSVPEGANASLNCTYSDKHSQGFFWYRQYSGKSPELIMSIYSNGDKQDGRFTAQLN KASQYVSLLIRDSQPSDSATYLCAVRGNEKLTFGTGTRLTIIPNIQNG GG SG GG G AIQMTQSPSSLSA SVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPE DFATYYCQQGNTLPWTFGQGTKVEIKEPKSSDKTHTCPPCPAPPVAGPGGEEPQNUKQNCELFEQ

[0397] SEQ ID NO: 21 is the full amino acid sequence of a second chain of an exemplary half-life extended bispecific molecule referred to herein as “Molecule C Dill v1 ”. The TOR alpha chain variable domain (Va) is underlined and is designated SEQ ID NO: 27. The CDRs (CDR1 , CDR2 and CDR3) within the Va are underlined and in bold and are designated SEQ ID NO: 28, 29 and 30. The light chain variable domain (VL) is in italics and is designated SEQ ID NO: 35. The CDRs (CDR1 , CDR2 and CDR3) within the VL are underlined and are designated SEQ ID NO: 36, 37 and 38. The Dill half-life extending (HLE) domain is dash-underlined and is designated SEQ ID NO: 44. A glycine-serine linker with the sequence of SEQ ID NO: 50, linking the Va and the VL, is shown in plain text. An IgG 1 / 2 hybrid hinge with the sequence of SEQ ID NO: 47, linked to the VL is shown in bold italics. A glycine linker (GG), linking the IgG 1 / 2 hybrid hinge and the HLE, is shown in plain text.

[0398] Molecule C Dill v2:

[0399] EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYAMNWVRQAPGKGLEWVALINPYKGVSTYNQKF KDRFTFSVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSGGGSGG GGNAGVTQTPKFRILKIGQSMTLQCTQDMNHSYMYWYRQDPGMGLKLIYYSVGVGFTDKGEVPNG YNVTRSTTEDFPLRLESAAPSQTSVYFCASAYMTGELFFGEGSRLTVLEDLKNEPKSSDKTHTCPPC KLVAASQAALGL (SEQ ID NO: 22)

[0400] SEQ ID NO: 22 is the full amino acid sequence of a first chain of an exemplary half-life extended bispecific molecule referred to herein as “Molecule C Dill v2”. Molecule C Dill v2 is a dual chain halflife extended bispecific molecule comprising (i) a soluble TOR, which comprises a TOR alpha chain variable domain (Va) and a TOR beta chain variable domain (Vp), (ii) an immune cell engaging domain, which comprises an antibody light chain variable domain (VL), and an antibody heavy chain variable domain (VH), and (iii) a Dill half-life extending domain (HLE). In Molecule C Dill v2, the soluble TOR binds to a peptide having the sequence of ALWGPDAAA (SEQ ID NO: 45) in complex with HLA-A*02 and the immune cell engaging domain binds to CD3. The heavy chain variable domain (VH) is in bold and is designated SEQ ID NO: 39. The CDRs (CDR1 , CDR2 and CDR3) within the VH are underlined and are designated SEQ ID NO: 40, 41 and 42. The TOR beta chain variable domain (Vp) is double underlined and is designated SEQ ID NO: 31 . The CDRs (CDR1 , CDR2 and CDR3) within Vp are double underlined and in bold and are designated SEQ ID NO: 32, 33 and 34. The Dill half-life extending (HLE) domain is dash-underlined and is designated SEQ ID NO: 44. A glycineserine linker with the sequence of SEQ ID NO: 50, linking the VH and Vp, is shown in plain text. An lgG1 / 2 hybrid hinge with the sequence of SEQ ID NO: 47, linked to the Vp, is shown in bold italics. A glycine linker (GG), linking the IgG 112 hybrid hinge and the HLE, is shown in plain text.

[0401] AKEVEQNSGPLSVPEGANASLNCTYSDKHSQGFFWYRQYSGKSPELIMSIYSNGDKQDGRFTAQLN KASQYVSLLIRDSQPSDSATYLCAVRGNEKLTFGTGTRLTIIPNIQNG GG SG GG G AIQMTQSPSSLSA SVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPE DFATYYCQQGNTLPWTFGQGTKVEIKEPKSSDKTHTCPPCPAPPVAGP (SEQ ID NO: 23)

[0402] SEQ ID NO: 23 is the full amino acid sequence of a second chain of an exemplary half-life extended bispecific molecule referred to herein as “Molecule C Dill v2”. The TOR alpha chain variable domain (Va) is underlined and is designated SEQ ID NO: 27. The CDRs (CDR1 , CDR2 and CDR3) within the Va are underlined and in bold and are designated SEQ ID NO: 28, 29 and 30. The light chain variable domain (VL) is in italics and is designated SEQ ID NO: 35. The CDRs (CDR1 , CDR2 and CDR3) within the VL are underlined and are designated SEQ ID NO: 36, 37 and 38. A glycine-serine linker with the sequence of SEQ ID NO: 50, linking the Va and the VL, is shown in plain text. An IgG 1 / 2 hybrid hinge with the sequence of SEQ ID NO: 47, linked to the VL is shown in bold italics.

[0403] Molecule C Dill v6:

[0404] EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYAMNWVRQAPGKGLEWVALINPYKGVSTYNQKF KDRFTFSVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSGGGSGG GGNAGVTQTPKFRILKIGQSMTLQCTQDMNHSYMYWYRQDPGMGLKLIYYSVGVGFTDKGEVPNG YNVTRSTTEDFPLRLESAAPSQTSVYFCASAYMTGELFFGEGSRLTVLEDLKNEPKSSDKTHTCPPC KLVAASQAALGLGGGGSAKEVEQNSGPLSVPEGANASLNCTYSDKHSQGFFWYRQYSGKSPELIMS IYSNGDKQDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVRGNEKLTFGTGTRLTIIPNIQNGGGS GGGGAIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFS GSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKEPKSSDKTHTCPPCPAPPVAGP (SEQ ID NO: 24)

[0405] SEQ ID NO: 24 is the full amino acid sequence of an exemplary half-life extended bispecific molecule referred to herein as “Molecule C Dill v6”. Molecule C Dill v6 is a single chain half-life extended bispecific molecule comprising (i) a soluble TOR, which comprises a TOR alpha chain variable domain (Va) and a TOR beta chain variable domain (Vp), (ii) an immune cell engaging domain, which comprises an antibody light chain variable domain (VL), and an antibody heavy chain variable domain (VH), and (iii) a Dill half-life extending domain (HLE). In Molecule C Dill v6, the soluble TOR binds to a peptide having the sequence of ALWGPDAAA (SEQ ID NO: 45) in complex with HLA-A*02 and the immune cell engaging domain binds to CD3. The heavy chain variable domain (VH) is in bold and is designated SEQ ID NO: 39. The CDRs (CDR1 , CDR2 and CDR3) within the VH are underlined and are designated SEQ ID NO: 40, 41 and 42. The TOR beta chain variable domain (Vp) is double underlined and is designated SEQ ID NO: 31. The CDRs (CDR1 , CDR2 and CDR3) within Vp are double underlined and in bold and are designated SEQ ID NO: 32, 33 and 34. The Dill half-life extending (HLE) domain is dash-underlined and is designated SEQ ID NO: 44. The TCR alpha chain variable domain (Va) is underlined and is designated SEQ ID NO: 27. The CDRs (CDR1 , CDR2 and CDR3) within the Va are underlined and in bold and are designated SEQ ID NO: 28, 29 and 30. The light chain variable domain (VL) is in italics and is designated SEQ ID NO: 35. The CDRs (CDR1 , CDR2 and CDR3) within the VL are underlined and are designated SEQ ID NO: 36, 37 and 38. A glycine-serine linker with the sequence of SEQ ID NO: 50, linking the VH and Vp, is shown in plain text. An lgG1 / 2 hybrid hinge with the sequence of SEQ ID NO: 47, linked to the Vp is shown in bold italics. A glycine linker (GG), linking the lgG1 / 2 hybrid hinge and HLE, is shown in plain text. A glycine-serine linker with the sequence of SEQ ID NO: 48, linking the HLE and Va, is shown in plain text. A glycine-serine linker with the sequence of SEQ ID NO: 50, linking the Va and VL, is shown in plain text. An IgG 1 / 2 hybrid hinge with the sequence of SEQ ID NO: 47, linked to the VL is shown in bold italics.

[0406] Molecule D1 Dill v1 :

[0407] AKEVEQNSGPLSVPEGANASLNCTYSDKHSQGFFWYRQYSGKSPELIMSIYSNGDKQDGRFTAQLN KASQYVSLLIRDSQPSDSATYLCAVRGNEKLTFGTGTRLTIIPNIQNGGGGSA / QMTQSPSSLSA S VG DRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFA TYYCQQG / VTLPI / I / TPGQGTK' / E / KGGGGSGGGGSGGGGSGGGGSGGGSEVQLVESGGGLVQPGG SLRLSCAASGYSFTGYAMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTFSVDKSKNTAYL QMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSGGGSGNAGVTQTPKFRILKIGQSM TLQCTQDMNHSYMYWYRQDPGMGLKLIYYSVGVGFTDKGEVPNGYNVTRSTTEDFPLRLESAAPS QTSVYFCASAYMTGELFFGEGSRLTVLEDLKNGGEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKK

[0408] SEQ ID NO: 25 is the full amino acid sequence of an exemplary half-life extended bispecific molecule referred to herein as “Molecule D1 Dill v1”. Molecule D1 Dill v1 is a single chain half-life extended bispecific molecule comprising (i) a soluble TOR, which comprises a TOR alpha chain variable domain (Va) and a TOR beta chain variable domain (Vp), (ii) an immune cell engaging domain, which comprises an antibody light chain variable domain (VL), and an antibody heavy chain variable domain (VH), and (iii) a Dill half-life extending domain (HLE). In Molecule D1 Dill v1 , the soluble TOR binds to a peptide having the sequence of ALWGPDAAA (SEQ ID NO: 45) in complex with HLA-A*02 and the immune cell engaging domain binds to CD3. The TOR alpha chain variable domain (Va) is underlined and is designated SEQ ID NO: 27. The CDRs (CDR1 , CDR2 and CDR3) within the Va are underlined and in bold and are designated SEQ ID NO: 28, 29 and 30. The light chain variable domain (VL) is in italics and is designated SEQ ID NO: 35. The CDRs (CDR1 , CDR2 and CDR3) within the VL are underlined and are designated SEQ ID NO: 36, 37 and 38. The heavy chain variable domain (VH) is in bold and is designated SEQ ID NO: 39. The CDRs (CDR1 , CDR2 and CDR3) within the VH are underlined and are designated SEQ ID NO: 40, 41 and 42. The TCR beta chain variable domain (Vp) is double underlined and is designated SEQ ID NO: 31 . The CDRs (CDR1 , CDR2 and CDR3) within Vp are double underlined and in bold and are designated SEQ ID NO: 32, 33 and 34. The Dill half-life extending (HLE) domain is dash-underlined and is designated SEQ ID NO: 44. A glycine-serine linker with the sequence of SEQ ID NO: 48, linking the Va and VL, is shown in plain text. A glycine-serine linker with the sequence of SEQ ID NO: 52, linking the VL and VH, is shown in plain text. A glycine- serine linker with the sequence of SEQ ID NO: 48, linking the VH and Vp, is shown in plain text. A glycine linker (GG), linking the Vp and HLE, is shown in plain text.

[0409] Molecule D2 Dill v1 :

[0410] EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYAMNWVRQAPGKGLEWVALINPYKGVSTYNQKF KDRFTFSVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSGGGGSN AGVTQTPKFRILKIGQSMTLQCTQDMNHSYMYWYRQDPGMGLKLIYYSVGVGFTDKGEVPNGYNVT RSTTEDFPLRLESAAPSQTSVYFCASAYMTGELFFGEGSRLTVLEDLKNGGSGGGGSGGGGSGGG GSTAKEVEQNSGPLSVPEGANASLNCTYSDKHSQGFFWYRQYSGKSPELIMSIYSNGDKQDGRFTA QLNKASQYVSLLIRDSQPSDSATYLCAVRGNEKLTFGTGTRLTIIPNIQNGGGSGA / QMTQSPSSLSA SVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPE

[0411] SEQ ID NO: 26 is the full amino acid sequence of an exemplary half-life extended bispecific molecule referred to herein as “Molecule D2 Dill v1 ”. Molecule D2 Dill v1 is a single chain half-life extended bispecific molecule comprising (i) a soluble TOR, which comprises a TOR alpha chain variable domain (Va) and a TOR beta chain variable domain (Vp), (ii) an immune cell engaging domain, which comprises an antibody light chain variable domain (VL), and an antibody heavy chain variable domain (VH), and (iii) a Dill half-life extending domain (HLE). In Molecule D2 Dill v1 , the soluble TOR binds to a peptide having the sequence of ALWGPDAAA (SEQ ID NO: 45) in complex with HLA-A*02 and the immune cell engaging domain binds to CD3. The heavy chain variable domain (VH) is in bold and is designated SEQ ID NO: 39. The CDRs (CDR1 , CDR2 and CDR3) within the VH are underlined and are designated SEQ ID NO: 40, 41 and 42. The TOR beta chain variable domain (Vp) is double underlined and is designated SEQ ID NO: 31. The CDRs (CDR1 , CDR2 and CDR3) within Vp are double underlined and in bold and are designated SEQ ID NO: 32, 33 and 34. The TOR alpha chain variable domain (Va) is underlined and is designated SEQ ID NO: 27. The CDRs (CDR1 , CDR2 and CDR3) within the Va are underlined and in bold and are designated SEQ ID NO: 28, 29 and 30. The light chain variable domain (VL) is in italics and is designated SEQ ID NO: 35. The CDRs (CDR1 , CDR2 and CDR3) within the VL are underlined and are designated SEQ ID NO: 36, 37 and 38. The Dill half-life extending (HLE) domain is dash-underlined and is designated SEQ ID NO: 44. A glycineserine linker with the sequence of SEQ ID NO: 48, linking the VH and Vp, is shown in plain text. A glycine-serine linker with the sequence of SEQ ID NO: 51 , linking the Vp and Va, is shown in plain text. A glycine-serine linker with the sequence of SEQ ID NO: 49, linking the Va and VL, is shown in plain text. A glycine linker (GG), linking the VL and HLE, is shown in plain text.

[0412] Molecule C mFc v4: GG.P.SVFCFPPKPKDTLMJSRTPEyTCywp SHEDPEyKFNVyYyD yys L LHQpyyLNGKEYKCKySNKALPAPJECTJSKAKGpCREP.QVYTSPPSRpELTKNQ SLRCH yKGF PSDlA EWESNGQPENNY^

[0413] HYTQKSLSLSPGKGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYAMNWVRQAPGKGLE WVALINPYKGVSTYNQKFKDRFTFSVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDV WGQGTLVTVSSGGGSGGGGNAGVTQTPKFRILKIGQSMTLQCTQDMNHSYMYWYRQDPGMGLKLI YYSVGVGFTDKGEVPNGYNVTRSTTEDFPLRLESAAPSQTSVYFCASAYMTGELFFGEGSRLTVLE DLKNEPKSSDKTHTCPPCPAPPVAGP (SEQ ID NO: 78)

[0414] SEQ ID NO: 78 is the full amino acid sequence of an exemplary half-life extended bispecific molecule referred to herein as “Molecule C mFc v4”. Molecule C mFc v4 is a dual chain half-life extended bispecific molecule comprising (i) a soluble TOR, which comprises a TOR alpha chain variable domain (Va) and a TOR beta chain variable domain (Vp), (ii) an immune cell engaging domain, which comprises an antibody light chain variable domain (VL), and an antibody heavy chain variable domain (VH), and (iii) a mFc half-life extending domain (HLE). In Molecule C mFc v4, the soluble TOR binds to a peptide having the sequence of ALWGPDAAA (SEQ ID NO: 45) in complex with HLA-A*02 and the immune cell engaging domain binds to CD3. The mFC half-life extending (HLE) domain is dash- underlined and is designated SEQ ID NO: 43. The heavy chain variable domain (VH) is in bold and is designated SEQ ID NO: 39. The CDRs (CDR1 , CDR2 and CDR3) within the VH are underlined and are designated SEQ ID NO: 40, 41 and 42. The TOR beta chain variable domain (Vp) is double underlined and is designated SEQ ID NO: 31. The CDRs (CDR1 , CDR2 and CDR3) within Vp are double underlined and in bold and are designated SEQ ID NO: 32, 33 and 34. A glycine-serine linker with the sequence of SEQ ID NO: 48, linking the HLE and VH, is shown in plain text. A glycine-serine linker with the sequence of SEQ ID NO: 50, linking the VH and Vp, is shown in plain text. An IgG 1 / 2 hybrid hinge with the sequence of SEQ ID NO: 47, linked to the Vp, is shown in bold italics.

[0415] AKEVEQNSGPLSVPEGANASLNCTYSDKHSQGFFWYRQYSGKSPELIMSIYSNGDKQDGRFTAQLN KASQYVSLLIRDSQPSDSATYLCAVRGNEKLTFGTGTRLTIIPNIQNG GG SG GG G AIQMTQSPSSLSA SVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPE DFATYYCQQGNTLPWTFGQGTKVEIKEPKSSDKTHTCPPCPAPPVAGP (SEQ ID NO: 79)

[0416] SEQ ID NO: 79 is the full amino acid sequence of an exemplary half-life extended bispecific molecule referred to herein as “Molecule C mFc v4”. The TOR alpha chain variable domain (Va) is underlined and is designated SEQ ID NO: 27. The CDRs (CDR1 , CDR2 and CDR3) within the Va are underlined and in bold and are designated SEQ ID NO: 28, 29 and 30. The light chain variable domain (VL) is in italics and is designated SEQ ID NO: 35. The CDRs (CDR1 , CDR2 and CDR3) within the VL are underlined and are designated SEQ ID NO: 36, 37 and 38. A glycine-serine linker with the sequence of SEQ ID NO: 50, linking the Va and VL, is shown in plain text. An IgG 1 / 2 hybrid hinge with the sequence of SEQ ID NO: 47, linked to the VL is shown in bold italics. Molecule E v1 :

[0417] GDAKTTQPNSMESNEGEPVHLPCQHSTISGTDYIHWYRQLPSQGPEYVIHGLTSNVNNRMASLAIAE DRKSSTLILHRATLRDAAVYYCILILGHSRLGNYIATFGKGTKLSVIPEPKSSDKTHTCPPCPAPPVAG

[0418] SEQ ID NO: 82 is the full amino acid sequence of a first chain of an exemplary half-life extended bispecific molecule referred to herein as “Molecule E v1”. Molecule E v1 is a triple chain half-life extended bispecific molecule comprising (i) a soluble TOR, which comprises a TOR alpha chain variable domain (Va) and a TOR beta chain variable domain (Vp), (ii) an immune cell engaging domain, which comprises an antibody light chain variable domain (VL), and an antibody heavy chain variable domain (VH), and (iii) an lgG1 Fc knob in hole half-life extending (HLE) domain, which comprises a first chain and a second chain. In Molecule E v1 , the soluble TCR binds to a peptide having the sequence of SLLQHLIGL (SEQ ID NO: 111) in complex with HLA-A*02 and the immune cell engaging domain binds to CD3. The TCR alpha chain variable domain (Va) is underlined and is designated SEQ ID NO: 91 . The CDRs (CDR1 , CDR2 and CD3) within the Va are underlined and in bold and are designated SEQ ID NO: 92, 93 and 94. The first chain of the IgG 1 Fc knob in hole halflife extending (HLE) domain is dash-underlined and is designated SEQ ID NO: 107. An lgG1 / 2 hybrid hinge with the sequence of SEQ ID NO: 109, linked to the Va and the first chain of the HLE, is shown in bold italics.

[0419] AIQMTQSPSSLSASVGDRVTITCRASQGIRKYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGS G TDFTL TISSL QPEDFA TYYCQQGNTLPWTFG QGTK VEIKEPKSSDKTHTCPPCPAPPVA GPS VFLF

[0420] SEQ ID NO: 83 is the full amino acid sequence of a second chain of an exemplary half-life extended bispecific molecule referred to herein as “Molecule E v1”. The light chain variable domain (VL) is in italics and is designated SEQ ID NO: 99. The CDRs (CDR1 , CDR2 and CDR3) within the VL are underlined and are designated SEQ ID NO: 100, 101 and 102. The second chain of the lgG1 Fc knob in hole half-life extending (HLE) domain is dash-underlined and is designated SEQ ID NO: 108. An lgG1 / 2 hybrid hinge with the sequence of SEQ ID NO: 109, linked to the VL and the second chain of the HLE, is shown in bold italics. EVQLLESGGGLVQPGGSLRLSCAASGYRFTGYLMNWVRQAPGKGLEWVSAINPYKGSTYYADSV KGRFTFSRDNSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDLWGRGTLVTVSSGGGSGG GGDGGITQSPKYLFRKEGQNVTLSCEQNLNHDAMYWYRQDPGQGLRLIYYSQIMGDEQKGDIAEGY SVSREKKESFPLTVTSAQKNHTAFYLCASSWWTGGASPIRFGPGTRLTVT (SEQ ID NO: 84)

[0421] SEQ ID NO: 84 is the full amino acid sequence of a third chain of an exemplary half-life extended bispecific molecule referred to herein as “Molecule E v1 ”. The heavy chain variable domain (VH) is in bold and is designated SEQ ID NO: 103. The CDRs (CDR1 , CDR2 and CDR3) within the VH are underlined and are designated SEQ ID NO: 104, 105 and 106. The TOR beta chain variable domain (Vp) is double underlined and is designated SEQ ID NO: 95. The CDRs (CDR1 , CDR2 and CDR3) within Vp are double underlined and in bold and are designated SEQ ID NO: 96, 97 and 98. A glycineserine linker with the sequence of SEQ ID NO: 50, linking the VH and Vp, is shown in plain text.

[0422] Molecule E v3:

[0423] EVQLLESGGGLVQPGGSLRLSCAASGYRFTGYLMNWVRQAPGKGLEWVSAINPYKGSTYYADSV KGRFTFSRDNSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDLWGRGTLVTVSSGGGSGG GGDGGITQSPKYLFRKEGQNVTLSCEQNLNHDAMYWYRQDPGQGLRLIYYSQIMGDEQKGDIAEGY SVSREKKESFPLTVTSAQKNHTAFYLCASSWWTGGASPIRFGPGTRLTVTGGSGGGGSGGGGSGG GGSTGDAKTTQPNSMESNEGEPVHLPCQHSTISGTDYIHWYRQLPSQGPEYVIHGLTSNVNNRMAS LAIAEDRKSSTLILHRATLRDAAVYYCILILGHSRLGNYIATFGKGTKLSVIPEPKSSDKTHTCPPCPAP NHYTQKSLSLSPGK (SEQ ID NO: 85)

[0424] SEQ ID NO: 85 is the full amino acid sequence of a first chain of an exemplary half-life extended bispecific molecule referred to herein as “Molecule E v3”. Molecule E v3 is a dual chain half-life extended bispecific molecule comprising (i) a soluble TOR, which comprises a TOR alpha chain variable domain (Va) and a TOR beta chain variable domain (Vp), (ii) an immune cell engaging domain, which comprises an antibody light chain variable domain (VL), and an antibody heavy chain variable domain (VH), and (iii) an lgG1 Fc knob in hole half-life extending (HLE) domain, which comprises a first chain and a second chain. In Molecule E v3, the soluble TCR binds to a peptide having the sequence of SLLQHLIGL (SEQ ID NO: 111) in complex with HLA-A*02 and the immune cell engaging domain binds to CD3. The heavy chain variable domain (VH) is in bold and is designated SEQ ID NO: 103. The CDRs (CDR1 , CDR2 and CDR3) within the VH are underlined and are designated SEQ ID NO: 104, 105 and 106. The TCR beta chain variable domain (Vp) is double underlined and is designated SEQ ID NO: 95. The CDRs (CDR1 , CDR2 and CDR3) within Vp are double underlined and in bold and are designated SEQ ID NO: 96, 97 and 98. The TCR alpha chain variable domain (Va) is underlined and is designated SEQ ID NO: 91 . The CDRs (CDR1 , CDR2 and CD3) within the Va are underlined and in bold and are designated SEQ ID NO: 92, 93 and 94. The first chain of the lgG1 Fc knob in hole half-life extending (HLE) domain is dash-underlined and is designated SEQ ID NO: 107. A glycine-serine linker with the sequence of SEQ ID NO: 50, linking the VH and Vp, is shown in plain text. A glycine-serine linker with the sequence of SEQ ID NO: 51 , linking the Vp and Va, is shown in plain text. An IgG 1 / 2 hybrid hinge with the sequence of SEQ ID NO: 109, linked to the Va and the first chain of the HLE, is shown in bold italics.

[0425] AIQMTQSPSSLSASVGDRVTITCRASQGIRKYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGS G TDFTL TISSL QPEDFA TYYCQQGNTLPWTFG QGTK VEIKEPKSSDKTHTCPPCPAPPVA GP SV F LF

[0426] SEQ ID NO: 86 is the full amino acid sequence of a second chain of an exemplary half-life extended bispecific molecule referred to herein as “Molecule E v3”. The light chain variable domain (VL) is in italics and is designated SEQ ID NO: 99. The CDRs (CDR1 , CDR2 and CDR3) within the VL are underlined and are designated SEQ ID NO: 100, 101 and 102. The second chain of the lgG1 Fc knob in hole half-life extending (HLE) domain is dash-underlined and is designated SEQ ID NO: 108. An lgG1 / 2 hybrid hinge with the sequence of SEQ ID NO: 109, linked to the VL and the second chain of the HLE, is shown in bold italics.

[0427] Molecule E v6:

[0428] GDAKTTQPNSMESNEGEPVHLPCQHSTISGTDYIHWYRQLPSQGPEYVIHGLTSNVNNRMASLAIAE DRKSSTLILHRATLRDAAVYYCILILGHSRLGNYIATFGKGTKLSVIPGGGGSGGGGSGGGGSEVQLL ESGGGLVQPGGSLRLSCAASGYRFTGYLMNWVRQAPGKGLEWVSAINPYKGSTYYADSVKGRFT FSRDNSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDLWGRGTLVTVSSGGGSGGGGDG GITQSPKYLFRKEGQNVTLSCEQNLNHDAMYWYRQDPGQGLRLIYYSQIMGDEQKGDIAEGYSVSR EKKESFPLTVTSAQKNHTAFYLCASSWWTGGASPIRFGPGTRLTVTEPKSSDKTHTCPPCPAPPVA GPSyFLFPPKPKDTL LSRTPEyTCyyyDySHEDPEyKFNVW

[0429] TQKSLSLSPGK (SEQ ID NO: 87)

[0430] SEQ ID NO: 87 is the full amino acid sequence of a first chain of an exemplary half-life extended bispecific molecule referred to herein as “Molecule E v6”. Molecule E v6 is a dual chain half-life extended bispecific molecule comprising (i) a soluble TOR, which comprises a TOR alpha chain variable domain (Va) and a TOR beta chain variable domain (Vp), (ii) an immune cell engaging domain, which comprises an antibody light chain variable domain (VL), and an antibody heavy chain variable domain (VH), and (iii) an lgG1 Fc knob in hole half-life extending (HLE) domain, which comprises a first chain and a second chain. In Molecule E v6, the soluble TCR binds to a peptide having the sequence of SLLQHLIGL (SEQ ID NO: 111) in complex with HLA-A*02 and the immune cell engaging domain binds to CD3. The TCR alpha chain variable domain (Va) is underlined and is designated SEQ ID NO: 91 . The CDRs (CDR1 , CDR2 and CD3) within the Va are underlined and in bold and are designated SEQ ID NO: 92, 93 and 94. The heavy chain variable domain (VH) is in bold and is designated SEQ ID NO: 103. The CDRs (CDR1 , CDR2 and CDR3) within the VH are underlined and are designated SEQ ID NO: 104, 105 and 106. The TCR beta chain variable domain (Vp) is double underlined and is designated SEQ ID NO: 95. The CDRs (CDR1 , CDR2 and CDR3) within Vp are double underlined and in bold and are designated SEQ ID NO: 96, 97 and 98. The first chain of the IgG 1 Fc knob in hole half-life extending (HLE) domain is dash-underlined and is designated SEQ ID NO: 107. A glycine-serine linker with the sequence of SEQ ID NO: 59, linking the Va and VH, is shown in plain text. A glycine-serine linker with the sequence of SEQ ID NO: 50, linking the VH and Vp, is shown in plain text. An IgG 1 / 2 hybrid hinge with the sequence of SEQ ID NO: 109, linked to the Vp and the first chain of the HLE, is shown in bold italics.

[0431] AIQMTQSPSSLSASVGDRVTITCRASQGIRKYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGS G TDFTL TISSL QPEDFA TYYCQQGNTLPWTFG QGTK VEIKEPKSSDKTHTCPPCPAPPVA GP SV F LF

[0432] SEQ ID NO: 88 is the full amino acid sequence of a second chain of an exemplary half-life extended bispecific molecule referred to herein as “Molecule E v6”. The light chain variable domain (VL) is in italics and is designated SEQ ID NO: 99. The CDRs (CDR1 , CDR2 and CDR3) within the VL are underlined and are designated SEQ ID NO: 100, 101 and 102. The second chain of the lgG1 Fc knob in hole half-life extending (HLE) domain is dash-underlined and is designated SEQ ID NO: 108. An lgG1 / 2 hybrid hinge with the sequence of SEQ ID NO: 109, linked to the VL and the second chain of the HLE, is shown in bold italics.

[0433] Molecule E v8:

[0434] EVQLLESGGGLVQPGGSLRLSCAASGYRFTGYLMNWVRQAPGKGLEWVSAINPYKGSTYYADSV KGRFTFSRDNSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDLWGRGTLVTVSSGGGSGG GGDGGITQSPKYLFRKEGQNVTLSCEQNLNHDAMYWYRQDPGQGLRLIYYSQIMGDEQKGDIAEGY SVSREKKESFPLTVTSAQKNHTAFYLCASSWWTGGASPIRFGPGTRLTVTGGGGSGGGGSGGGGS AIQMTQSPSSLSASVGDRVTITCRASQGIRKYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGS G TDFTL TISSL QPEDFA TYYCQQGNTLPWTFG QGTK VEIKEPKSSDKTHTCPPCPAPPVA GP SV F LF

[0435] SEQ ID NO: 89 is the full amino acid sequence of a first chain of an exemplary half-life extended bispecific molecule referred to herein as “Molecule E v8”. Molecule E v8 is a dual chain half-life extended bispecific molecule comprising (i) a soluble TOR, which comprises a TOR alpha chain variable domain (Va) and a TOR beta chain variable domain (Vp), (ii) an immune cell engaging domain, which comprises an antibody light chain variable domain (VL), and an antibody heavy chain variable domain (VH), and (iii) an lgG1 Fc knob in hole half-life extending (HLE) domain, which comprises a first chain and a second chain. In Molecule E v8, the soluble TCR binds to a peptide having the sequence of SLLQHLIGL (SEQ ID NO: 111) in complex with HLA-A*02 and the immune cell engaging domain binds to CD3. The heavy chain variable domain (VH) is in bold and is designated SEQ ID NO: 103. The CDRs (CDR1 , CDR2 and CDR3) within the VH are underlined and are designated SEQ ID NO: 104, 105 and 106. The TCR beta chain variable domain (Vp) is double underlined and is designated SEQ ID NO: 95. The CDRs (CDR1 , CDR2 and CDR3) within Vp are double underlined and in bold and are designated SEQ ID NO: 96, 97 and 98. The light chain variable domain (VL) is in italics and is designated SEQ ID NO: 99. The CDRs (CDR1 , CDR2 and CDR3) within the VL are underlined and are designated SEQ ID NO: 100, 101 and 102. The first chain of the IgG 1 Fc knob in hole half-life extending (HLE) domain is dash-underlined and is designated SEQ ID NO: 107. A glycine-serine linker with the sequence of SEQ ID NO: 50, linking the VH and Vp, is shown in plain text. A glycine-serine linker with the sequence of SEQ ID NO: 59, linking the Vp and VL, is shown in plain text. An lgG1 / 2 hybrid hinge with the sequence of SEQ ID NO: 109, linked to the VL and the first chain of the HLE, is shown in bold italics.

[0436] GDAKTTQPNSMESNEGEPVHLPCQHSTISGTDYIHWYRQLPSQGPEYVIHGLTSNVNNRMASLAIAE DRKSSTLILHRATLRDAAVYYCILILGHSRLGNYIATFGKGTKLSVIPEPKSSDKTHTCPPCPAPPVAG .QKSLSLSPGK (SEQ ID NO: 90)

[0437] SEQ ID NO: 90 is the full amino acid sequence of a second chain of an exemplary half-life extended bispecific molecule referred to herein as “Molecule E v8”. The TCR alpha chain variable domain (Va) is underlined and is designated SEQ ID NO: 91 . The CDRs (CDR1 , CDR2 and CD3) within the Va are underlined and in bold and are designated SEQ ID NO: 92, 93 and 94. The second chain of the IgG 1 Fc knob in hole half-life extending (HLE) domain is dash-underlined and is designated SEQ ID NO: 108. An lgG1 / 2 hybrid hinge with the sequence of SEQ ID NO: 109, linked to the Va and the second chain of the HLE, is shown in bold italics. Examples

[0438] The invention will be more fully understood by reference to the following examples. They should not, however, be construed as limiting the scope of the invention. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the purview of this application and scope of the appended claims.

[0439] Example 1 - Materials and Methods

[0440] Molecule construction, expression, and purification

[0441] Expression constructs for bispecific molecules were made by cloning the fragment of interest into a vector based on pCDNA3.1 . Proteins were expressed in mammalian cells using the ExpiCHO transient expression system and purified using Protein L affinity capture followed by size exclusion chromatography.

[0442] Cell-based functional assays

[0443] IFNy ELISpot assays were performed according to the manufacturer’s instructions (BD Biosciences). Briefly, target cells were plated at ~5x104cells / per well and incubated with PBMC effector cells at a donor-dependant density. Test molecules were added at concentrations between 1 pM and 10 nM and plates were incubated overnight at 37°C / 5% CO2. IFNy-release was quantified using the BD ELISpot reader (Immunospot Series 5 Analyzer, Cellular Technology Ltd). Data were plotted using PRISM software and EC50 values were calculated from the curves.

[0444] Binding kinetics

[0445] SPR analysis was carried out using a BIAcore 8K with single cycle kinetic analysis. Kinetic parameters were calculated using BIAevaluation® software. The dissociation phase was fitted to a single exponential decay equation enabling calculation of half-life. The equilibrium constant (KD) was calculated from koff / kon. Measurements were performed at 25°C, unless otherwise indicated, in Dulbecco’s PBS buffer, supplemented with 0.005% P20.

[0446] Example 2 -Bispecific molecules with improved potency

[0447] A panel of 24 bispecific molecules was designed according to Figure 1. Each molecule comprised four domains; a TCR alpha chain variable domain, a TCR beta variable domain (together forming the soluble TCR domain) and antibody VL domain and an antibody VH domain (together forming the immune cell engaging domain). The molecular weight of each molecule was approximately 50 kDa.

[0448] The soluble TCR of each molecule was designed to bind to a defined peptide HLA complex, while the immune cell engaging domain was designed to bind CD3. The molecules were grouped into 4 different format types (including single chain and dual chain formats), and different domain arrangements were constructed within each format.

[0449] Molecules were expressed and purified from mammalian cells and tested for their ability to activate T cells in the presence of antigen presenting cells. T cell activation was determined by measuring interferon gamma release in an ELISpot assay. The Ec50 value was used as a measure of the potency of T cell activation and compared to the Ec50 obtained with a corresponding molecule in ImmTAC™ format. ImmTAC™ molecules are known in the art and additionally comprise TCR constant domains. The molecule weight of an ImmTAC™ molecule is approximately 75 kDa. In this example, the TCR variable domains bound to a HLA-A*02 restricted peptide from either preproinsulin (PPI) (ALWGPDPAAA (SEQ ID NO: 45)) or CT83 (KLVELEHTL (SEQ ID: 112)). The TCR variable domains included mutations relative to a native TCR sequence that resulted in increased affinity for antigen and improved stability. Antigen binding affinity for each TCR was confirmed using surface plasmon resonance (SPR). Amino acid sequences of the PPI TCR Va and Vp variable domains are provided in SEQ ID NOs: 27 and 34, respectively, and amino acid sequences of the VL and VH domain of an antiCD3 antibody are provided in SEQ ID NOs: 35 and 39, respectively. Other suitable TCRs and antiCD3 antibodies are known in the art.

[0450] Results

[0451] Five of the 24 bispecific molecules demonstrated increased potency (Ec50) of T cell activation in the presence of antigen positive cells, relative to the equivalent ImmTAC™ molecule incorporating the same soluble TCR and immune cell engaging domains. All other molecules resulted in lower potency compared to ImmTAC™. As shown in Figure 2 all five molecules with improved potency (labelled ‘molecules A-D’) have the VH of the immune cell engaging domain linked to the N-terminus of the beta chain. Similar data were obtained with molecules comprising either the PPI TCR or the CT83 TCR, demonstrating that the results are independent of the soluble TCR sequence (Figure 3). Full amino acid sequences of molecules A-D, comprising the PPI TCR variable domains, are provided (SEQ ID Nos: 1-7). The binding affinity of molecules A-D for ALWGPDPAAA (SEQ ID NO 45) HLA- A*02 complex was determined by SPR and shown in the table below, along with Ec50 values obtained from the graphs: These data demonstrate that bispecific molecules that are below 50 kDa and that have the immune cell engaging domain linked to the N-terminus of the beta chain demonstrate improved function in vitro compared to a larger TCR bispecific molecule (ImmTAC™).

[0452] Example 3 - Half-life extended bispecific molecules with improved potency

[0453] Bispecific molecules may be further engineered to comprise additional domains designed to extend the half life of the molecule in vivo. It is appreciated in the art that a longer half-life may provide a therapeutic advantage by reducing dosing frequency. Suitable half-life extending (HLE) domains are known in the art and include immunoglobulin Fc domains and fragments thereof (including monomeric Fc domains), HSA domains, or FcRn binding peptides. It has previously been shown that attaching a HLE domain to an ImmTAC™ molecule results in a reduction in potency in in vitro cell assays.

[0454] To investigate whether the molecules identified in Example 2 could be further modified to incorporate a HLE domain and retain a high degree of potency in vitro, a further panel of 18 molecules were made in which the HLE domain was located at different sites as shown in Figure 4. In this example, a monomeric Fc domain (mFc) (SEQ ID NO: 43) or the Dill domain of Human Serum Albumun (HSA Dill) (SEQ ID NO: 44) were used as the HLE domain. Full sequences of exemplary HLE variants comprising the PPI TCR are provided (SEQ ID Nos: 8-26 and 78-79).

[0455] Results

[0456] SPR measurements confirmed pH dependent binding of mFc and HSA Dill to FcRn. Figure 5-8 show concentration dependent T cell activation for molecules comprising both mFc and HSA Dill. In each case Ec50 is similar, or in some cases greater then, than the equivalent ImmTAC™ (without HLE domain).

[0457] These data provide improved TCR bispecific molecules with desirable therapeutic properties.

[0458] Example 4 - Further half-life extended bispecific molecules with improved potency

[0459] A panel of 8 bispecific molecules was designed according to Figure 9. Each molecule comprised five domains; a TCR alpha chain variable domain, a TCR beta variable domain (together forming the soluble TCR domain), an antibody VL domain and an antibody VH domain (together forming the immune cell engaging domain), and an immunoglobulin Fc domain as the HLE domain. The molecular weight of each molecule (excluding the HLE domain) was approximately 50 kDa.

[0460] The soluble TCR of each molecule was designed to bind to a defined peptide HLA complex, while the immune cell engaging domain was designed to bind CD3. The molecules were in either dual chain (Molecules E v2-8) or triple chain format (Molecule E v1), and different domain arrangements were constructed within each format. Molecules E v1 and E v3 have the VH of the immune cell engaging domain linked to the N-terminus of the beta chain.

[0461] Molecules E v1 and E v3 were expressed and purified from mammalian cells and tested for their ability to activate T cells in the presence of antigen presenting cells. T cell activation was determined by measuring interferon gamma release in an ELISpot assay. The Ec50 value was used as a measure of the potency of T cell activation. In this example, the TCR variable domains bound to a HLA-A*02 restricted peptide from PRAME (SLLQHLIGL (SEQ ID NO: 111)). The TCR variable domains included mutations relative to a native TCR sequence that resulted in increased affinity for antigen and improved stability. Antigen binding affinity for each TCR was confirmed using surface plasmon resonance (SPR). Amino acid sequences of the PRAME TCR Va and Vp variable domains are provided in SEQ ID NOs: 91 and 95, respectively, and amino acid sequences of the VL and VH domain of an antiCD3 antibody are provided in SEQ ID NOs: 99 and 103, respectively. Full sequences of exemplary HLE variants comprising the PRAME TCR are provided (SEQ ID Nos: 82- 90). Other suitable TCRs and antiCD3 antibodies are known in the art.

[0462] Results

[0463] The binding affinity of molecules E v1 and E v3 for SLLQHLIGL (SEQ ID NO: 111) HLA-A*02 complex was determined by SPR and shown in the table below, along with Ec50 values obtained from the graphs:

[0464] SPR measurements confirmed pH dependent binding of Fc to FcRn. In each case Ec50 is similar to the Ec50 value calculated for the equivalent ImmTAC™ molecule. Figures 10-11 show concentration dependent T cell activation for Molecules E v1 and E v3. In each case KD is less than 100 pM.

[0465] These data demonstrate that bispecific molecules that are below 50 kDa and that have the immune cell engaging domain linked to the N-terminus of the beta chain, and that further comprise a HLE, retain a high level of potency in vitro, with Ec50 values in the low pM range in vitro.

[0466] These data provide improved TCR bispecific molecules with desirable therapeutic properties, combining pM potency with the potential for extended in vivo half life.

Claims

Claims1 . A bispecific molecule comprising a soluble TCR and an immune cell engaging domain, wherein said soluble TCR binds to a peptide-major histocompatibility complex (pMHC) and comprises a TCR alpha chain variable domain (Va) and a TCR beta chain variable domain (Vp); wherein said immune cell engaging domain comprises an antibody light chain variable domain (VL), and an antibody heavy chain variable domain (VH); wherein said bispecific molecule is not greater than 60 kDa in size; and wherein said VH is linked to the N-terminus of the Vp.

2. The bispecific molecule of claim 1 , wherein the soluble TCR binds to the pMHC with a KD of less than 1 nM.

3. The bispecific molecule of claim 1 or 2, wherein the soluble TCR binds to the pMHC with a KD of less than 100 pM.

4. The bispecific molecule of any one of claims 1-3, wherein said Va is linked to said VL.

5. The bispecific molecule of any one of claims 1-4, wherein said bispecific molecule is a single chain molecule.

6. The bispecific molecule of claim 5, wherein said bispecific molecule is in a format selected from the group consisting of:(i) VL-VH-Vp-Va;(ii) VH-Vp-Va-VL; and(iii) Va -VL-VH-Vp.

7. The bispecific molecule of any one of claims 1-4, wherein said bispecific molecule is a dual chain molecule.

8. The bispecific molecule of claim 7, wherein said bispecific molecule comprises:(i) a first chain comprising VH-Vp; and(ii) a second chain comprising Va-VL.

9. The bispecific molecule of any one of claims 1-8, wherein two or more of the Va, Vp, VH and VL are linked via a linker and / or an IgG hinge sequence.

10. The bispecific molecule of any one of claims 7-9, wherein said Vp is linked to said VL via a disulphide bond.

11. The bispecific molecule of any one of claims 1-10, wherein the soluble TCR has the property of binding to a ALWGPDAAA (SEQ ID NO: 45) HLA-A*02 complex.

12. The bispecific molecule of claim 11 , wherein:(a) the Va comprises a CDR1 , a CDR2 and a CDR3 comprising the following sequences: CDR1 - DKHSQG (SEQ ID NO: 28), optionally with one, two or three mutations therein, CDR2 - IYSQGD (SEQ ID NO: 80), optionally with one, two or three mutations therein, CDR3 - AVRGNEKLT (SEQ ID NO: 30), optionally with one, two or three mutations therein, and / or(b) the Vp comprises a CDR1 , a CDR2 and a CDR3 comprising the following sequences: CDR1 - LQHSY (SEQ ID NO: 81), optionally with one, two or three mutations therein, CDR2 - SVGVGF (SEQ ID NO: 33), optionally with one, two or three mutations therein, CDR3 - ASAYMTGELF (SEQ ID NO: 34), optionally with one, two or three mutations therein; optionally wherein the soluble TCR comprises a Va comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or is 100% identical to SEQ ID NO: 27 and a Vp comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or is 100% identical to SEQ ID NO: 31.

13. The bispecific molecule of any one of claims 1-10, wherein the soluble TCR has the property of binding to a SLLQHLIGL (SEQ ID NO: 111) HLA-A*02 complex.

14. The bispecific molecule of claim 13, wherein:(a) the Va comprises a CDR1 , a CDR2 and a CDR3 comprising the following sequences: CDR1 - TISGTDY (SEQ ID NO: 92), optionally with one, two or three mutations therein, CDR2 - GLTSN (SEQ ID NO: 93), optionally with one, two or three mutations therein, CDR3 - CILILGHSRLGNYIATF (SEQ ID NO: 94), optionally with one, two or three mutations therein, and / or(b) the Vp comprises a CDR1 , a CDR2 and a CDR3 comprising the following sequences: CDR1 - LNHDA (SEQ ID NO: 96), optionally with one, two or three mutations therein, CDR2 - SQIMGD (SEQ ID NO: 97), optionally with one, two or three mutations therein, CDR3 - CASSWWTGGASPIRF (SEQ ID NO: 98), optionally with one, two or three mutations therein; optionally wherein the soluble TCR comprises a Va comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or is 100% identical to SEQ ID NO: 91 and a Vp comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or is 100% identical to SEQ ID NO: 95.

15. The bispecific molecule of any one of claims 1-14, wherein the immune cell engaging domain binds to a T cell surface antigen.

16. The bispecific molecule of claim 15, wherein said T cell surface antigen is CD3.

17. The bispecific molecule of claim 16, wherein the immune cell engaging domain comprises:(a) a VL comprising a CDR1 , a CDR2 and a CDR3 comprising the following sequences:CDR1 - QDIRNY (SEQ ID NO: 36),CDR2 - YTS (SEQ ID NO: 37),CDR3 - QQGNTLPWT (SEQ ID NO: 38); and / or(b) a VH comprising a CDR1 , a CDR2 and a CDR3 comprising the following sequences:CDR1 - GYSFTGYA (SEQ ID NO: 40),CDR2 - INPYKGVS (SEQ ID NO: 41),CDR3 - ARSGYYGDSDWYFDV (SEQ ID NO: 42); optionally wherein the immune cell engaging domain comprises a VL comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or is 100% identical to SEQ ID NO: 35 and a VH comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or is 100% identical to the sequence of SEQ ID NO: 39.

18. The bispecific molecule of claim 16, wherein the immune cell engaging domain comprises:(a) a VL comprising a CDR1 , a CDR2 and a CDR3 comprising the following sequences:CDR1 - QGIRKY (SEQ ID NO: 100),CDR2 - AAS (SEQ ID NO: 101),CDR3 - QQGNTLPWT (SEQ ID NO: 102); and / or(b) a VH comprising a CDR1 , a CDR2 and a CDR3 comprising the following sequences:CDR1 - GYRFTGYL (SEQ ID NO: 104),CDR2 - INPYKGST (SEQ ID NO: 105),CDR3 - ARSGYYGDSDWYFDL (SEQ ID NO: 106); optionally wherein the immune cell engaging domain comprises a VL comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or is 100% identical to SEQ ID NO: 99 and a VH comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, or is 100% identical to the sequence of SEQ ID NO: 103.

19. A half-life extended bispecific molecule comprising the bispecific molecule of any one of claims 1- 20 and a half-life extending domain (HLE).

20. The half-life extended bispecific molecule of claim 19, wherein said half-life extended bispecific molecule is a single chain molecule and in a format selected from the group consisting of:(i) A / -VL-VH-Vp-Va-HLE-C;(ii) A / -HLE-VL-VH-Vp-Va-C;(iii) A / -Va-VL-VH-Vp-HLE-C;(iv) A / -HLE-Va-VL-VH-Vp-C;(v) A / -VH-Vp-Va-VL-HLE-C;(vi) / V-HLE-VH-Vp-Va-VL-C;(vii) / V-Va-VL-HLE-VH-Vp-C; and(viii) / V-VH-Vp-HLE-Va-VL-C.

21. The half-life extended bispecific molecule of claim 19, wherein said half-life extended bispecific molecule is a dual chain molecule comprising or consisting of:(i) a first chain having the format A / -VH-Vp-C and a second chain having the format N- Va-VL-HLE-C;(ii) a first chain having the format A / -VH-Vp-HLE-C and a second chain having the format / V-Va-VL-C;(iii) a first chain having the format A / -VH-Vp-C and a second chain having the format N- HLE-Va-VL-C; or(iv) a first chain having the format A / -HLE-VH-Vp-C and a second chain having the format M-Va-VL-C;.

22. The half-life extended bispecific molecule of any one of claims 19-21 , wherein said HLE is selected from the group consisting of (i) monomeric Fc; (ii) monomeric Fc CH2; (iii) monomeric Fc CH3; (iv) HSA Dill; (v) HSA Dll IA; (vi) HSA DI I lb; (vii) an FcRn binding peptide; (viii) a scFv that binds to FcRn; and (ix) a VHH that binds to FcRn.

23. The half-life extended bispecific molecule of claim 22, wherein said HLE is monomeric Fc.

24. The half-life extended bispecific molecule of claim 23, wherein said monomeric Fc has the sequence of SEQ ID NO: 43.

25. The half-life extended bispecific molecule of claim 22, wherein said HLE is HSA DHL26. The half-life extended bispecific molecule of claim 25, wherein said HSA Dill has the sequence ofSEQ ID NO: 44.

27. The half-life extended bispecific molecule of claim 19, wherein said HLE is an IgG-Fc, and wherein said IgG-Fc comprises or consists of a first domain (FC1) and a second domain (FC2).

28. The half-life extended bispecific molecule of claim 27, wherein said half-life extended bispecific molecule is a triple chain molecule comprising or consisting of a first chain having (i) a first chain having the format A / -Va-FC1-C, (ii) a second chain having the format A / -VL-FC2-C, and (iii) a third chain having the format A / -VH-Vp-C.

29. The half-life extended bispecific molecule of claim 27, wherein said half-life extended bispecific molecule is a dual chain molecule comprising or consisting of:(i) a first chain having the format A / -VH-Vp-Va-FC1-C and a second chain having the format A / -VL-FC2-C;(ii) a first chain having the format A / -Va-VH-Vp-FC1-C and a second chain having the format A / -VL-FC2-C; or(iii) a first chain having the format A / -VH-Vp-VL-FC1-C and a second chain having the format A / -Va-FC2-C.

30. The half-life extended bispecific molecule of any one of claims 27-29, wherein the FC1 comprises or consists of the sequence of SEQ ID NO: 107 and the FC2 comprises or consists of the sequence of SEQ ID NO: 108, or wherein the FC1 comprises or consists of the sequence of SEQ ID NO: 108 and the FC2 comprises or consists of the sequence of SEQ ID NO: 107.

31. The half-life extended bispecific molecule of any one of claims 19-30, wherein said HLE is linked to the VH, VL, Va or Vp via a linker and / or an IgG hinge sequence.

32. The bispecific molecule of any one of claims 9-18 or the half-life extended molecule of any one of 19-31 , wherein the linker has the sequence of any one of SEQ ID NOs: 46, 48-61 and / or the amino acid sequence GG; and / or the IgG hinge sequence has the sequence of any one of SEQ ID NOs: 47, 62-68 or 109-110.

33. The bispecific molecule of any one of claims 1-6, 9-12 or 15-17, wherein the bispecific molecule is a single chain molecule comprising or consisting of the sequence of any one of SEQ ID NOs: 1 , 6 and 7.

34. The bispecific molecule of any one of claims 1-4, 7-12 or 15-17, wherein the bispecific molecule is a dual chain molecule comprising or consisting of the sequences of: (i) SEQ ID NOs: 2-3, or (ii) SEQ ID NOs: 4-5.

35. The half-life extended bispecific molecule of any one of claims 19-20 or 22-26, wherein the halflife extended bispecific molecule is a single chain molecule comprising or consisting of the sequence of SEQ ID NO: 8, 9, 16, 17, 18, 19, 24, 25 or 26.

36. The half-life extended bispecific molecule of any one of claims 19 or 21-26, wherein the half-life extended bispecific molecule is a dual chain molecule comprising or consisting of the sequences of: (i) SEQ ID NOs: 10-11 ; (ii) SEQ ID NOs: 12-13; (iii) SEQ ID NOs: 14-15; (iv) SEQ ID NOs: 20-21 ; (v) SEQ ID NOs: 22-23; or (vi) SEQ ID NOs: 78-79.

37. The half-life extended triple chain molecule bispecific molecule of any one of claims 19 or 27-28 comprising or consisting of the sequence of any one of SEQ ID NOs: 82-84.

38. The half-life extended dual chain molecule bispecific molecule of any one of claims 19, 27 or 29 comprising or consisting of the sequences of (i) SEQ ID NOs: 85-86; (ii) SEQ ID NOs: 87-88; or (iii) SEQ ID NOs: 89-90.

39. A nucleic acid encoding the bispecific molecule of any one of claims 1-18 or the half-life extended bispecific molecule of any one of claims 19-38.

40. An expression vector comprising the nucleic acid of claim 39.41 . A cell harbouring:(a) the nucleic acid of claim 39;(b) the expression vector of claim 40;(c) a first expression vector comprising a nucleic acid encoding a first chain of a dual chain bispecific molecule of any one of claims 7-18 or 34, and a second expression vector comprising a nucleic acid encoding a second chain of the dual chain bispecific molecule; or(d) a first expression vector comprising a nucleic acid encoding a first chain of a dual chain half-life extended bispecific molecule of any one of claims 19, 21-27, 29-32, 36 or 38, and a second expression vector comprising a nucleic acid encoding a second chain of the dual chain half-life extended bispecific molecule; or(e) a first expression vector comprising a nucleic acid encoding a first chain of a triple chain half-life extended bispecific molecule of any one of claims 28, 30-32 or 37, a second expression vector comprising a nucleic acid encoding a second chain of the triple chain halflife extended bispecific molecule, and a third expression vector comprising a nucleic acid encoding a third chain of the triple chain half-life extended bispecific molecule.

42. The cell of claim 41 , wherein the cell is a mammalian cell expressing one or more chains of a bispecific molecule of any one of claims 1-18 or a half-life extended bispecific molecule of any one of claims 19-38, optionally wherein the mammalian cell is a CHO cell.

43. A pharmaceutical composition comprising the bispecific molecule of any one of claims 1-18, the half-life extended bispecific molecule of any one of claims 19-38, the nucleic acid of claim 39, the expression vector of claim 40, and / or the cell of claim 41 or 42, together with one or more pharmaceutically acceptable carriers or excipients.

44. A method of treating a disease comprising administering the bispecific molecule of any one of claims 1-18, the half-life extended bispecific molecule of any one of claims 19-38, the nucleic acid of claim 39, the expression vector of claim 40, the cell of claim 41 or 42, and / or the pharmaceutical composition of claim 43 to a subject in need thereof.

45. The method of claim 44, wherein the disease to be treated is cancer, an infectious disease and / or an autoimmune disease.

46. A method of targeting a tumor and / or redirecting T cells to a tumor comprising administering the bispecific molecule of any one of claims 1-18, the half-life extended bispecific molecule of any one of claims 19-38, the nucleic acid of claim 39, the expression vector of claim 40, the cell of claim 41 or 42, and / or the pharmaceutical composition of claim 43 to a subject in need thereof.

47. The bispecific molecule of any one of claims 1-18, the half-life extended bispecific molecule of any one of claims 19-38, the nucleic acid of claim 39, the expression vector of claim 40, the cell of claim 41 or 42, and / or the pharmaceutical composition of claim 43 for use in a method of treating a disease, comprising administering the bispecific molecule, the nucleic acid, the expression vector, the cell, and / or the pharmaceutical composition to a subject in need thereof.

48. The bispecific molecule, half-life extended bispecific molecule, nucleic acid, expression vector, cell, and / or pharmaceutical composition for use according to claim 47, wherein the disease is cancer, an infectious disease or an autoimmune disease.

49. A method of producing the bispecific molecule of any one of claims 1-18 or the half-life extended bispecific molecule of any one of claims 19-38, the method comprising:(a) maintaining the cell of claim 41 or 42 under optimal conditions for expression of the bispecific molecule or the half-life extended bispecific molecule; and(b) isolating the bispecific molecule or the half-life extended bispecific molecule.

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