Multi-domain binding molecules

Abstract

The invention relates to a multi-domain single chain binding molecule. The molecule comprises i) a peptide-major histocompatibility complex (pMHC) binding domain comprising a first variable region (VC1) linked to a constant region and a second variable region (VC2) linked to a constant region; ii) a T cell-engaging immune effector domain comprising an antibody light chain variable region (TCE-VL) and an antibody heavy chain variable region (TCE-VH); and iii) a half-life extending domain comprising a first IgG Fc region (FC1) and a second IgG Fc region (FC2) wherein the FC1 region and the FC2 region are dimerized to form an Fc domain. The binding molecules can be used to treat diseases, such as cancer and infectious diseases.

Description

[0001] Sequence Listing

[0002] This application contains a Sequence Listing, the entire contents of which are incorporated herein by reference. The XML copy was created on August 2, 2023, named P206512WO_ST.26Sequence Listing.xml, and is 55320 bytes in size. Background Art

[0003] Many protein-based therapies, including antibody fragments and fusion proteins, are rapidly cleared from the body after administration. Their short circulatory half-life is usually attributed to their small size, which allows for efficient clearance by renal filtration, and the lack of protection against intracellular degradation. In such cases, frequent administration or prolonged infusion is required to maintain an effective concentration of the drug over a longer period. To improve drug delivery, several strategies have been adopted to extend the circulatory half-life. These include increasing the hydrodynamic radius of the protein by attaching flexible hydrophilic molecules such as carbohydrates or PEG (polyethylene glycol), and taking advantage of recycling via the neonatal Fc receptor (FcRn) by attaching an antibody Fc domain or serum albumin (Konnteman, Curr Opin Biotechnol. 2011 Dec; 22(6):868-76).

[0004] The strategy of using FcRn-mediated recycling is particularly attractive because the risk of inducing immunogenicity in vivo is low and longer half-life extensions can be achieved. For example, it has been reported that after attaching an Fc domain, the half-life of a T cell-engaging bispecific antibody in the form of exceeds 200 h (Lorenczewski et al., Blood 2017.130 (suppl 1), 2815). Similarly, it has been reported that a bispecific antibody in the form of

[0005] Fusion proteins containing a soluble T cell receptor (TCR) fused to an anti-CD3 antibody fragment are a relatively new class of immune cell (e.g., T cell)-engaging bispecific fusion proteins with an in vivo half-life in the range of 6 - 8 h (Sato et al., 2018 J Clin Onc 2018 36, no. 15, suppl 9521 - 9521; Middleton et al., J Clin Onc 2016 34, no. 15, suppl 3016 - 3016). This is far shorter than that of conventional monoclonal antibodies, which typically have a half-life in the range of 260 - 720 hours (Ovacik & Lin, 2018 Clin Transl Sci, 11:540). In addition, TCR - anti-CD3 fusion proteins have demonstrated favorable therapeutic properties, including picomolar potency (Lowe et al., 2019 Cancer treatment reviews, vol. 77 35 - 43). Thus, there is a need to identify suitable methods for extending the half-life of TCR - immune cell - engaging domain fusion proteins, such as TCR - anti-CD3 fusion proteins and other TCR - containing proteins, to reduce the dosing frequency and maintain effective concentrations over an extended period without compromising other therapeutic properties.

[0006] Unlike conventional antibodies, TCRs are designed to recognize short peptides derived from intracellular antigens and presented on the cell surface by human leukocyte antigens (peptide - HLA). The formation of an effective immune synapse between the peptide - HLA complex on antigen - presenting cells and the corresponding receptors on immune cells such as T cells depends on, for example, carefully designed interactions that can be disrupted by an increased intermembrane distance (Choudhuri et al., 2005 Nature Jul 28; 436(7050):578 - 82; Holland et al., J Clin Invest. 2020; 130(5):2673 - 2688). Thus, fusion methods for increasing the half-life of TCR - containing proteins, such as attaching an antibody Fc domain or serum albumin, are highly challenging due to the risk of disrupting the interaction geometry required for TCR binding. Similar challenges also apply to fusion proteins containing antibodies that bind to the peptide - HLA complex, and these antibodies are referred to as TCR - like or TCR - mimicking antibodies.

[0007] WO 2020 / 157211 describes a method for extending the half-life of a TCR-antibody CD3 fusion protein by fusing the TCR-antibody CD3 fusion protein to an immunoglobulin Fc domain or an albumin-binding domain. However, such multi-domain binding molecules are large and complex proteins, and thus there are multiple possible forms, i.e., the possible combinations of the positions and orientations of each domain (and each region within each domain) on one or more polypeptide chains. The position and orientation of each domain (and its regions) in the molecule and the number of polypeptide chains present may affect the characteristics of the binding molecule, such as activity, half-life, and manufacturability. Accordingly, there remains a need to identify favorable forms of such multi-domain binding molecules. Summary of the Invention

[0008] The present invention generally relates to multi-domain binding molecules. In particular, the present invention relates to multi-domain binding molecules comprising: i) a peptide-major histocompatibility complex (pMHC) binding domain comprising a first variable region (VC1) linked to a constant region and a second variable region (VC2) linked to a constant region; ii) an immune cell engaging (ICE) domain (such as a T cell engaging immune effector domain) comprising, for example, an antibody light chain variable region (TCE-VL) and an antibody heavy chain variable region (TCE-VH); and iii) a half-life extending domain comprising a first IgG Fc region (FC1) and a second IgG Fc region (FC2), wherein the FC1 region and the FC2 region dimerize to form an Fc domain. The binding molecule can be used to treat diseases such as cancer, infectious diseases, and autoimmune diseases. Detailed Description

[0009] The present invention tested more than 35 different forms (i.e., the orientation and position of each domain in the polypeptide) of multi-domain binding molecules comprising a pMHC binding domain, a T cell engaging immune effector domain, and a half-life extending domain. In so doing, they found that in many forms, fusing a TCR-antibody CD3 fusion protein to an Fc domain resulted in a substantial loss of in vitro potency. However, the inventors surprisingly identified a form of such molecules that can be expressed as a single polypeptide chain, has a significantly enhanced half-life, and retains the high potency of the original molecule. Example 8 further shows that the identified form functions advantageously with TCRs that bind different targets.

[0010] In a first aspect, there is provided a multi-domain single-chain binding molecule comprising:

[0011] i) A peptide-major histocompatibility complex (pMHC) binding domain, which comprises a first variable region (VC1) linked to a constant region and a second variable region (VC2) linked to a constant region, wherein VC1 and VC2 dimerize to form the pMHC binding domain;

[0012] ii) An immune cell engaging (ICE) domain; and

[0013] iii) A half-life extension domain, which comprises a first IgG Fc region (FC1) and a second IgG Fc (FC2), wherein the FC1 region and the FC2 region dimerize to form an Fc domain;

[0014] wherein the ICE domain is linked to the N-terminus of VC1, VC1 is linked to the N-terminus of the FC1 region through its C-terminus, the FC1 region is linked to the N-terminus of VC2 through its C-terminus, and VC2 is linked to the N-terminus of the FC2 region through its C-terminus; and

[0015] wherein the pMHC binding domain and the T cell engaging immune effector domain are capable of binding to a pMHC complex and an immune cell, respectively.

[0016] In one embodiment, there is provided a multi-domain single-chain binding molecule, which comprises:

[0017] i) A peptide-major histocompatibility complex (pMHC) binding domain, which comprises a first variable region (VC1) linked to a constant region and a second variable region (VC2) linked to a constant region, wherein VC1 and VC2 dimerize to form the pMHC binding domain;

[0018] ii) A T cell engaging immune effector domain, which comprises an antibody light chain variable region (TCE-VL) and an antibody heavy chain variable region (TCE-VH); and

[0019] iii) A half-life extension domain, which comprises a first IgG Fc region (FC1) and a second IgG Fc region (FC2), wherein the FC1 region and the FC2 region dimerize to form an Fc domain;

[0020] wherein the T cell engaging immune effector domain is linked to the N-terminus of VC1, VC1 is linked to the N-terminus of the FC1 region through its C-terminus, the FC1 region is linked to the N-terminus of VC2 through its C-terminus, and VC2 is linked to the N-terminus of the FC2 region through its C-terminus; and

[0021] wherein the pMHC binding domain and the T cell engaging immune effector domain are capable of binding to a pMHC complex and a T cell, respectively.

[0022] In another aspect, there is provided a multi-domain single-chain binding molecule comprising:

[0023] i) a soluble TCR comprising a first variable region (VC1) linked to a constant region and a second variable region (VC2) linked to a constant region, wherein VC1 comprises a TCRβ variable and constant region having the amino acid sequence provided in SEQ ID NO:16, or a sequence that is at least 90%, at least 95% or at least 98% identical thereto, and VC2 comprises a TCRα variable and constant region having the amino acid sequence provided in SEQ ID NO:14, or a sequence that is at least 90%, at least 95% or at least 98% identical thereto;

[0024] ii) an anti-CD3 scFv comprising an antibody light chain variable region (TCE-VL) having the amino acid sequence provided in SEQ ID NO:31, or a sequence that is at least 90%, at least 95% or at least 98% identical thereto, and an antibody heavy chain variable region (TCE-VH) having the amino acid sequence provided in SEQ ID NO:32, or a sequence that is at least 90%, at least 95% or at least 98% identical thereto; and

[0025] iii) a half-life extending domain comprising a first IgG Fc region (FC1) having the amino acid sequence provided in SEQ ID NO:42, or a sequence that is at least 90%, at least 95% or at least 98% identical thereto, and a second IgG Fc region (FC2) having the amino acid sequence provided in SEQ ID NO:43, or a sequence that is at least 90%, at least 95% or at least 98% identical thereto, wherein the FC1 region and the FC2 region dimerize to form an FC domain;

[0026] wherein the T cell-engaging immune effector domain is linked to the N-terminus of VC1, VC1 is linked to the N-terminus of the FC1 region through its C-terminus, the FC1 region is linked to the N-terminus of VC2 through its C-terminus, and VC2 is linked to the N-terminus of the FC2 region through its C-terminus; and

[0027] wherein the pMHC-binding domain and the T cell-engaging immune effector domain are capable of binding to a pMHC complex and a T cell, respectively.

[0028] In another aspect, there is provided a multi-domain single-chain binding molecule comprising the amino acid sequence provided in SEQ ID NO:45.

[0029] In a further aspect, a nucleic acid encoding a multi-domain binding molecule is provided. An expression vector comprising the nucleic acid of this aspect is also provided. In addition, a host cell comprising the nucleic acid or vector of this aspect is provided.

[0030] In a further aspect, a method for preparing the multi-domain binding molecule is also provided, which comprises maintaining the host cell described above under optimal conditions for expressing the nucleic acid and isolating the multi-domain binding molecule.

[0031] In a further aspect, a pharmaceutical composition comprising the multi-domain binding molecule is provided.

[0032] The multi-domain binding molecule, nucleic acid, vector, host cell or pharmaceutical composition of any one of the above aspects can be used for treating diseases such as cancer, infectious diseases and autoimmune diseases. Thus, in a further aspect, a multi-domain binding molecule, nucleic acid, vector, host cell or pharmaceutical composition for use as a drug is also provided. In yet a further aspect, a method of treatment is provided, which comprises administering a multi-domain binding molecule, nucleic acid, vector, host cell or pharmaceutical composition to a patient in need thereof.

[0033] Peptide-major histocompatibility complex (pMHC) binding domain

[0034] As used herein, a "pMHC binding domain" is a protein domain capable of binding to a peptide-MHC complex. A first variable region (VC1) linked to a constant region and a second variable region (VC2) linked to a constant region dimerize to form a pMHC binding domain. In this context, "VC1" refers to the pMHC binding domain sequence region that contains the first variable region linked to the constant region, and "VC2" refers to the region containing the second variable region linked to the constant region. The pMHC binding site is within the variable regions of VC1 and VC2. Suitable variable region and constant region sequences include TCR or antibody variable and constant regions. As used herein, the terms "MHC" and "HLA" may be used interchangeably.

[0035] The pMHC binding domain may comprise at least a portion of the TCRα and TCRβ chains. For example, the variable regions of VC1 and VC2 may be TCR variable regions. VC1 may comprise a TCRα or TCRβ variable region and VC2 may comprise the other of the TCRα and TCRβ variable regions. For example:

[0036] (i) VC1 may comprise (a) a TCRα variable and constant region or (b) a TCRβ variable and constant region; and

[0037] (ii) VC2 may comprise the other of (a) or (b). Preferably, VC1 comprises the variable and constant regions of TCRβ and VC2 comprises the variable and constant regions of TCRα.

[0038] The pMHC binding domain can be a T cell receptor (TCR) comprising the variable and constant regions of the TCR, such as a soluble TCR. The TCR sequences defined herein are described with reference to the IMGT nomenclature, which is well known and accessible to those skilled in the TCR art. 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, the TCR consists of two disulfide-linked chains. Each chain (α and β) generally has two extracellular regions, a variable region and a constant region. A short connecting region links the variable region and the constant region and is generally considered part of the α variable region. In addition, the β chain usually contains a short diversity region adjacent to the connecting region, which is also generally considered part of the β variable region. The variable region of each chain of the telecommunication TCR is located at the N-terminus and contains three complementarity determining regions (CDRs) embedded in the framework sequences. The CDRs contain the recognition sites for peptide-MHC binding.

[0039] Alternatively, the pMHC binding domain can contain the variable region of an antibody. The VC1 and VC2 variable regions can be the variable regions of the heavy or light chains of an antibody. For example, VC1 can comprise the variable region of the heavy or light chain of an antibody, and VC2 can comprise the other of the variable regions of the heavy or light chain of an antibody. In this regard, the pMHC binding domain can be a TCR-like antibody, also known as a "TCR mimicking antibody" (TCRm-Ab). For example, the pMHC binding domain can comprise the variable region of a TCR-like antibody. Antibodies do not naturally recognize pMHC complexes. However, it is known that antibodies can be engineered to be specific for pMHC, as described in Chang et al., Expert Opin Biol Ther. 2016 Aug;16(8):979-87 and Dahan et al., Expert Rev Mol Med. 2012 Feb 24;14:e6.

[0040] The pMHC binding domain can comprise at least one immunoglobulin constant region. For example, the constant regions in VC1 and VC2 can be immunoglobulin constant regions. The constant region can correspond to the constant region from the TCRα chain or TCRβ chain (TRAC or TRBC, respectively). Alternatively, the constant region of the pMHC binding domain can be the constant region from an antibody light or heavy chain (CL, CH1, CH2, CH3, or CH4). The constant region can be full-length or can be truncated. The TCR constant region can be truncated to remove the transmembrane domain and cytoplasmic tail. In cases where the constant region is truncated, preferably only the membrane-associated and cytoplasmic portions are removed from the C-terminus. In cases where the pMHC binding domain comprises TCRα or TCRβ chain sequences, VC1 and VC2 can comprise the TCR variable region and TCR constant region, respectively. Preferably, VC1 and VC2 do not comprise transmembrane or cytoplasmic domains, i.e., preferably the pMHC binding domain is soluble. Other mutations can be introduced into the amino acid sequence of the constant region relative to the native constant region. The constant region can also comprise naturally occurring or introduced residues that allow dimerization, for example, by a disulfide bond between two cysteine residues.

[0041] If present, the TCR portion of the molecule of the invention can be an αβ heterodimer. The α-β heterodimeric TCR portion of the molecule of the invention can comprise the TRAC constant region sequence of the α chain and / or the TRBC1 or TRBC2 constant region sequence of the β chain. As described above, the constant region can be in a soluble form (i.e., without transmembrane or cytoplasmic domains). One or both of the constant regions can contain mutations, substitutions, or deletions relative to the native TRAC and / or TRBC1 / 2 sequences. The terms TRAC and TRBC1 / 2 also encompass naturally occurring polymorphic variants, such as the change from N to K at position 4 of TRAC (Bragado et al., International immunology. 1994 Feb;6(2):223-30).

[0042] The α-chain and β-chain constant region sequences can be modified by truncation or substitution to lack the native disulfide bond between Cys4 of exon 2 of TRAC and Cys2 of exon 2 of TRBC1 or TRBC2. The α- and / or β-chain constant region sequences can have introduced disulfide bonds between residues of their respective constant domains, as described, for example, in WO 2003 / 020763, WO 2004 / 033685, and WO2006 / 000830, and also, for example, in U.S. Patent Nos. 7,329,731, 7,569,664, and 8,361,794, the contents of each of which are incorporated herein by reference. The α- and β-chain constant regions can be modified by substituting the cysteine residues at position Thr48 of TRAC and position Ser 57 of TRBC1 or TRBC2, which cysteines form a disulfide bond between the α constant region and the β constant region of the TCR. TRBC1 or TRBC2 can additionally contain a cysteine to alanine mutation at position 75 of the constant region and an asparagine to aspartic acid mutation at position 89 of the constant region. One or both of the extracellular constant regions present in the αβ heterodimer can be truncated at the C-terminus, for example, truncated by up to 15, or up to 10, or up to 8 or fewer amino acids. The C-terminus of the α-chain extracellular constant region can be truncated by 8 amino acids.

[0043] The amino acid sequences of the VC1 and VC2 variable and constant regions can correspond to those found in nature, or they can contain one or more mutations relative to the native protein. Such mutations can be made to increase the affinity of the pMHC binding domain for a given antigen. Additionally or alternatively, mutations can be introduced to improve stability and manufacturability. The VC1 and VC2 sequences can be derived from human sequences.

[0044] The VC1 and VC2 sequences can contain one or more engineered cysteine residues in the constant regions to form non-native disulfide bonds between VC1 and VC2. Suitable positions for introducing disulfide bonds between residues of the respective constant regions are described in WO 2003 / 020763 and WO 2004 / 033685. Single-chain TCRs are further described in WO2004 / 033685; W098 / 39482; WO01 / 62908; Weidanz et al., (1998) J Immunol Methods 221(1-2):59-76; Hoo et al., (1992) Proc Natl Acad Sci U S A 89(10):4759-4763; Schodin (1996) Mol Immunol 33(9):819-829.

[0045] VC1 can comprise the TCRα or TCRβ variable region, and VC2 can comprise the other of the TCRα and TCRβ variable regions. Preferably:

[0046] (i) The TCRα variable region comprises CDRs that are SEQ ID NO:3, 4, and 5 as CDR1, CDR2, and CDR3, respectively; and

[0047] (ii) The TCRβ variable region comprises CDRs that are SEQ ID NO:9, 10, and 11 as CDR1, CDR2, and CDR3, respectively.

[0048] Alternatively, the TCRα and TCRβ CDR sequences can each optionally have one, two, three, or four amino acid substitutions relative to the sequences listed above.

[0049] The TCRα variable region can comprise CDRs that are at least 90%, at least 95%, at least 98%, or at least 99% identical to the sequences of SEQ ID NO:3, 4, and 5 as CDR1, CDR2, and CDR3, respectively, and / or the TCRβ variable region can comprise CDRs that are at least 90%, at least 95%, at least 98%, or at least 99% identical to the sequences of SEQ ID NO:9, 10, and 11 as CDR1, CDR2, and CDR3, respectively.

[0050] The TCRα variable region can comprise CDRs corresponding to the sequences of SEQ ID NO:3, 4, and 5, and comprise FRs that are at least 90%, at least 95%, at least 98%, or at least 99% identical to the sequences of SEQ ID NO:27, 6, 7, and 28, and / or the TCRβ variable region can comprise CDRs corresponding to the sequences of SEQ ID NO:9, 10, and 11, and comprise FRs that are at least 90%, at least 95%, at least 98%, or at least 99% identical to the sequences of SEQ ID NO:29, 12, 13, and 30.

[0051] The TCRα variable region can be at least 80% identical to the sequence of SEQ ID NO:2 and the TCRβ variable region can be at least 80% identical to the sequence of SEQ ID NO:8. The TCRα variable region can be at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO:2 and the TCRβ variable region can be at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO:8. Preferably, the TCRα variable region has the sequence provided in SEQ ID NO:2 and the TCRβ variable region has the sequence provided in SEQ ID NO:8.

[0052] VC1 may comprise the TCRα or TCRβ constant region and VC2 may comprise the other of the TCRα and TCRβ constant regions. The TCRα constant region may be at least 80% identical to the sequence of SEQ ID NO:15 and the TCRβ constant region may be at least 80% identical to the sequence of SEQ ID NO:19. The TCRα constant region may be at least 90%, at least 95% or at least 98% identical to the sequence of SEQ ID NO:15 and the TCRβ constant region may be at least 90%, at least 95% or at least 98% identical to the sequence of SEQ ID NO:19. Preferably, the TCRα constant region has the sequence provided in SEQ ID NO:15 and the TCRβ constant region has the sequence provided in SEQ ID NO:19.

[0053] VC1 may comprise one of the TCRα variable and constant regions or the TCRβ variable and constant regions, and VC2 may comprise the other of the TCRα and TCRβ variable and constant regions. The TCRα variable and constant region may comprise or consist of an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO:14, and the TCRβ variable and constant region may comprise or consist of an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO:16. The TCRα variable and constant region may comprise or consist of an amino acid sequence that is at least 90%, at least 95% or at least 98% identical to the sequence of SEQ ID NO:14, and the TCRβ variable and constant region may comprise or consist of an amino acid sequence that is at least 90%, at least 95% or at least 98% identical to the sequence of SEQ ID NO:16. Preferably, the TCRα variable and constant region comprises or consists of the amino acid sequence provided in SEQ ID NO:14, and the TCRβ variable and constant region comprises or consists of the amino acid sequence provided in SEQ ID NO:16.

[0054] Those skilled in the art will appreciate that the forms of the multi-domain binding molecules of the present invention can equally be applied to TCR sequences other than the TCR sequences described above. For example, other suitable TCR chain amino acid sequences are provided in WO2011001152, WO2017109496, WO2017175006 and WO2018234319 and also, for example, in US Patent Nos. 8,519,100, 11,639,374, 11,505,590 and 11,427,624, the content of each of which is incorporated herein by reference.

[0055] As is well known in the art, post-translational modifications can be made to protein molecules. Glycosylation is one such modification, which involves the covalent attachment of an oligosaccharide moiety to specific amino acids in the TCR or antibody chain. For example, asparagine residues or serine / threonine residues are well-known sites for oligosaccharide attachment. The glycosylation state of a particular protein depends on many factors, including the protein sequence, protein conformation, and the availability of certain enzymes. In addition, the glycosylation state (i.e., the type of oligosaccharide, the covalent linkage, and the total number of attachments) can affect protein function. Therefore, controlled glycosylation is often desirable when producing recombinant proteins. Controlled glycosylation has been used to improve antibody-based therapies (Jefferis et al., (2009) Nat Rev Drug Discov Mar; 8(3):226-34.). For example, glycosylation can be controlled by using specific cell lines (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 because glycosylation can improve pharmacokinetics, reduce immunogenicity, and more closely mimic natural human proteins (Sinclair and Elliott, (2005) Pharm Sci. Aug; 94(8):1626-35). Alternatively, glycosylation can lead to a lack of consistency during production, which is undesirable for therapeutic molecules. Residues at high risk of glycosylation (such as asparagine) can be replaced with alternative amino acids (such as glutamine).

[0056] VC1 and / or VC2 can contain one or more amino acid substitutions, where one or more amino acid substitutions remove one or more glycosylation sites. In this case, the substitutions are relative to the native (e.g., wild-type) sequence or the unmodified sequence. For example:

[0057] (i) VC1 or VC2 can contain the TCRα variable and constant regions, which contain one or more amino acid substitutions at positions selected from: N24, N148, N182, and N193, numbered according to SEQ ID NO:14; and / or

[0058] (ii) The other of VC1 and VC2 can contain the TCRβ variable and constant regions, which contain an amino acid substitution at position N184, numbered according to SEQ ID NO:16. The substitution can be an Asn to Gln (i.e., N to Q) substitution. Preferably, the TCRα variable and constant regions contain the N24Q, N148Q, N182Q, and N193Q substitutions, numbered according to SEQ ID NO:14, and the TCRβ variable and constant regions contain the N184Q substitution, numbered according to SEQ ID NO:16.

[0059] The pMHC binding domain may be incompletely deglycosylated, i.e., the pMHC may retain one or more glycosylation sites from its native sequence. For example, the pMHC binding domain 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 variable region of VC1 or VC2. The single glycosylation site may be at position N18 in the TCRβ variable region, numbered according to SEQ ID NO:16. Advantageously, the inventors have identified that, compared to other glycosylation and / or deglycosylation variants, the multi-domain binding protein having this single glycosylation site has better manufacturability (e.g., protein yield, resistance to heat stress and aggregation) in addition to retaining the affinity for peptide-MHC binding and the target cell killing efficacy.

[0060] The pMHC binding domain binds to MHC complexed with a peptide antigen. The peptide antigen may be a disease-related antigen. The pMHC binding domain may bind to a tumor-associated antigen complexed with MHC. For example, the peptide antigen may be a peptide derived from GP100, NYESO, MAGEA4, or PRAME, as described in WO2011001152, WO2017109496, WO2017175006, and WO2018234319. The tumor-associated antigen may be PIWIL1. For example, the pMHC binding domain may bind to the SLSNRLYYL (SEQ ID NO:56) HLA-A*02 complex. The tumor-associated antigen may be PRAME. Preferably, the pMHC binding domain binds to the SLLQHLIGL (SEQ ID NO:1) HLA-A*02 complex.

[0061] Immune cell engagement domain

[0062] As used herein, an "immune cell engaging domain" is a protein domain capable of binding to a target on an immune cell and / or modifying an immune response, e.g., promoting or inhibiting an immune response such as T cell activation. The immune cell engaging domain is also referred to herein as an "ICE" domain.

[0063] In some embodiments, the immune cell engaging domain comprises an antibody light chain variable region (ICE-VL) and an antibody heavy chain variable region (ICE-VH). As used herein, "ICE-VL" and "ICE-VH" refer to the light chain variable region and the heavy chain variable region of the immune cell engaging domain, respectively. "ICE-VL" and "ICE-VH" may also be referred to herein as "ICEVL" and "ICEVH". Thus, the immune cell engaging domain may comprise an antigen binding site. The antibody may also be a single domain antibody ("ICE-SD"), such as the variable region of a heavy chain antibody (e.g., VHH).

[0064] The immune cell engaging domain can be a T cell engaging immune effector domain. As used herein, a "T cell engaging immune effector domain" is a protein domain capable of binding to a target on a T cell to promote an immune response. The T cell engaging immune effector domain can comprise an antibody light chain variable region (TCE-VL) and an antibody heavy chain variable region (TCE-VH). As used herein, "TCE-VL" and "TCE-VH" refer to the light chain variable region and heavy chain variable region of the T cell engaging immune effector domain. "TCE-VL" and "TCE-VH" may also be referred to herein as "TCEVL" and "TCEVH".

[0065] The T cell engaging immune effector domain can bind to a protein expressed on the cell surface of a T cell to promote T cell activation. For example, the T cell engaging immune effector domain can be a CD3 effector domain. The T cell engaging immune effector domain can bind to, for example, specifically bind to CD3 (i.e., the T cell engaging immune effector domain can be a CD3 binding protein). The T cell engaging effector can be an antibody or a functional fragment thereof, such as a single-chain variable fragment (scFv) or an antibody-like scaffold of similar size, or any other binding protein that activates T cells by interacting with CD3 and / or the TCR / CD3 complex. The antibody can also be a single-domain antibody, such as the variable region of a heavy chain antibody (e.g., VHH).

[0066] Alternatively, the immune cell engaging domain can be an immunosuppressor. As used herein, the term "immunosuppressor" refers to any molecule, e.g., a protein, capable of inhibiting an immune response, such as inhibiting T cell activation. The immunosuppressor can bind to a target (e.g., an antigen). For example, the immunosuppressor can be an immune checkpoint agonist, i.e., a molecule that induces immune checkpoint signal transduction. The immunosuppressor can comprise an antigen-binding portion capable of binding to an antigen. The antigen of the immunosuppressor can be located on an immune cell (such as a T cell). The binding molecule can comprise an antibody or an antigen-binding fragment thereof. For example, the antibody can also be a single-domain antibody, such as the variable region of a heavy chain antibody. Alternatively, the antibody can be a single-chain variable fragment (scFv), or an antibody-like scaffold of similar size, or any other binding protein that inhibits T cells by inducing immune checkpoint signal transduction. Such immunosuppressors are described below.

[0067] The immune cell engaging domain can comprise an antigen-binding portion capable of binding to an antigen. The antigen of the immune cell engaging domain can be located on an immune cell such as a T cell. The binding molecule can comprise an antibody or an antigen-binding fragment thereof. As used herein, the term “antibody” is intended to include conventional / natural antibodies and engineered antibodies, particularly functional antibody fragments, single-chain antibodies, single-domain antibodies, and bispecific or multispecific antibodies. In this context, “natural” or “conventional” refers to having the same type of domains and domain arrangements as antibodies found in nature and comprising the CDR and FR sequences from the antibody source. In a natural / conventional four-chain (e.g., human) antibody, the 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 the light chain (VL) and the heavy chain (VH) determine the binding recognition and specificity for the antigen. A conventional antibody binding site is composed of residues mainly from the “antibody complementarity determining regions” (CDRs) or hypervariable regions. Occasionally, residues from non-hypervariable or framework regions (FRs) affect the overall domain structure and thus the binding site. CDRs refer to the amino acid sequences that together define the binding affinity and specificity of the natural antibody binding site. The light chain and heavy chain of a conventional four-chain antibody each have three CDRs, named CDR1-L, CDR2-L, CDR3-L and CDR1-H, CDR2-H, CDR3-H, respectively. Thus, the conventional four-chain antibody antigen binding site includes six CDRs, which comprise the CDRs from each VH and VL.

[0068] “Engineered” antibody forms include functional antibody fragments, single-chain antibodies, single-domain antibodies, and chimeric, humanized, bispecific or multispecific antibodies. Engineered antibody forms further include constructs in which CDRs from a TCR (optionally including additional 3, 2 or 1 N- and / or C-terminal framework residues) or the entire variable domain from a TCR are grafted onto an antibody heavy or light chain. A “functional antibody fragment” refers to a portion of a full-length antibody or a protein similar to a portion of a full-length antibody that retains the ability to bind to its target antigen, particularly the antigen-binding region or variable region of a full-length antibody. Examples of functional antibody “fragments” include Fv, Fab, F(ab’)2, Fab’, dsFv, (dsFv)2, scFv, sc(Fv)2, and diabodies. For example, the binding molecule of the present invention can comprise an scFv.

[0069] The antibody can also be a single-domain antibody, such as the variable region of a heavy-chain antibody. In this regard, the term "single-domain antibody" refers to an antibody consisting of a single antibody variable domain (e.g., a heavy-chain variable domain). Thus, the immunocyte-engaging domain can comprise, for example, a VHH (i.e., the variable domain of a heavy-chain antibody). As is well known in the art, the antigen-binding site of a single-domain antibody (such as a VHH) can comprise three CDRs (as opposed to six in a conventional four-chain antibody). As used herein, the term "antigen-binding portion of an antibody" encompasses such binding sites. Alternatively or additionally, the binding molecule can comprise Fab or Fv fragments. The term "Fab" ("fragment antigen-binding") denotes the antigen-binding fragment of an antibody that comprises the antibody light chain (VL-CL) and the variable and CH1 domains of the antibody heavy chain (VH-CH1). Fab fragments typically have a molecular weight of approximately 50,000 daltons. An Fv fragment is the N-terminal portion of an antibody Fab fragment and consists of the variable portions of one light chain (VL) and one heavy chain (VH).

[0070] The immunocyte-engaging domain can comprise the variable region of the antibody heavy chain (VH) and the variable region of the antibody light chain (VL), which bind to form an antigen-binding portion capable of binding an antigen. Thus, the antigen-binding portion can comprise VH and VL. For example, the immunocyte-engaging domain can comprise a scFv containing VH and VL.

[0071] Other suitable antigen-binding portions are heavy-chain antibodies (hcAb), single-domain antibodies (sdAb), minibodies (Tramontano et al., (1994) J. Mol. Recognition 7, 9-24), variable domains of camel heavy-chain antibodies (VHH), variable domains of new antigen receptors (VNAR), affibodies (Nygren P.A. (2008) FEBS J. 275, 2668-2676), alphabodies (see WO2010066740), designed ankyrin repeat domains (DARPin) (Stumpp et al., (2008) Drug Discovery Today 13, 695-701), anticalins (anti-carrier proteins) (Skerra et al., (2008) FEBS J. 275, 2677-2683), knottins (Kolmar et al., (2008) FEBS J. 275, 2684-2690) and engineered CH2 domains (nanobodies, see Dimitrov D.S. (2009) mAbs 1, 26-28).

[0072] The antigen-binding portion can be or comprise a heavy chain variable domain, which comprises, consists of, or consists essentially of: four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively); or any suitable fragment of such a heavy chain variable domain that retains the antigen-binding site. The antigen-binding portion can be a heavy chain antibody. The antigen-binding portion can be the heavy chain variable domain sequence of an antibody derived from a conventional four-chain antibody, such as but not limited to the VH sequence derived from a human antibody. Preferably, the antigen-binding portion is or comprises the variable domain of a heavy chain antibody (e.g., a camelid antibody), such as VHH (also referred to herein as a "VHH domain"). Preferably, the antigen-binding portion is a VHH.

[0073] As described herein, the immune cell engaging domain can comprise an antigen-binding portion (e.g., an antibody antigen-binding portion) that binds to an antigen located on an immune cell. In the context of the present invention, an "immune cell" can refer to, for example, a T cell or a B cell. In particular, the antigen of the antigen-binding portion can be a T cell surface antigen.

[0074] The immune cell engaging domain can be a single-chain variable fragment (scFv). A "single-chain Fv" (also abbreviated as "sFv" or "scFv") is an antibody fragment that comprises the VH and VL antibody domains joined into a single polypeptide chain. The scFv polypeptide can also comprise a polypeptide linker between the VH and VL domains, which enables the scFv to form the required structure for antigen binding. For a review of scFv, see Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenberg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0075] The immune cell engaging domain can be a CD3 effector. CD3 effectors include, but are not limited to, anti-CD3 antibodies or antibody fragments, particularly anti-CD3 scFv or antibody-like scaffolds. The immune cell engaging domain can be a T cell engaging immune effector domain, which can be an anti-CD3 scFv. Other immune effectors include, but are not limited to, antibodies that bind to antigens on T cells, including fragments, derivatives, and variants thereof. Such antigens include CD28, 4-1BB (CD137), or CD16 or any molecule that functions at the immune synapse. Particularly preferred immune effectors are anti-CD3 antibodies or functional fragments or variants of said anti-CD3 antibodies. 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 / analogs suitable for use in the compositions and methods described herein include minibodies, Fab fragments, F(ab’) 2 fragments, dsFv, and scFv fragments.

[0076] Preferably, the immune cell engaging domain is a T cell engaging immune effector domain comprising:

[0077] (i) a VL region that comprises CDRs of SEQ ID NO: 33, 34, and 35 as CDR1, CDR2, and CDR3, respectively; and

[0078] (ii) a VH region that comprises CDRs of SEQ ID NO: 36, 37, and 38 as CDR1, CDR2, and CDR3, respectively.

[0079] Alternatively, the immune cell engaging domain can be a T cell engaging immune effector domain comprising:

[0080] (i) a VL region that comprises CDRs of SEQ ID NO: 33, 34, and 35 as CDR1, CDR2, and CDR3, respectively; and

[0081] (ii) a VH region that comprises CDRs of SEQ ID NO: 48, 37, and 38 as CDR1, CDR2, and CDR3, respectively.

[0082] The above VL and VH CDR sequences can optionally have one, two, three, or four amino acid substitutions relative to the sequences described above.

[0083] Alternatively, TCE-VL may comprise CDRs as CDR1, CDR2, and CDR3 that are at least 90%, at least 95%, at least 98%, or at least 99% identical to the sequences of SEQ ID NO:33, 34, and 35, and / or TCE-VH may comprise CDRs as CDR1, CDR2, and CDR3 that are at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO:36, 37, and 38.

[0084] Alternatively, TCE-VL may comprise CDRs as CDR1, CDR2, and CDR3 that are at least 90%, at least 95%, at least 98%, or at least 99% identical to the sequences of SEQ ID NO:33, 34, and 35, and / or TCE-VH may comprise CDRs as CDR1, CDR2, and CDR3 that are at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO:48, 37, and 38.

[0085] TCE-VL may comprise or consist of an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO:31, and TCE-VH may comprise or consist of an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO:32. TCE-VL may comprise or consist of an amino acid sequence that is at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO:31, and TCE-VH may comprise or consist of an amino acid sequence that is at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO:32. Preferably, TCE-VL comprises or consists of the amino acid sequence provided in SEQ ID NO:31 and TCE-VH comprises or consists of the amino acid sequence provided in SEQ ID NO:32.

[0086] Alternatively, TCE-VL may comprise or consist of an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO:31, and TCE-VH may comprise or consist of an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO:41. TCE-VL may comprise or consist of an amino acid sequence that is at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO:31, and TCE-VH may comprise or consist of an amino acid sequence that is at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO:41. For example, TCE-VL comprises or consists of the amino acid sequence provided in SEQ ID NO:31 and TCE-VH comprises or consists of the amino acid sequence provided in SEQ ID NO:41.

[0087] As described above, the immune cell engaging domain or the T cell engaging immune effector domain can be a scFv. The immune cell engaging domain or the T cell engaging immune effector domain can be a scFv comprising or consisting of an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 17 or 40. The scFv can comprise or consist of an amino acid sequence that is at least 90%, at least 95% or at least 98% identical to the sequence of SEQ ID NO: 17 or 40. Preferably, the scFv comprises or consists of the amino acid sequence provided in SEQ ID NO: 17. Alternatively, the scFv can comprise or consist of the amino acid sequence provided in SEQ ID NO: 40.

[0088] The immune cell engaging domain can alternatively be an immune checkpoint inhibitor. For example, the target of the immune checkpoint inhibitor can 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 known as CD276), B7-H4 (B7 homolog 4, also known as VTCN1), BTLA (B and T lymphocyte attenuator, also known as CD272), CTLA-4 (cytotoxic T lymphocyte associated protein 4, also known as 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 NADHPH oxidase subtype 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 known as CD328) and SIGLEC9 (sialic acid-binding immunoglobulin-type lectin 9, also known as CD329).

[0089] In this regard, the immune checkpoint inhibitor can be an agonist of one or more of the above immune checkpoint molecules. Thus, the immune checkpoint inhibitor can be an immune checkpoint agonist (i.e., to inhibit immune activation). Suitable immune checkpoint agonists include natural ligands and antibodies, as reviewed, for example, in Paluch et al., Front Immunol, 2018, 9:2306.

[0090] The immunosuppressor can comprise one of a receptor-ligand pair, rather than the antigen-binding portion of an antibody, and is capable of binding to the other of the receptor-ligand pair. The target ligand or receptor can be located on an immune cell. For example, the immunosuppressor can comprise the ligand of the immune checkpoint molecule described above. In particular, the immunosuppressor can comprise a portion of PD-L1 that is capable of binding to PD-1 (e.g., the soluble extracellular region). Such an immunosuppressor can engage an immune cell by binding to PD-1 and stimulating PD-1 signal transduction.

[0091] Alternatively, the immunosuppressor can comprise an agonist antibody that binds to an immune checkpoint molecule and can stimulate its signal transduction. For example, the immunosuppressor can be or comprise a PD-1 agonist antibody (e.g., a single-domain antibody). Preferably, such a PD-1 agonist does not compete with PD-L1 for binding to PD-1. The PD-1 agonist can be a full-length antibody or a fragment thereof, such as an scFv antibody or a Fab fragment, or a single-domain antibody. Examples of such antibodies are provided in WO2011110621 and WO2010029434 and WO2018024237. Thus, the antigen of the immunosuppressor can be PD-1 and the antigen-binding portion of the immunosuppressor can be a PD-1 agonist. The antigen-binding portion of the immunosuppressor can comprise a single-domain antibody, optionally a VHH. For example, the immunosuppressor can be a PD-1 agonist VHH.

[0092] As described above, the immunosuppressor can be a PD-1 agonist. As used herein, the term "PD-1 agonist" refers to any molecule capable of binding to PD-1 and activating PD-1 signal transduction, including, for example, PD-1 ligands, PD-L1, and PD-1 agonist antibodies. Activation of the PD-1 pathway downregulates immune activity, promotes immune tolerance, and prevents autoimmunity (Keir et al., Annu Rev Immunol, 26:677-704, 2008; Okazaki et al., Int Immunol 19:813-824, 2007).

[0093] Half-life extension domain

[0094] As used herein, the "half-life extension domain" refers to a protein domain for extending the half-life of a multi-domain binding protein relative to a multi-domain binding protein lacking a half-life extension domain. The half-life extension domain comprises a first IgG Fc region (FC1) and a second IgG Fc region (FC2), wherein the FC1 region and the FC2 region dimerize to form an Fc domain. As used herein, the term "Fc region" is used to refer to the region of a single polypeptide chain that comprises at least the CH2 domain and CH3 domain sequences, while the term "Fc domain" refers to the dimer of two Fc regions (i.e., FC1 and FC2).

[0095] As used herein, the term "half-life" refers to the pharmacokinetic property of a binding molecule, which is an indicator of the average residence time of the binding molecule after its administration. The half-life of a binding molecule can be expressed as, for example, the time required to eliminate 50% of a known amount of the binding molecule from the patient's body (or other mammalian) or its specific compartment as measured in serum (i.e., the circulating half-life) or other tissues.

[0096] An increase in half-life can reduce the amount of drug administered to a patient and decrease the frequency of administration. For example, for the treatment of cancer, infectious diseases or immune diseases or conditions, an increase in half-life may be advantageous. Binding molecules with increased half-lives can also be generated by modifying amino acid residues identified as being involved in the interaction between the Fc and FcRn receptors. Compared to binding molecules containing a native Fc region, binding molecules containing an Fc region comprising one or more modifications that promote binding to FcRn can 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. Compared to binding molecules containing a native Fc region, binding molecules containing an Fc region comprising one or more modifications that promote binding to FcRn can 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 between 2-fold and 10-fold, or between 5-fold and 25-fold, or between 15-fold and 50-fold.

[0097] WO 2020 / 157211 describes a method for extending the half-life by fusing a TCR-antibody CD3 fusion protein to an IgG Fc domain. The inventors have surprisingly found that the multi-domain binding molecules of the present invention retain the extended half-life provided by the Fc domain in the form disclosed in WO2020 / 157211 and, moreover, have significantly higher potency.

[0098] The immunoglobulin Fc domain can 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"), each of which has two or three heavy chain constant domains (referred to as CH2, CH3, and CH4), and optionally a hinge region. The two Fc region chains can be linked by one or more disulfide bonds within the hinge region. Fc domains from immunoglobulin subclasses IgG1, IgG2, and IgG4 bind to FcRn and undergo FcRn-mediated recycling, providing a longer circulatory half-life (3 - 4 weeks), thereby extending the half-life of the multi-domain binding molecule of the method. The interaction of IgG with FcRn is localized within the Fc region, covering portions 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 IgG1 or IgG4. For example, the Fc domain can be an IgG1 Fc domain, i.e., the FC1 and FC2 regions can be IgG1 Fc regions. The Fc domain can be derived from a human sequence.

[0099] The FC1 region can comprise or consist of an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 42 and the FC2 region can comprise or consist of an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 43. The FC1 region can comprise or consist of an amino acid sequence that is at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 42 and the FC2 region can comprise or consist of an amino acid sequence that is at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 43. Preferably, the FC1 region comprises or consists of the amino acid sequence provided in SEQ ID NO: 42 and the FC2 region comprises or consists of the amino acid sequence provided in SEQ ID NO: 43. As will be appreciated by those skilled in the art, the sequences provided above for FC1 and FC2 are equally applicable vice versa. For example, the FC1 region comprises or consists of the amino acid sequence provided in SEQ ID NO: 43 and the FC2 region comprises or consists of the amino acid sequence provided in SEQ ID NO: 42.

[0100] The Fc region can contain mutations relative to a wild-type or unmodified Fc sequence. Mutations include substitutions, insertions, and deletions. Such mutations can be made to introduce desired therapeutic properties. For example, to promote heterodimerization, knobs into holes (KiH) mutations can be engineered into the CH3 domain. Thus, the half-life extending domain can contain one or more amino acid substitutions that promote dimerization of the FC1 and FC2 regions. Such substitutions include "knobs into holes" substitutions. In this case, one chain (i.e., one of the FC1 or FC2 regions) is engineered to contain a large 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 can be constructed by replacing a small amino acid side chain with a large side chain. A hole can be constructed by replacing a large amino acid side chain with a smaller side chain. Without wishing to be bound by theory, this is thought to stabilize the heterodimer of the FC1 and FC2 regions by promoting the formation of heterodimers rather than other species (e.g., homopolymers of FC1 and FC2), thereby enhancing the stability and manufacturability of the multi-domain binding molecules of the invention.

[0101] Suitable positions and substitutions for KiH mutations, and other mutations that promote dimerization of the Fc region are well known in the art and include those described in Merchant et al., Nat Biotechnol 16:677 (1998) and Ridgway et al., ProtEngineering 9:617 (1996) and Atwell et al., J Mol Biol 270,1 (1997):26 - 35. For example, forming substitutions corresponding to knobs and holes in two Fc regions can correspond to one or more pairs provided in the table below:

[0102] CH3 of one of FC1 and FC2 regions CH3 of the other of FC1 and FC2 regions T366Y Y407T T366W Y407A T366W T366S:L368A:Y407V F405A T394W Y407T T366Y T366Y:F405A T394W:Y407T T366W:F405W T394S:Y407A F405W:Y407A T366W:T394S F405W T394S

[0103] The substitutions in the table above are represented as follows: the original residue, followed by the position using the EU numbering system and then the introduced residue (all residues are given in single-letter amino acid code). Multiple substitutions are separated by colons.

[0104] The FC1 and FC2 regions can contain one or more of the substitutions in the table above. For example:

[0105] (i) One of the FC1 and FC2 regions can contain one or more amino acid substitutions selected from: T366S, L368A, T394S, F405A, Y407A, Y407T, and Y407V, according to the EU numbering scheme; and

[0106] (ii) Another one of the FC1 region and the FC2 region may contain one or more amino acid substitutions selected from the following: T366W, T366Y, T366W, T394W, and F405W, according to the EU numbering scheme. The substitutions in (i) and (ii) are substitutions in the pestle form and the mortar form, respectively. The FC1 region may contain one or more substitutions in (i) and the FC2 region may contain one or more substitutions in (ii).

[0107] For example:

[0108] (i) One of the FC1 region and the FC2 region may contain one or more amino acid substitutions selected from the following: T366S, L368A, and Y407V, according to the EU numbering scheme; and

[0109] (ii) Another one of the FC1 region and the FC2 region may contain the T366W amino acid substitution, according to the EU numbering scheme. The FC1 region may contain one or more substitutions in (i) and the FC2 region may contain the substitution in (ii).

[0110] Preferably, (i) one of the FC1 region and the FC2 region contains the T366S, L368A, and Y407V amino acid substitutions, according to the EU numbering scheme; and (ii) another one of the FC1 region and the FC2 region contains the T366W amino acid substitution, according to the EU numbering scheme. For example, the FC1 region may contain the T366S, L368A, and Y407V amino acid substitutions, according to the EU numbering scheme; and the FC2 region may contain the T336W amino acid substitution, according to the EU numbering scheme.

[0111] The Fc domain may also comprise one or more mutations that attenuate the effector function of the Fc domain. Exemplary effector functions include, but are not limited to, complement-dependent cytotoxicity (CDC) and / or antibody-dependent cytotoxicity (ADCC). Modifications that attenuate effector function may be modifications that alter the glycosylation pattern of the Fc domain, e.g., modifications that result in an aglycosylated Fc domain. Alternatively, modifications that attenuate effector function may be modifications that do not alter the glycosylation pattern of the Fc domain. Modifications that attenuate effector function may be those that reduce or eliminate binding to human effector cells, binding to one or more Fc receptors, and / or binding to cells expressing Fc receptors. For example, the half-life extension domain may comprise one or more amino acid substitutions selected from: S228P, E233P, L234A, L235A, L235E, L235P, G236R, G237A, P238S, F241A, V264A, D265A, H268A, D270A, N297A, N297G, N297Q, E318A, K322A, L328R, P329G, P329A, A330S, A330L, P331A, and P331S, according to the EU numbering scheme. Specific modifications include N297G or N297A substitutions (EU numbering) in the Fc region of human IgG1. Other suitable modifications include L234A, L235A, and P329G substitutions (EU numbering) in the Fc region of human IgG1, which result in attenuation of effector function. The Fc region in the multi-domain binding molecule of the present invention may comprise a substitution at residue N297 according to the EU index numbering. 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.

[0112] An Fc variant with reduced effector function refers to an Fc variant in which the effector function (e.g., activities such as CDC, ADCC, and / or binding to FcR) is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or more compared to the effector function achieved by a wild-type Fc region (e.g., an Fc region that does not have mutations that reduce effector function, but may have other mutations). An Fc variant with reduced effector function may be an Fc variant that eliminates all detectable effector function compared to the wild-type Fc region. Assays for measuring effector function are well known in the art and are described below.

[0113] In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / depletion of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the Fc region or fusion protein lacks FcγR binding (and thus may lack ADCC activity), but retains the ability to bind to FcRn. The primary cell NK cells that mediate ADCC only express FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. The FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for evaluating the ADCC activity of a target molecule are described in U.S. Patent No. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Nat’l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat’l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)).

[0114] Substitutions that eliminate or reduce binding to Fcy receptors and / or increase binding to FcRn, and / or prevent Fab arm exchange and / or remove protease sites can be introduced into the FC1 and FC2 regions. In this regard, the half-life extending domain can also include one or more amino acid substitutions that prevent or reduce binding to activating receptors. The half-life extending domain can include one or more amino acid substitutions that prevent or reduce binding to FcγR. For example, the FC1 region and / or the FC2 region can include the N297G amino acid substitution, according to the EU numbering scheme. Both the FC1 region and the FC2 region can include the N297G amino acid substitution.

[0115] Compared to an unmodified half-life extending domain, the half-life extending domain can comprise one or more amino acid substitutions, wherein the one or more amino acid substitutions promote the binding of the Fc domain to FcRn. Methods for measuring binding to FcRn are well known (see, e.g., Ghetie and Ward, Immunol. Today 18:(12):592-8 (1997); Ghetie et al., Nature Biotechnology 15(7):637-40 (1997); Hinton et al., J. Biol. Chem. 279(8):6213-6 (2004); WO 2004 / 92219 (Hinton et al.)). For example, binding to FcRn in vivo and the serum half-life of a polypeptide with a variant Fc region can be determined in transgenic mice expressing human FcRn or transfected human cell lines, or in primates administered a polypeptide with a variant Fc region. WO 2004 / 42072 (Presta) describes antibody substitutions that increase or decrease binding to FcR. See also, e.g., Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001). In particular, Mackness et al., MAbs. 11:1276-1288 (2019) describe suitable amino acid substitutions in the Fc region of an antibody for enhancing binding to FcRn.

[0116] Modifications (e.g., amino acid substitutions, amino acid insertions, or amino acid deletions) in the Fc region that promote the binding of the Fc domain to FcRn can be at one or more positions selected from: 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 247, 251, 252, 254, 255, 256, 262, 263, 264, 265, 266, 267, 268, 269, 279, 280, 284, 292, 296, 297, 298, 299, 305, 313, 316, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 339, 341, 343, 370, 373, 378, 392, 416, 419, 421, 440, and 443, as shown by the EU index numbering in Kabat. Optionally, the Fc region can contain non-naturally occurring amino acid residues at other and / or alternative positions well known in the art.

[0117] More specifically, the Fc region may contain at least one substitution selected from the following: 234D, 234E, 234N, 234Q, 234T, 234H, 234Y, 234I, 234V, 234F, 235A, 235D, 235R, 235W, 235P, 235S, 235N, 235Q, 235T, 235H, 235Y, 235I, 235V, 235F, 236E, 239D, 239E, 239N, 239Q, 239F, 239T, 239H, 239Y, 240I, 240A, 240T, 240M, 241W, 241L, 241Y, 241E, 241R.243W, 243L, 243Y, 243R, 243Q, 244H, 245A, 247L, 247V, 247G, 251F, 252Y, 254T, 255L, 256E, 256M, 262I, 262A, 262T, 262E, 263I, 263A, 263T, 263M, 264L, 264I, 264W, 264T, 264R, 264F, 264M, 264Y, 264E, 265G, 265N, 265Q, 265Y, 265F, 265V, 265I, 265L, 265H, 265T, 266I, 266A, 266T, 266M, 267Q, 267L, 268E, 269H, 269Y, 269F, 269R, 270E, 280A, 284M, 292P, 292L, 296E, 296Q, 296D, 296N, 296S, 296T, 296L, 2961, 296H, 269G, 297S, 297D, 297E, 298H, 298I, 298T, 298F, 299I, 299L, 299A, 299S, 299V, 299H, 299F, 299E, 305I, 313F, 316D, 325Q, 325L, 325I, 325D, 325E, 325A, 325T, 325V, 325H, 327G, 327W, 327N, 327L, 328S, 328M, 328D, 328E, 328N, 328Q, 328F, 328I, 328V, 328T, 328H, 328A, 329F, 329H, 329Q, 330K, 330G, 330T, 330C, 330L, 330Y, 330V, 330I, 330F, 330R, 330H, 331G, 331A, 331L, 331M, 331F, 331W, 331K, 331Q, 331E, 331S, 331V, 331I, 331C, 331Y, 331H, 331R, 331N, 331D, 331T, 332D, 332S, 332W, 332F, 332E, 332N, 332Q, 332T, 332H, 332Y, 332A, 339T, 370E, 370N, 378D, 392T, 396L, 416G, 419H, 421K, 440Y and 434W, as by the EU index numbers shown in Kabat. Optionally, the Fc region may contain non-naturally occurring amino acid residues at other and / or alternative positions known in the art.

[0118] Modifications in the Fc region that promote binding to FcRn (e.g., amino acid substitutions, amino acid insertions, or amino acid deletions) can be at one or more positions selected from: 234, 235, and 331, as numbered by the EU index as shown in Kabat. For example, the Fc region can contain at least one substitution selected from: 234F, 235F, 235Y, and 331S, as numbered by the EU index as shown in Kabat.

[0119] Modifications in the Fc region that promote binding to FcRn (e.g., amino acid substitutions, amino acid insertions, or amino acid deletions) can be at one or more positions selected from: 239, 330, and 332, as numbered by the EU index as shown in Kabat. For example, the Fc region can contain at least one substitution selected from: 239D, 330L, and 332E, as numbered by the EU index as shown in Kabat.

[0120] Modifications in the Fc region that promote binding to FcRn (e.g., amino acid substitutions, amino acid insertions, or amino acid deletions) can be at one or more positions selected from: 252, 254, and 256, as numbered by the EU index as shown in Kabat. For example, the Fc region can contain at least one substitution selected from: 252Y, 254T, and 256E, as numbered by the EU index as shown in Kabat, as described in U.S. Patent No. 7,083,784, the entire content of which is incorporated herein by reference. The Fc region can contain all of the following substitutions: 252Y, 254T, and 256E, as numbered by the EU index as shown in Kabat.

[0121] The substitutions that promote binding to FcRn listed above are relative to the corresponding wild-type Fc region (e.g., human IgG1 or IgG4 Fc region) and can be present in one or preferably both of the FC1 and FC2 portions of the Fc domain. In other words, a substitution refers to an amino acid that is not normally present in the corresponding wild-type Fc region (e.g., human IgG1 or IgG4 Fc region). In this regard, as used herein, a "substitution" means the presence of a listed amino acid in a polypeptide and does not necessarily require the replacement of one amino acid with another. In one embodiment, the FC1 and / or FC2 region contains the 252Y, 254T, and 256E amino acid substitutions (numbered according to the EU numbering scheme).

[0122] Additionally or alternatively, mutations can be made for preparative reasons, e.g., to remove or replace amino acids that can undergo post-translational modifications such as glycosylation as described herein. The immunoglobulin Fc can be fused to other domains (i.e., VC1 or VC2) in the molecule of the invention via a linker and / or hinge sequence as described herein. Alternatively, a linker can be omitted.

[0123] The FC1 region can comprise or consist of an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO:57 and the FC region can comprise or consist of an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO:58. The FC1 region can comprise or consist of an amino acid sequence that is at least 90%, at least 95% or at least 98% identical to the sequence of SEQ ID NO:57 and the FC region can comprise or consist of an amino acid sequence that is at least 90%, at least 95% or at least 98% identical to the sequence of SEQ ID NO:58. The FC1 region can comprise or consist of the amino acid sequence provided in SEQ ID NO:57, and the FC2 region can comprise or consist of the amino acid sequence provided in SEQ ID NO:58. As will be appreciated by those skilled in the art, the sequences provided above for FC1 and FC2 are also suitable vice versa. For example, the FC1 region can comprise or consist of the amino acid sequence provided in SEQ ID NO:57, and the FC2 region can comprise or consist of the amino acid sequence provided in SEQ ID NO:58.

[0124] The two Fc regions in the molecules of the present invention can comprise all or part of the CH2 and CH3 constant domains and the hinge sequence. The hinge sequence can substantially or partially correspond to the hinge region from IgG1, IgG2, IgG3 or IgG4. The hinge sequence can be an IgG1 hinge sequence, such as the amino acid sequence provided in SEQ ID NO:44. The hinge can comprise all or part of the core hinge domain and all or part of the lower hinge region.

[0125] Suitable half-life extended forms of the multi-domain binding molecules of the present invention are also described in the application filed together therewith, the invention name of which is "Multi-domain binding molecules", which claims the priority of US Provisional Application No. 63 / 371,863 filed on August 18, 2022, the content of which is incorporated herein by reference.

[0126] Form and linker

[0127] As used herein, the term "form" refers to the position and orientation of each domain (and each region within each domain) in the multi-domain binding molecules of the present invention, as well as the number of polypeptide chains. In Figure 1 a schematic diagram of the form of an exemplary multi-domain binding molecule is provided. In Figure 1The immunocyte-engaging domain in the exemplary binding molecule shown in [Figure 0] is a T cell-engaging immune effector domain comprising VH (TCE-VH) and VL (TCE-VL). Other types of immunocyte-engaging domains (e.g., single-domain antibodies, VHHs, etc.) are also suitable, as described above herein. The pMHC-binding domain and the immunocyte-engaging domain of such molecules are capable of binding to the pMHC complex and immunocytes, respectively. In this regard, the pMHC-binding domain and the immunocyte-engaging domain may be capable of binding to the pMHC complex and immunocytes simultaneously, respectively.

[0128] In the form of the multi-domain binding molecule of the present invention, the immunocyte-engaging domain is linked to the N-terminus of VC1, VC1 is linked to the N-terminus of the FC1 region by its C-terminus, the FC1 region is linked to the N-terminus of VC2 by its C-terminus, and VC2 is linked to the N-terminus of FC2 by its C-terminus. Each region is covalently linked in a single polypeptide chain. The form can be represented as: N-ICE-VC1-FC1-VC2-FC2-C. The inventors have identified that, among more than 35 different forms tested, the molecule of this form has the highest activity (i.e., potency and selectivity) and yield.

[0129] When the immunocyte-engaging domain is a T cell-engaging immune effector domain comprising VH and VL, the form can be represented as: N-(TCEVL-TCEVH or TCEVH-TCEVL)-VC1-FC1-VC2-FC2-C. The inventors have identified that, among more than 35 different forms tested, the molecule of this form has the highest activity (i.e., potency and selectivity) and yield.

[0130] The multi-domain binding molecule of the present invention is in a single-chain form. In this context, "single-chain" is used to describe a multi-domain binding molecule represented by a single polypeptide chain, which comprises a pMHC-binding domain, an immunocyte-engaging domain, and a half-life extending domain.

[0131] Preferably, VC1 comprises the variable and constant regions of TCRβ, VC2 comprises the variable and constant regions of TCRα, the immunocyte-engaging domain is an anti-CD3 scFv, and the Fc domain is an IgG1 Fc domain.

[0132] Two or more of the ICE, TCE-VH, TCE-VL, VC1, VC2, FC1, and / or FC2 regions can be connected to each other via a linker and / or an IgG hinge sequence. The linker sequence can be flexible as it is mainly composed of amino acids such as glycine, alanine, and serine, which do not have bulky side chains that may limit flexibility. Such linkers include "glycine-serine" linkers, which refer to linkers that contain only or mainly glycine and serine residues, such as (GGGGS)n. Alternatively, a linker with greater rigidity may be desired. Examples of more rigid linkers include α-helical form linkers with the (EAAAK)n sequence. The usable or optimal length of the linker sequence can be easily determined. Generally, the length of the linker sequence will be less than about 15, such as less than 10, or from 2 to 10 amino acids. The length of the linker can 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. Examples of suitable linkers that can be used in multi-domain binding molecules are well known in the art and include those described in WO2010 / 133828 and Chen et al., Adv Drug Deliv Rev. 2013;65(10):1357-1369. For example, the linker present in the multi-domain binding protein of the present invention can have a sequence selected from the following: GGGGS (SEQ ID NO:18), GGGSG (SEQ ID NO:20), GGSGG (SEQ ID NO:21), GSGGG (SEQ ID NO:22), GSGGGP (SEQ ID NO:23), GGEPS (SEQ ID NO:24), GGEGGGP (SEQ ID NO:25), GGEGGGSEGGGS (SEQ ID NO:26), GGGSGGGG (SEQ ID NO:47), GGGGSGGGGSGGGGSGGGGSGGGS (SEQ ID NO:39), GGGGSGGGGSGGGGS (SEQ ID NO:49), EAAAK (SEQ ID NO:50), and EAAAKEAAAKEAAAK (SEQ ID NO:51).

[0133] Suitable IgG hinge sequences are well known in the art and include the exemplary IgG1 hinge sequence provided in SEQ ID NO:44. Other suitable IgG hinge sequences include the truncated IgG1 hinge sequence provided in SEQ ID NO:52 and the IgG4 hinge provided in SEQ ID NO:53.

[0134] The ICE domain can be linked to the N-terminus of VC1 via its C-terminus. In this regard, the multi-domain binding molecule of the present invention can have the following form: N-ICE-VC1-FC1-VC2-FC2-C. When the immune cell engaging domain is a T cell engaging immune effector domain comprising VH and VL, the TCE-VL region can be linked to the N-terminus of the TCE-VH region via its C-terminus, and the TCE-VH region can be linked to the N-terminus of VC1 via its C-terminus. In this regard, the multi-domain binding molecule of the present invention can have the following form: N-TCEVL-TCEVH-VC1-FC1-VC2-FC2-C.

[0135] VC1 can comprise the variable and constant regions of TCRβ, and VC2 can comprise the variable and constant regions of TCRα. Thus, VC1 and VC2 can dimerize to form a soluble TCR. In this regard, preferably, the multi-domain binding molecule of the present invention has the following form: N-ICE-TCRβ-FC1-TCRα-FC2-C or N-TCEVL-TCEVH-TCRβ-FC1-TCRα-FC2-C (where "TCRβ" refers to the variable and constant regions of TCRβ, and "TCRα" refers to the variable and constant regions of TCRα).

[0136] If present, the TCE-VL region can be linked to the TCE-VH region via a sequence comprising a glycine-serine linker. Preferably, the sequence linking the TCE-VL region to the TCE-VH region is the amino acid sequence provided in SEQ ID NO:39.

[0137] The ICE domain or the TCE-VH region can be linked to the VC1 region via a sequence comprising or consisting of a glycine-serine linker. Preferably, the sequence linking the ICE domain or the TCE-VH region to VC1 is the amino acid sequence provided in SEQ ID NO:18.

[0138] VC1 can be linked to the FC1 region via a sequence comprising an IgG hinge sequence and / or VC2 can be linked to the FC2 region via a sequence comprising an IgG hinge sequence. The IgG hinge sequence can be at least 80% identical to SEQ ID NO:44. Preferably, the IgG hinge sequence is at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO:44.

[0139] The sequence connecting VC1 to the FC1 region may further comprise a glycine-serine linker and / or the sequence connecting VC2 to the FC2 region may further comprise a glycine-serine linker. Preferably, the glycine-serine linker has the sequence provided in SEQ ID NO:47. Preferably, these sequences are in the form of VC1-GS linker-IgG hinge-FC1 and VC2-GS linker-IgG hinge-FC2 from the N-terminus to the C-terminus.

[0140] The FC1 region may be connected to VC2 by a sequence comprising a glycine-serine linker. Preferably, the glycine-serine linker connecting the FC1 region to the VC2 region has the sequence provided in SEQ ID NO:47.

[0141] The multi-domain binding molecule of the present invention is a single polypeptide chain (see Figure 1 ). The multi-domain binding molecule may be soluble and / or recombinant and / or isolated. The complete amino acid sequences of two exemplary multi-domain binding molecules are provided in SEQ ID NO:45 and SEQ ID NO:46.

[0142] The multi-domain binding molecule may have an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO:45. The multi-domain binding molecule may have an amino acid sequence that is at least 90%, at least 95% or at least 98% identical to the sequence of SEQ ID NO:45. Preferably, the multi-domain binding molecule comprises or consists of the amino acid sequence provided in SEQ ID NO:45.

[0143] The multi-domain binding molecule may have an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO:46. The multi-domain binding molecule may have an amino acid sequence that is at least 90%, at least 95% or at least 98% identical to the sequence of SEQ ID NO:46. Preferably, the multi-domain binding molecule may comprise or consist of the amino acid sequence provided in SEQ ID NO:46.

[0144] Optionally, the above multi-domain binding molecule sequence may be further fused with one or more other polypeptide sequences.

[0145] The above sequence relates to a multi-domain binding molecule comprising a TCR chain that binds to the HLA-A*02 complex with SLLQHLIGL (SEQ ID NO:1). Those skilled in the art can make these sequences suitable for another target by substituting the TCR chain sequences in SEQ ID NO:45 and SEQ ID NO:46 with the sequences of different TCRs of interest. Similarly, those skilled in the art can substitute the anti-CD3 scFv sequences (i.e., the T cell-engaging immune effector domain) in SEQ ID NO:45 and SEQ ID NO:46 with another T cell-engaging immune effector domain (e.g., a different anti-CD3 scFv sequence).

[0146] Preferably:

[0147] a) VC1 comprises the TCRβ variable and constant regions,

[0148] b) VC2 comprises the TCRα variable and constant regions,

[0149] c) The immune cell-engaging domain is a T cell-engaging immune effector domain, which is an anti-CD3 scFv,

[0150] d) FC1 has the amino acid sequence provided in SEQ ID NO:42, or an amino acid sequence that is at least 90%, or at least 95%, or at least 98% identical thereto, and

[0151] e) FC2 has the amino acid sequence provided in SEQ ID NO:43, or an amino acid sequence that is at least 90%, or at least 95%, or at least 98% identical thereto.

[0152] The multi-domain binding molecule preferably comprises the following amino acid sequence, in the order from the N-terminus to the C-terminus:

[0153] a) The amino acid sequence of the anti-CD3 scFv (TCE-VL and TCE-VH), optionally followed by the linker sequence provided in SEQ ID NO:18;

[0154] b) The amino acid sequence of the TCRβ variable and constant regions (VC1);

[0155] c) The linker sequence provided in SEQ ID NO:47, followed by the IgG hinge sequence provided in SEQ ID NO:44;

[0156] d) The Fc region (FC1) having the sequence provided in SEQ ID NO:42;

[0157] e) The linker sequence provided in SEQ ID NO:47;

[0158] f) Amino acid sequence of the TCRα variable and constant regions (VC2);

[0159] g) The linker sequence provided in SEQ ID NO:47, followed by the IgG hinge sequence provided in SEQ ID NO:44; and

[0160] h) An Fc region (FC2) having the sequence provided in SEQ ID NO:43.

[0161] The TCRβ constant region may have the amino acid sequence provided in SEQ ID NO:19 and / or the TCRα constant region may have the amino acid sequence provided in SEQ ID NO:15. The multi-domain binding molecule may not contain amino acid sequences other than those in a) to h) above.

[0162] The anti-CD3 scFv may comprise or consist of the amino acid sequence provided in SEQ ID NO:17 or the amino acid sequence provided in SEQ ID NO:40.

[0163] Amino acid sequence

[0164] Within the scope of the present invention are phenotypically silent variants of any of the molecules disclosed herein. As used herein, the term "phenotypically silent variant" is understood to refer to a variant that, in addition to those described above, contains one or more other amino acid changes, including substitutions, insertions, and deletions, and that has a similar phenotype to the corresponding molecule without such changes. For the purposes of this application, phenotype includes binding affinity (K D and / or binding half-life) and specificity. The phenotype of the soluble multi-domain binding molecule may include immunostimulatory potency and purification yield, as well as binding affinity and specificity.

[0165] Phenotypically silent variants may contain one or more conservative substitutions and / or one or more tolerated substitutions. Tolerated substitutions refer to substitutions that are not within the conservative definitions described below but are phenotypically silent. Those skilled in the art know that various amino acids have similar properties and are thus "conservative". One or more such amino acids of a protein, polypeptide, or peptide can generally be replaced by one or more other such amino acids without eliminating the desired activity of the protein, polypeptide, or peptide.

[0166] Thus, the amino acids glycine, alanine, valine, leucine, and isoleucine can generally be substituted for one another (amino acids with aliphatic side chains). Among these possible substitutions, preferably, glycine and alanine are used to substitute for one another (because they have relatively short side chains), and valine, leucine, and isoleucine are used to substitute for one another (because they have larger hydrophobic aliphatic side chains). Other amino acids that can generally be substituted for one another include: phenylalanine, tyrosine, and tryptophan (amino acids with aromatic side chains); lysine, arginine, and histidine (amino acids with basic side chains); aspartic acid and glutamic acid (amino acids with acidic side chains); asparagine and glutamine (amino acids with amide side chains); and cysteine and methionine (amino acids with sulfur-containing side chains). It should be appreciated that amino acid substitutions within the scope of the present invention can be prepared using either naturally occurring or non-naturally occurring amino acids. For example, the present concept contemplates substituting the methyl group on alanine with an ethyl group, and / or making minor alterations to the peptide backbone. Whether natural or synthetic amino acids are used, preferably only L-amino acids are present.

[0167] Substitutions of this nature are generally referred to as "conservative" or "semi-conservative" amino acid substitutions. Thus, the present invention extends to the use of specific binding molecules comprising any of the above amino acid sequences, but having one or more conservative substitutions and / or one or more tolerated substitutions in such sequences, such that the amino acid sequence of the molecule or any of its domains or regions has at least 90% identity with the sequences disclosed herein, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

[0168] "Identity", as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences 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 matching between strings of such sequences. While there are many methods for measuring identity between two polypeptide sequences or two polynucleotide sequences, the methods commonly used to determine identity are encoded in computer programs. Preferred computer programs for determining identity between two sequences include, but are not limited to, the GCG program package (Devereux et al., Nucleic Acids Research, 12, 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul et al., J. Molec. Biol. 215, 403 (1990)).

[0169] Programs such as the CLUSTAL program can be used to compare amino acid sequences. The program compares amino acid sequences and finds the optimal alignment by inserting spaces in either sequence as appropriate. Amino acid identity or similarity (identity plus conservation of amino acid type) can be calculated for the optimal alignment. Programs such as BLASTx align the longest stretches of similar sequences and assign a score to the fit. Thus, a comparison can be obtained where several similar regions are found, each with a different score. Two types of identity analysis are considered in the present invention.

[0170] The percent identity between two amino acid sequences or two nucleic acid sequences is determined by aligning the sequences for optimal comparison (e.g., introducing gaps in the first sequence to best align with the sequence) and comparing the amino acid residues or nucleotides at the corresponding positions. "Optimal alignment" is the alignment of two sequences that gives 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 × 100).

[0171] The determination of percent identity between two sequences can be accomplished using mathematical algorithms known to those of skill in the art. An example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, which was improved in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. The BLASTn and BLASTp programs of Altschul et al., (1990) J. Mol. Biol. 215:403-410 have incorporated this algorithm. The BLASTn program can be used to determine the percent identity between two nucleotide sequences. The BLASTp program can be used to determine the percent identity between two protein sequences. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast can be used to perform an iterative search to detect distant relationships between molecules (Id.). When using the 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 can include, for example, font size = 3, expectation threshold = 10. Parameters can be selected to automatically adjust for short input sequences. Another example of a mathematical algorithm for sequence comparison is the algorithm of Myers and Miller, CABIOS (1989). The ALIGN program (version 2.0), which is part of the CGC sequence alignment software package, has incorporated this algorithm. Other algorithms for sequence analysis known in the art include ADVANCE and ADAM as described in Torellis and Robotti (1994) Comput. Appl. Biosci., 10:3-5; and FASTA as described in Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-8. In FASTA, ktup is a control option that sets the sensitivity and speed of the search. For the purpose of evaluating percent identity in the present invention, BLASTp with default parameters is used as the comparison method. Additionally, when the percent identity provides a non-integer value for amino acids (i.e., a sequence of 25 amino acids with 90% sequence identity provides a value of "22.5"), the resulting value is rounded down to the next integer, i.e., "22").Thus, in the provided example, a sequence having 22 matches out of 25 amino acids is within 90% sequence identity.

[0172] It will be apparent to those skilled in the art that the sequences provided at its C-terminus and / or N-terminus can be truncated or extended by 1, 2, 3, 4, 5 or more residues, with substantially no effect on the functional characteristics of the molecule, such as the TCR moiety. The sequences provided at its C-terminus and / or N-terminus can be truncated or extended by 1, 2, 3, 4 or 5 residues. The present invention encompasses all such variants.

[0173] Mutations (including conservative and tolerated substitutions, insertions and deletions) can be introduced into the provided sequences using any suitable method, including but not limited to methods based on polymerase chain reaction (PCR), restriction enzyme-based cloning or ligation-independent cloning (LIC) steps. These methods are described in detail in many standard molecular biology texts. For more details on polymerase chain reaction (PCR) and restriction enzyme-based cloning, see Sambrook & Russell, (2001) Molecular Cloning – A Laboratory Manual (3rd Edition) CSHL Press. More information on ligation-independent cloning (LIC) steps can be found in Rashtchian, (1995) Curr Opin Biotechnol 6(1):30-6. The protein sequences provided herein can be obtained from recombinant expression, solid-phase synthesis or any other suitable method known in the art.

[0174] Evaluating the binding characteristics and activities of multi-domain binding molecules

[0175] Methods for determining binding affinity (inversely proportional to the equilibrium constant K D and binding half-life (expressed as T 1 / 2 )) are known to those skilled in the art. Binding affinity and binding half-life are determined using surface plasmon resonance (SPR) or biolayer interferometry (BLI), respectively, for example using a BIAcore instrument or an Octet instrument, respectively. It will be appreciated that doubling the affinity of a specific binding molecule results in a halving of K D . T off is calculated according to ln2 divided by the dissociation rate (k 1 / 2 ). Thus, doubling T 1 / 2 results in a halving of koff. The K D and k offValues are typically measured on soluble forms of the TCR, i.e., those forms that are truncated to remove cytoplasmic and transmembrane domain residues. To account for variation between independent measurements, particularly for interactions with dissociation times exceeding 20 hours, the binding affinity and / or binding half-life of a given specific binding molecule can be measured several times (e.g., 3 or more times) using the same assay protocol, and the 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 preferred to make the measurements using the same assay conditions (e.g., temperature). The measurement methods described for the TCR can also be applied to the multi-domain binding molecules described herein.

[0176] Certain multi-domain binding molecules of the invention are capable of generating potent T cell responses in vitro against antigen-positive cells, particularly those cells presenting low levels of typical cancer cell antigens (i.e., about 5 - 100, such as 50 antigens / cell (Bossi et al., (2013) Oncoimmunol. 1; 2(11):e26840; Purbhoo et al., (2006). J Immunol 176(12):7308 - 7316.)). Such TCRs can be adapted to be introduced into the multi-domain binding molecules described herein. The T cell response measured can be the release of T cell activation markers such as interferon γ or granzyme B, or target cell killing, or other indicators of T cell activation such as T cell proliferation. A potent response can be an EC 50 value in the nM - pM range, such as 500 nM or lower, preferably 1 nM or lower, or 500 pM or lower.

[0177] Alternatively, certain binding molecules of the invention can generate potent anti-inflammatory responses, such as CD8+ cell killing and / or CD4+ inflammation inhibition. Such binding molecules can be in soluble form and can comprise an immune cell engaging domain that is an immunosuppressor, such as a PD-1 agonist or an interleukin or cytokine, such as IL-2, IL-4, IL-10 or IL-13. The anti-inflammatory response measured can be CD8+ cell killing and / or CD4+ inflammation inhibition, and / or inhibition of the CD8+ T cell signal transduction pathway. Suitable methods for assessing anti-inflammatory responses will be well known in the art and include the Jurkat NFAT cell reporter assay. Preferably, a potent response is an IC 50 value in the pM range, i.e., 1000 pM or lower. Preferably, the maximum inhibition obtained in the reporter assay is greater than 50%, such as 80% or higher.

[0178] The molecules covered by the present invention can have an improved half-life. Methods for determining whether a protein has an improved half-life will be apparent to those skilled in the art. For example, the ability of the protein to bind to the neonatal Fc receptor (FcRn) is evaluated. In this regard, an increased binding affinity for FcRn increases the serum half-life of the protein (see, for example, Kim et al., Eur J Immunol., 24:2429, 1994).

[0179] The protein half-life of the present disclosure can also be measured by pharmacokinetic studies, for example, according to the method described by Kim et al., Eur J of Immunol 24:542, 1994. According to this method, a radiolabeled protein is injected intravenously into mice, and its plasma concentration is measured regularly as a function of time, for example, from 3 minutes to 72 hours after injection. Alternatively, an unlabeled protein of the present disclosure can be injected and its plasma concentration measured regularly using ELISA. The clearance curve obtained thereby should be biphasic, i.e., the α-phase and the β-phase. To determine the in vivo half-life of the protein, the clearance rate in the β-phase is calculated and compared with that of the wild-type or unmodified protein.

[0180] Nucleic acids, vectors and host cells

[0181] The present invention provides a nucleic acid encoding the multi-domain binding protein of the present invention. The nucleic acid can be cDNA. The nucleic acid can be mRNA. The nucleic acid can be non-naturally occurring and / or purified and / or engineered. The nucleic acid sequence can be codon-optimized according to the expression system used. As is well known to those skilled in the art, the expression system can include bacterial cells, such as Escherichia coli, or yeast cells, or mammalian cells, or insect cells, or it can be a cell-free expression system.

[0182] The present invention also provides constructs in the form of plasmids, vectors, transcription or expression cassettes comprising at least one nucleic acid as described above. The present invention also provides a recombinant host cell comprising one or more constructs as described above. As mentioned, the nucleic acid encoding the specific binding molecule of the present invention forms one aspect of the present invention, and the method for preparing the specific binding molecule including the nucleotide expression encoding the specific binding molecule of the present invention also forms the present invention. Expression can be conveniently achieved by culturing the recombinant host cell containing the nucleic acid under appropriate conditions. After production by expression, the specific binding molecule can be isolated and / or purified using any suitable technique and then used as appropriate.

[0183] Systems for polypeptide cloning and expression in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, yeast, and baculovirus systems. Mammalian cell lines that can be used for expressing heterologous polypeptides in the art include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, NSO mouse melanoma cells, and many others. A commonly preferred bacterial host is Escherichia coli. Expression of antibodies and antibody fragments in prokaryotic cells such as E. coli has been well established in the art. For a review, see, e.g., Plückthun, Bio / Technology 9:545-551 (1991). Those skilled in the art can also choose to express in cultured eukaryotic cells as an option for producing specific binding molecules. See recent reviews, e.g., Reff, Curr. Opinion Biotech. 4:573-576 (1993); Trill et al., Curr. Opinion Biotech. 6:553-560 (1995).

[0184] Suitable vectors containing appropriate regulatory sequences can be selected or constructed, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes, and other sequences as needed. The vector can be any suitable vector known in the art, including plasmid or viral vectors as needed (e.g., "phage" or "phagemid"). For other details, see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual: 2nd Edition, Cold Spring Harbor Laboratory Press (1989). Many known techniques and protocols for manipulating nucleic acids, such as in nucleic acid construct preparation, mutagenesis, sequencing, introducing DNA into cells and gene expression, and protein analysis, are described in detail in Ausubel et al., Short Protocols in Molecular Biology, 2nd Edition, John Wiley & Sons (1992).

[0185] The present invention also provides a host cell containing the nucleic acid as described herein. In addition, the present invention provides a method that includes introducing such nucleic acid into a host cell. The introduction can be carried out using any available technique. For eukaryotic cells, suitable techniques can include calcium phosphate transfection, DEAE-dextran, electroporation, liposome-mediated transfection, and transduction using retroviruses or other viruses, such as vaccinia or baculovirus for insect cells. For bacterial cells, suitable techniques can include calcium chloride transformation, electroporation, and transfection using phage. After introduction, expression can then be induced or allowed from the nucleic acid, e.g., by culturing the host cell under conditions for gene expression.

[0186] Suitable host cells for the cloning or expression of the polynucleotides and / or vectors of the present invention are well known in the art. Suitable host cells for the expression of (glycosylated) proteins are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. A number of baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for the transfection of Spodoptera frugiperda cells. Plant cell cultures can also be used as hosts. See, for example, US 5,959,177, US 6,040,498, US 6,420,548, US 7,125,978, and US 6,417,429 (describing PLANTIBODIES for the production of antibodies in transgenic plants). TM(techniques). Vertebrate cells can also be used as hosts. For example, mammalian cells adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are the SV40-transformed monkey kidney CV1 cell line (COS-7); human embryonic kidney cell lines (e.g., 293 or 293T cells as described in Graham, F.L. et al., J. Gen Virol. 36 (1977) 59-74); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells as described in Mather, J.P., Biol. Reprod. 23 (1980) 243-252); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); dog 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 (e.g., as described in Mather, J.P. et al., Annals N.Y. Acad. Sci. 383 (1982) 44-68); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980) 4216-4220); and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for protein production, see, e.g., Yazaki, P. and Wu, A.M., Methods in Molecular Biology, Vol. 248, Lo, B.K.C. (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268. The host cell can be a eukaryotic cell, e.g., Chinese hamster ovary (CHO) cells or lymphocytes (e.g., Y0, NS0, and Sp20 cells).

[0187] The nucleic acids of the present invention can be integrated into the genome (e.g., chromosome) of a host cell. Integration can be facilitated by introducing sequences that promote recombination with the genome according to standard techniques.

[0188] Methods for preparing multi-domain binding molecules

[0189] Also provided herein are methods for preparing the multi-domain binding molecules described herein. The methods include maintaining the host cells of the present invention under optimal conditions for nucleic acid expression or vector expression of the present invention and isolating the multi-domain binding molecules.

[0190] Methods for producing recombinant proteins are well known in the art. Nucleic acids encoding the protein can be cloned into an expression construct or vector and then transfected into a host cell that would not otherwise produce the protein, such as Escherichia coli cells, yeast cells, insect cells or mammalian cells, such as simian COS cells, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells or myeloma cells. Exemplary mammalian cells for protein expression are CHO cells, myeloma cells or HEK cells. The molecular cloning techniques for achieving these purposes are well known in the art and are described, for example, in Ausubel et al. (eds.), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date) or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989). There are a variety of cloning and in vitro propagation methods suitable for the construction of recombinant nucleic acids. Methods for producing recombinant antibodies are also well known in the art, see, for example, US4816567 or US5530101.

[0191] The nucleic acid can be inserted to be operably linked to a promoter in an expression construct or expression vector for further cloning (DNA amplification) or expression in a cell-free system or in a cell. As used herein, the term "promoter" should be understood in its broadest context and includes transcriptional regulatory sequences of genomic genes, including the TATA box or promoter elements, which are necessary for accurate transcription initiation, with or without other regulatory elements, such as upstream activating sequences, transcription factor binding sites, enhancers and silencers, that respond to, for example, developmental and / or external stimuli or alter nucleic acid expression in a tissue-specific manner. In the context of the present invention, the term "promoter" is also used to describe a recombinant, synthetic or fusion nucleic acid or derivative that confers, activates or enhances the expression of the nucleic acid to which it is operably linked. Exemplary promoters can contain additional copies of one or more specific regulatory elements to further enhance the expression of the nucleic acid and / or alter its spatial and / or temporal expression. As used herein, the term "operably linked to" refers to the positioning of the promoter relative to the nucleic acid such that the expression of the nucleic acid is under the control of the promoter.

[0192] Many vectors for expression in cells are commercially available. Vector components typically include, but are not limited to, one or more of the following: signal sequences, sequences encoding proteins (e.g., derived from the information provided herein), enhancer elements, promoters, and transcription termination sequences. Those skilled in the art are generally aware of suitable sequences for protein expression. Exemplary signal sequences include prokaryotic secretion signals (e.g., pelB, alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II), yeast secretion signals (e.g., invertase leader sequence, factor leader sequence, or acid phosphatase leader sequence), or mammalian secretion signals (e.g., herpes simplex virus gD signal).

[0193] Exemplary promoters active in mammalian cells include the cytomegalovirus immediate early promoter (CMV-IE), the human elongation factor 1-a promoter (EF1), the small nuclear RNA promoters (U1a and U1b), the α-myosin heavy chain promoter, the simian virus 40 promoter (SV40), the Rous sarcoma virus promoter (RSV), the adenovirus major late promoter, the β-actin promoter; hybrid regulatory elements containing the CMV enhancer / β-actin promoter or immunoglobulin promoters, or active fragments thereof. Examples of useful mammalian host cell lines are the monkey kidney CV1 cell line transformed by SV40 (COS-7, ATCC CRL 1651); the human embryonic kidney cell line (subcloned 293 or 293 cells grown in suspension medium); the baby hamster kidney cells (BHK, ATCC CCL 10); or the Chinese hamster ovary cells (CHO).

[0194] Typical promoters suitable for expression in yeast cells, such as yeast cells selected from, for example, Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe (S. pombe), include, but are not limited to, the ADH1 promoter, the GAL1 promoter, the GAL4 promoter, the CUP1 promoter, the PHO5 promoter, the nmt promoter, the RPR1 promoter, or the TEF1 promoter.

[0195] Host cells for protein production can be cultured in a variety of media, depending on the cell type used. Commercially available media, such as Ham’s F10 (Sigma), minimum essential medium (MEM) (Sigma), RPMI-1640 (Sigma), and Dulbecco’s modified Eagle medium (DMEM) (Sigma), are suitable for culturing mammalian cells. Media for culturing other cell types discussed herein are well known in the art.

[0196] Methods for separating proteins are well known in the art. In the case where the protein is secreted into the culture medium, the supernatant from such an expression system can first be concentrated using a commercially available protein concentration filter (e.g., Amicon or Millipore Pellicon ultrafiltration unit). Protease inhibitors (such as PMSF) can be included in any of the foregoing steps to inhibit proteolysis, and antibiotics can be included to prevent the growth of accidental contaminants. Alternatively or additionally, the supernatant can be filtered and / or separated from the cells expressing the protein, for example, using continuous centrifugation.

[0197] Proteins prepared from 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 affinity chromatography), or any combination of the foregoing.

[0198] These methods are well known in the art and are described, for example, in WO99 / 57134 or Ed Harlow and David Lane (eds.) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988).

[0199] Those skilled in the art will also appreciate that the protein can be modified to include tags that facilitate purification or detection, such as polyhistidine tags, hexahistidine tags, influenza virus hemagglutinin (HA) tags, simian virus 5 (V5) tags, LLAG tags, or glutathione S-transferase (GST) tags. The resulting protein is then purified using methods well known in the art, such as affinity purification. For example, a protein containing a hexahistidine tag is purified by contacting a sample containing the protein with nickel-nitrilotriacetic acid (Ni-NTA) that specifically binds to the hexahistidine tag immobilized on a solid or semi-solid support, washing the sample to remove unbound protein, and subsequently eluting the bound protein. Alternatively or additionally, a ligand or antibody that binds to the tag is used in the affinity purification method.

[0200] The molecules of the present invention can be purified with high yield. The yield can 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 solubilized material obtained prior to refolding), and / or the yield can 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 a yield greater than 1% or greater than 5% or higher. High yield means a yield greater than 1 mg / ml, or greater than 3 mg / ml, or greater than 5 mg / ml or higher.

[0201] Pharmaceutical compositions and medical methods

[0202] For administration to a patient, the molecules, nucleic acids, expression vectors or cells of the invention can be provided as part of a pharmaceutical composition together with one or more pharmaceutically acceptable carriers or excipients. The pharmaceutical composition can be in any suitable form (e.g., depending on the method required for its administration to the patient). It can be provided in unit dosage form and will generally be provided in a sealed container and can be provided as part of a kit. Such kits will usually (although not necessarily) include instructions for use. It can include a plurality of said unit dosage forms.

[0203] The pharmaceutical composition can be adapted for administration by any appropriate route, such as parenterally (including subcutaneous, intramuscular, intrathecal or intravenous), enterally (including oral or rectal), inhalation or intranasal routes. Such compositions can be prepared by any method known in the art of pharmacy, for example by mixing the active ingredient with one or more carriers or excipients under sterile conditions. Methods for formulating proteins into a form suitable for administration to a subject (e.g., a pharmaceutical composition) are known in the art and include, for example, those described in Remington's Pharmaceutical Sciences (18th Edition, Mack Publishing Co., Easton, Pa., 1990) and the United States Pharmacopeia: National Formulary (Mack Publishing Company, Easton, Pa., 1984).

[0204] The pharmaceutical composition generally contains a solution of the multi-domain binding molecule of the invention (or the nucleic acid, cell or vector of the invention) dissolved in a pharmaceutically acceptable carrier (e.g., an aqueous carrier). A variety of aqueous carriers can be used, for example, buffered saline, etc. The composition can contain pharmaceutically acceptable auxiliary substances required for close to physiological conditions, such as pH regulators and buffers, toxicity regulators, etc., for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of the molecules of the invention in these formulations can vary widely and will be selected mainly based on fluid volume, viscosity, body weight, etc., depending on the particular mode of administration selected and the needs of the patient. Exemplary carriers include water, saline, Ringer's solution, dextrose solution and 5% human serum albumin. Non-aqueous carriers can also be used, such as a mixture of oils and ethyl oleate. Liposomes can also be used as carriers. The carrier can contain a small amount of additives that enhance isotonicity and chemical stability, such as buffers and preservatives.

[0205] The molecules of the present invention can have desirable safety properties for use as therapeutic agents. Desirable safety properties mean that in addition to exhibiting good specificity, the molecules of the present invention may have passed further preclinical safety tests. Examples of such tests include whole blood assays, which demonstrate minimal cytokine release in the presence of whole blood and thus a lower risk of inducing a possible cytokine release syndrome in vivo, and alloreactivity tests to confirm a lower likelihood of recognizing alternative HLA types.

[0206] The dosage of the molecules of the present invention can vary within a wide range, depending on the disease or disorder being treated, the age and condition of the individual to be treated, etc. The physician will ultimately determine the appropriate dosage to be used.

[0207] The multi-domain binding molecules, pharmaceutical compositions, carriers, nucleic acids and cells of the present invention can 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.

[0208] The multi-domain binding molecules of the present invention can be further conjugated to therapeutic agents. Therapeutic agents that can be conjugated to the molecules of the present invention include immunomodulators and effectors, radioactive compounds, enzymes (e.g., perforin), or chemotherapeutic agents (e.g., cisplatin). To ensure toxic effects at the desired location, toxins can be encapsulated within liposomes conjugated to the multi-domain binding molecules described herein such that the compounds are slowly released. This will prevent harmful effects during in vivo transport and ensure maximum effect of the toxin after the multi-domain binding molecules described herein bind to the relevant antigen-presenting cells.

[0209] Examples of suitable therapeutic agents include, but are not limited to:

[0210] · Small molecule cytotoxins, i.e., compounds with a molecular weight less than 700 daltons that are capable of killing mammalian cells. Such compounds may also contain toxic metals that can have cytotoxic effects. In addition, it should be understood that these small molecule cytotoxins also include prodrugs, i.e., compounds that decay or transform under physiological conditions to release a cytotoxic agent. Examples of such agents include cisplatin, maytansine derivatives, calicheamicin, cactinomycin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, porfimer sodium Photofrin II, temozolomide, topotecan, trimetrexate glucuronate, auristatin E, vincristine and doxorubicin;

[0211] · Peptide cytotoxins, i.e., proteins or fragments thereof that have the ability to kill mammalian cells. For example, ricin, diphtheria toxin, Pseudomonas aeruginosa exotoxin A, Dnase and Rnase;

[0212] · Radioactive nuclides, i.e., unstable isotopes of elements that decay and simultaneously emit one or more of α or β particles or γ rays. For example, iodine 131, rhenium 186, indium 111, yttrium 90, bismuth 210 and 213, actinium 225 and astatine 213; Chelating agents can be used to facilitate the binding of these radioactive nuclides to the multi-domain binding molecule;

[0213] · Immunostimulants, i.e., immune effector molecules that stimulate an immune response. For example, cytokines such as IL-2 and IFN-γ,

[0214] · Superantigens and their mutants;

[0215] · TCR-HLA fusions, for example, fused to a peptide-HLA complex, wherein the peptide is derived from a common human pathogen such as Epstein-Barr virus (EBV);

[0216] · Chemokines such as IL-8, platelet factor 4, melanoma growth stimulatory protein, etc.;

[0217] · Antibodies or fragments thereof, including anti-T cell or NK cell determinant antibodies (e.g., anti-CD3, anti-CD28 or anti-CD16);

[0218] · Antibodies or fragments thereof that bind to molecules located at the immunological synapse;

[0219] · Alternative protein scaffolds with antibody-like binding characteristics;

[0220] · Complement activators;

[0221] · Xenoprotein domains, alloprotein domains, viral / bacterial protein domains, viral / bacterial peptides.

[0222] The multi-domain binding molecules, nucleic acids, vectors, pharmaceutical compositions and cells of the present invention can be used to treat diseases such as cancer, especially cancers associated with the expression of tumor-associated antigens. For example, cancers can be associated with the expression of GP100, NYESO, MAGEA4 or PRAME, as described in WO2011001152, WO2017109496, WO2017175006 and WO2018234319, and for example corresponding to U.S. Patent Nos. 8,519,100, 11,639,374, 11,505,590 and 11,427,624, the contents of each of which are incorporated herein by reference.

[0223] The cancer to be treated can be a cancer associated with PRAME expression. "Associated with PRAME expression" means that the cancer comprises cancer cells that express PRAME. In this regard, the cancer can be a PRAME-positive cancer. The cancer can be known to be associated with the expression of PRAME and thus PRAME expression may not be evaluated. Alternatively, PRAME expression can be evaluated using any method known in the art, including, for example, histological methods. However, the present invention is not intended to be limited to treating cancers in which PRAME expression can be detected by histological methods. Cancers associated with PRAME expression include, but are not limited to, melanoma, lung cancer, breast cancer, ovarian cancer, endometrial cancer, esophageal cancer, bladder cancer, head and neck cancer, uterine cancer, acute myeloid leukemia, chronic myeloid leukemia, and non-Hodgkin lymphoma. For example, the cancer associated with PRAME expression can be melanoma. The melanoma can be uveal melanoma or cutaneous melanoma. The lung cancer can be non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC). The breast cancer can be triple-negative breast cancer (TNBC). The bladder cancer can be urothelial carcinoma. The esophageal cancer can be gastroesophageal junction (GEJ) adenocarcinoma. The ovarian cancer can be epithelial ovarian cancer, such as high-grade serous ovarian cancer.

[0224] The multi-domain binding molecules, nucleic acids, vectors, pharmaceutical compositions, and cells of the present invention can be used to treat infectious diseases. The infectious diseases can be caused by bacterial, viral, fungal, or parasitic pathogens. Any infection by a pathogen that causes antigen-presenting cells to present MHCs bound to peptides from the pathogen can be suitable for treatment using the multi-domain binding molecules of the present invention. The multi-domain binding molecules of the present invention are particularly well-suited for infections in which antigen-presenting cells present pathogen peptides at levels lower than the optimal level for the innate immune system to clear the infection without additional treatment. The infectious diseases can be chronic infections. Exemplary infectious diseases include hepatitis B virus (HBV) infection and human immunodeficiency virus (HIV) infection.

[0225] The multi-domain binding molecules of the present invention can be used in methods for treating autoimmune diseases (such as type 1 diabetes). Given the potentially significant adverse events associated with systemic immunosuppression, organ-specific immunosuppression rather than systemic immunosuppression can be a beneficial approach to treatment. In autoimmunity, there is also increasing evidence that impairment of the PD-1 pathway 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 patients with type 1 diabetes and Crohn's disease. Thus, engaging PD-1 on activated autoreactive lymphocytes may be a mechanism for treating autoimmune diseases. Effective therapeutic agents for treating autoimmune diseases include those with favorable risk profiles (e.g., high levels of target engagement and tissue specificity) and that can be administered at a lower frequency.

[0226] The present invention also provides:

[0227] · The multi-domain binding molecules, nucleic acids, vectors, pharmaceutical compositions, or cells of the present invention for use as a medicament, preferably in methods for treating cancer or tumors or infectious diseases;

[0228] · The multi-domain binding molecules, nucleic acids, vectors, pharmaceutical compositions, or cells of the present invention for use as a medicament, preferably in methods for treating cancer or tumors or autoimmune diseases or infectious diseases;

[0229] · Use of the multi-domain binding molecules, nucleic acids, vectors, pharmaceutical compositions, or cells of the present invention in the preparation of a medicament for treating cancer or tumors or infectious diseases;

[0230] · Use of the multi-domain binding molecules, nucleic acids, vectors, pharmaceutical compositions, or cells of the present invention in the preparation of a medicament for treating cancer or tumors or autoimmune diseases or infectious diseases;

[0231] · A method for treating cancer or tumors or infectious diseases in a patient, which comprises administering to the patient the multi-domain binding molecules, nucleic acids, vectors, pharmaceutical compositions, or cells of the present invention;

[0232] · A method for treating cancer or tumors or autoimmune diseases or infectious diseases in a patient, which comprises administering to the patient the multi-domain binding molecules, nucleic acids, vectors, pharmaceutical compositions, or cells of the present invention; and

[0233] · An injectable preparation for administration to a human subject, comprising the multi-domain binding molecules, nucleic acids, vectors, pharmaceutical compositions, or cells of the present invention.

[0234] The treatment method may further comprise administering other anti-neoplastic agents alone, in combination, or sequentially. Examples of such agents are well known in the art and may include immune activators and / or T cell modulators.

[0235] Kits and articles

[0236] In another aspect, a kit or article is provided that contains materials for treating and / or preventing the diseases described above.

[0237] The kit may comprise (a) a container that contains a molecule, nucleic acid, vector, or cell of the present invention, optionally in a pharmaceutically acceptable carrier or diluent; and (b) a package insert that has 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 other therapeutically active compound or drug.

[0238] The package insert may be on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container may be formed from a variety of materials such as glass or plastic. The container holds or contains a composition that includes a molecule, nucleic acid, vector, or cell of the present invention and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). At least one active agent in the composition is a molecule, nucleic acid, vector, or cell of the present invention. The label or package insert indicates that the composition is for treating a subject who meets the treatment criteria, e.g., an individual who has or is susceptible to the diseases described herein, and there are specific instructions regarding the dosage amount and time interval for administration of the provided composition and any other drugs. The kit may further comprise other containers that contain pharmaceutically acceptable diluent buffers such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and / or dextrose solution. The kit may further comprise other materials that are desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0239] The kit optionally further comprises a container that contains a second drug, where the molecule, nucleic acid, vector, or cell of the present invention is the first drug, and the kit further comprises instructions on the package insert for treating the subject with the second drug in an effective amount.

[0240] In addition to those shown and described herein, various modifications of the present invention will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. The preferred features of each aspect of the present invention are the same as those shown in other aspects, with necessary modifications in details. The documents mentioned herein are incorporated by reference to the maximum extent permitted by law. BRIEF DESCRIPTION OF THE DRAWINGS

[0241] Figure 1 Schematic of an exemplary multi-domain single-chain binding molecule of the present invention. Figure 1 a shows a diagram of the domain arrangement from the N-terminus to the C-terminus, and Figure 1 b shows a putative diagram of the molecular fold.

[0242] Figure 2 Shows the results of an ELISpot assay using IFNγ as a readout for T cell activation. For comparison, the same TCR was used to construct multi-domain molecules in the form previously disclosed in WO 2020 / 157211 and detected in parallel with the Figure 1 single-chain form shown in. The schematic of each form is placed in Figure 2 to indicate the corresponding data points.

[0243] Figure 3 Shows a graph of surface plasmon resonance experiments used to evaluate the binding of mol093v9 and mol093v11 to each of pHLA, CD3, and FcRn.

[0244] Figure 4 Shows the pharmacokinetic properties evaluated in Tg32 SCID mice. Mice were dosed by IV bolus at 1 mg / Kg and blood was sampled continuously over a 21-day period. Samples were detected in serum by electrochemiluminescent immunoassay. The graph shows serum concentrations over time for 4 individual mice.

[0245] Figure 5 Presents a graph showing the results of an ELISPot assay in which the T cell activation of mol093v9 and mol093v11 was evaluated in vitro.

[0246] Figure 6 Presents a graph showing the results of an ELISPot assay in which the T cell activation of mol093v9 was directly compared to an alternative molecule (WO 2018 / 234319) that targets the same PRAME peptide but does not contain a half-life extended Fc domain. Figure 6 Shows that the two molecules drive similar potent T cell responses.

[0247] Figure 7 Shows a graph demonstrating the real-time killing of antigen-positive cells determined using the xCELLigence platform in the presence of mol093v9 and mol093v11.

[0248] Figure 8Presents a graph showing the results of a T cell killing assay in which mol093v9 was directly compared to an alternative molecule (WO 2018 / 234319) targeting the same PRAME peptide that does not contain a half-life extended Fc domain. Figure 8 Shows that mol093v9 showed killing data comparable to the non-HLE form of the molecule (“Mol001”).

[0249] Figure 9 Shows data from an ELISPOT T cell activation assay obtained using two normal cell batches (heart cells (HCM27) and lung epithelial cells (HSAEpiC9)) for one PBMC effector. The lowest T cell activation against normal cells was observed for mol093v9 and mol093v11 concentrations up to and including 1.1 nM of the fusion molecule.

[0250] Figure 10 Presents a graph showing the results of an ELISPOT T cell activation assay in which the reactivity of normal cells to mol093v9 was directly compared to the reactivity to an alternative molecule (WO 2018 / 234319) targeting the same PRAME peptide that does not contain a half-life extended Fc domain. Figure 10 Shows that both molecules showed similar lack of reactivity against normal cells from skin (melanocytes) and kidney (renal proximal tubules).

[0251] Figure 11 Is a graph showing T cell activation measured by IFNγ release against the TCR anti-CD3 fusion molecule a40b23U28-mol93 in the presence of antigen-positive and antigen-negative cancer cell lines.

[0252] Figure 12 Is a graph showing T cell activation measured by IFNγ release against the half-life extended TCR anti-CD3 fusion molecules a40b23U28-mol93 and a40b23U28-mol14 in the presence of antigen-positive cells.

[0253] Sequence description

[0254] SEQ ID NO:1 HLA-A*02-restricted peptide: SLLQHLIGL

[0255] SEQ ID NO:2 Amino acid sequence of the variable domain of the TCRα chain of an exemplary TCR. CDRs (CDR1, CDR2, and CDR3) are underlined and named SEQ ID NO:3, 4, and 5 respectively, and framework regions (FR1, FR2, FR3, and FR4) are italicized and named SEQ ID NO:27, 6, 7, and 28 respectively. This sequence contains an N24Q mutation (double underlined), which removes the N-linked glycosylation site.

[0256]

[0257] SEQ ID NO:8 Amino acid sequence of the variable domain of the TCRβ chain of an exemplary TCR. CDRs (CDR1, CDR2, and CDR3) are underlined and named SEQ ID NO:9, 10, and 11 respectively, and framework regions (FR1, FR2, FR3, and FR4) are italicized and named SEQ ID NO:29, 12, 13, and 30 respectively.

[0258]

[0259] SEQ ID NO:14 Amino acid sequence of the TCRα chain of an exemplary TCR. CDRs (CDR1, CDR2, and CDR3) are underlined and named SEQ ID NO:3, 4, and 5 respectively, and framework regions (FR1, FR2, FR3, and FR4) are italicized and named SEQ ID NO:27, 6, 7, and 28 respectively. The constant region is shown in bold and named SEQ ID NO:15. Within the constant region, a non-native cysteine residue is double underlined (at position 48 in the constant region), which is introduced to form an interchain disulfide bond. This sequence also contains N24Q, N146Q, N180Q, and N191Q substitutions (double underlined), each of which removes an N-linked glycosylation site.

[0260]

[0261] SEQ ID NO:16 Amino acid sequence of the TCRβ chain of an exemplary TCR. CDRs (CDR1, CDR2, and CDR3) are underlined and named SEQ ID NO:9, 10, and 11 respectively, and framework regions (FR1, FR2, FR3, and FR4) are italicized and named SEQ ID NO:29, 12, 13, and 30 respectively. The constant region is shown in bold (non-underlined) and named SEQ IDNO:19. Within the constant region, a non-native cysteine residue is shaded (at position 57 in the constant region), which is introduced to form an interchain disulfide bond. This sequence also contains an N184Q substitution (double underlined), which removes an N-linked glycosylation site.

[0262]

[0263] SEQ ID NO:17 is an exemplary anti-CD3 scFv (T cell-engaging immune effector domain), referred to herein as "U0". The light chain variable domain (VL) is shown in italics and designated SEQ ID NO:31. The light chain CDRs (CDR1, CDR2, and CDR3) are underlined and designated SEQ ID NO:33, 34, and 35. The heavy chain variable domain (VH) is shown in bold and designated SEQ ID NO:32. The heavy chain CDRs (CDR1, CDR2, and CDR3) are underlined and designated SEQ ID NO:36, 37, and 38. The glycine-serine linker connecting VL and VH is shown in plain text and designated SEQ ID NO:39.

[0264]

[0265] SEQ ID NO:40 is another exemplary anti-CD3 scFv (T cell-engaging immune effector domain), referred to herein as "U28". This sequence is identical to SEQ ID NO:17 above, except for two substitutions that are double-underlined (T164A and I201F). The light chain variable domain (VL) is shown in italics and designated SEQ ID NO:31. The light chain CDRs (CDR1, CDR2, and CDR3) are underlined and designated SEQ ID NO:33, 34, and 35. The heavy chain variable domain (VH) is shown in bold and designated SEQ ID NO:41. The heavy chain CDRs (CDR1, CDR2, and CDR3) are underlined and designated SEQ ID NO:48, 37, and 38. The glycine-serine linker connecting VL and VH is shown in plain text and designated SEQ ID NO:39.

[0266]

[0267] SEQ ID NO: 54 Unmodified human IgG1 Fc region (CH2 and CH3 domains) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0268] SEQ ID NO:42 Exemplary IgG1 Fc region sequence. Relative to the unmodified IgG1 Fc sequence above (SEQ ID NO:54), this sequence has four substitutions, double underlined. These are the N297G substitution for inhibiting binding to FcγRs and the T366S, L368A and Y407V substitutions (hole-forming substitutions) for enhancing dimerization with another Fc region (e.g., SEQ ID NO:43) containing the T366W substitution (knob-forming substitution). The numbering of the substitutions in this sequence is according to the EU numbering scheme.

[0269]

[0270]

[0271] SEQ ID NO:43 Another exemplary IgG1 Fc region sequence. This sequence has two substitutions, double underlined, relative to the above unmodified IgG1 Fc sequence (SEQ ID NO:54). These are the N297G substitution for inhibiting binding to FcγRs, and the T366W substitution (knob-forming substitution) for enhancing dimerization with another Fc region (e.g., SEQ ID NO:42) containing the T366S, L368A, and Y407V substitutions (hole-forming substitutions).

[0272] The numbering of substitutions in this sequence is according to the EU numbering scheme.

[0273]

[0274] SEQ ID NO:57 exemplary IgG1 Fc region sequence is shown below. This sequence has 7 substitutions (in bold) relative to the above unmodified IgG1 Fc sequence (SEQ ID NO:54). These are N297G substitutions for inhibiting binding to FcγR, T366S, L368A and Y407V substitutions (substitutions forming a hole) for enhancing dimerization with another Fc region (e.g., SEQ ID NO:58) containing a T366W substitution (substitution forming a knob), and substitutions M252Y, S254T and T256E for enhancing binding to FcRn. The numbering of substitutions in this sequence is according to the EU numbering scheme.

[0275]

[0276] SEQ ID NO:58 A further exemplary IgG1 Fc region sequence is shown below. This sequence has 5 substitutions (in bold) relative to the above unmodified IgG1 Fc sequence (SEQ ID NO:54). These are N297G substitutions for inhibiting binding to FcγRs, T366W substitutions (knob-forming substitutions) for enhancing dimerization with another Fc region (e.g., SEQ ID NO:57) containing T366S, L368A, and Y407V substitutions (hole-forming substitutions), and substitutions M252Y, S254T, and T256E for enhancing binding to FcRn. The numbering of substitutions in this sequence is according to the EU numbering scheme.

[0277]

[0278]

[0279] SEQ ID NO:44 Exemplary IgG1 hinge sequence (relative to native human IgG1 sequence, containing a C to S substitution at position 5 numbered according to SEQ ID NO:44): EPKSSDKTHTCPPCP

[0280] SEQ ID NO:52 Truncated IgG1 hinge sequence:

[0281] DKTHTCPPCP

[0282] SEQ ID NO:53 IgG4 hinge sequence:

[0283] ESKYGPPCPSCP

[0284] The complete amino acid sequence of the exemplary multi-domain single-chain binding molecule named "mol093v11" as SEQ ID NO:45. The T cell-engaging immune effector domain (underlined) is the anti-CD3 scFv sequence ("U0") provided in SEQ ID NO:17. The pMHC-binding domain is double-underlined and contains the TCRβ chain sequence (which is "VC1" in this case) (double-underlined, plain text) provided in SEQ ID NO:16 and the TCRα chain sequence (which is "VC2" in this case) (double-underlined, bold text) provided in SEQ ID NO:14. The half-life extension domain is an Fc domain, which is a dimer formed between the Fc region sequence provided in SEQ ID NO:42 (italic) (which is the FC1 region in this case) and the Fc region sequence provided in SEQ ID NO:43 (italic and bold) (which is the FC2 region in this case).

[0285]

[0286]

[0287] The complete amino acid sequence of the exemplary multi-domain single-chain binding molecule named "mol093v9" as SEQ ID NO:46. The T cell-engaging immune effector domain (underlined) is the anti-CD3 scFv sequence ("U28") provided in SEQ ID NO:40. The pMHC-binding domain is double-underlined and contains the TCRβ chain sequence (which is "VC1" in this case) (double-underlined, plain text) provided in SEQ ID NO:16 and the TCRα chain sequence (which is "VC2" in this case) (double-underlined, bold text) provided in SEQ ID NO:14. The half-life extension domain is an Fc domain, which is a dimer formed between the Fc region sequence provided in SEQ ID NO:42 (italic) (which is the FC1 region in this case) and the Fc region sequence provided in SEQ ID NO:43 (italic and bold) (which is the FC2 region in this case).

[0288]

[0289] The complete amino acid sequence of the exemplary multi-domain single-chain binding molecule named "a40b23U28-mol93" as SEQ ID NO:55. The T cell engaging immune effector domain (underlined) is the anti-CD3 scFv sequence ("U28") provided in SEQ ID NO:40. The pMHC binding domain in this molecule binds to the human PIWIL1 (PIWI-like protein 1) peptide-MHC complex. The pMHC binding domain is double-underlined and contains the TCRβ chain sequence (which is "VC1" in this case) (double-underlined, plain text) and the TCRα chain sequence (which is "VC2" in this case) (double-underlined, bold text). The half-life extension domain is an Fc domain, which is a dimer formed between the Fc region sequence provided in SEQ ID NO:42 (italicized) (which is the FC1 region in this case) and the Fc region sequence provided in SEQ ID NO:43 (italicized and bold) (which is the FC2 region in this case).

[0290]

[0291] The amino acid sequence of the PIWIL1 peptide (when in complex with MHC) bound by a40b23U28-mol93 as SEQ ID NO:56.

[0292] SLSNRLYYL

[0293] Other linker sequences:

[0294] GGGGS (SEQ ID NO:18), GGGSG (SEQ ID NO:20), GGSGG (SEQ ID NO:21), GSGGG (SEQ ID NO:22), GSGGGP (SEQ ID NO:23), GGEPS (SEQ ID NO:24), GGEGGGP (SEQ ID NO:25), GGEGGGSEGGGS (SEQ ID NO:26), GGGSGGGG (SEQ ID NO:47), GGGGSGGGGSGGGGSGGGGSGGGS (SEQ ID NO:39), GGGGSGGGGSGGGGS (SEQ ID NO:49), EAAAK (SEQ ID NO:50) and EAAAKEAAAKEAAAK (SEQ ID NO:51).

[0295] Examples

[0296] The present invention will be more fully understood by reference to the following examples. However, it should not be construed as limiting the scope of the present invention. It should be understood that the embodiments and implementations described herein are for illustrative purposes only, and various revisions or changes will be suggested to those skilled in the art and will be included within the scope of the present application and the appended claims.

[0297] Example 1 - Multi-domain Fc fusion molecules with improved potency

[0298] Multidomain molecules comprising a TCR - anti - CD3 fusion protein and incorporating a half - life - extended Fc domain have been previously described in WO 2020 / 157211 and their functionality has been demonstrated. However, it was subsequently found that such molecules have a substantially reduced ability to activate T cells in vitro relative to the non - Fc - fused form of the molecule and are not considered to be the optimal choice for therapeutic use. Further engineering was carried out to identify novel molecular forms with improved therapeutic properties.

[0299] Construct a multidomain molecule in which each functional domain is arranged on a single polypeptide chain. Figure 1 a shows a schematic diagram of the domain arrangement and Figure 1 b shows a putative illustration of the molecular fold.

[0300] In a first embodiment, as previously described (WO 2018 / 234319), the TCR domain of the multidomain single - chain molecule was designed to recognize the HLA - A*02 - restricted peptide SLLQHLIGL (SEQ ID NO:1) derived from PRAME. Using an ELISpot assay with IFNγ as the readout for T - cell activation, the ability of the molecule to drive T - cell activation in the presence of antigen - positive cancer cells was studied. For comparison, a multidomain molecule was constructed with the same TCR in the form previously disclosed in WO 2020 / 157211 and tested in parallel with the Figure 1 single - chain form presented in.

[0301] Figure 2 The data presented in show that the single - chain molecule is able to drive a significantly improved T - cell response against antigen - positive cancer cells compared to the previously disclosed form.

[0302] Thirty - seven other molecular forms with alternative domain arrangements were prepared and tested for in vitro potency. None of these forms performed better than the Figure 1 molecule presented in. Little or no response was observed in 17 forms; low - level responses were observed in 15 forms and medium - level responses were observed in 2 forms. The remaining three forms exhibited increased cross - reactivity against antigen - negative cell lines in addition to lower performance against antigen - positive cells.

[0303] Example 2 - Preparation of single-chain multi-domain molecules targeting PRAME

[0304] Use Figure 1 Prepare two multi-domain molecules (referred to as mol093v9 and mol093v11) in the form shown. The TCR regions of the two molecules are designed to recognize the HLA-A*02-restricted peptide SLLQHLIGL derived from PRAME. The two molecules differ in the amino acid sequence of the anti-CD3 scFv fragment. The complete amino acid sequences of mol093v9 and mol093v11 are provided in SEQ ID NO:46 and 45, respectively.

[0305] Express

[0306] Use Thermo ExpiCHO TM Transient expression protocol to express Mol093v9 and mol093v11 in Cho cells. Briefly, before transfection, the cultured cells were diluted to a concentration of 6x10 6 . Cells were harvested on day 14 after transfection, where the temperature was changed to 32 °C on day 1 after transfection. Feed additions were made on days 1 and 5 after transfection. Clarification was performed by two consecutive centrifugation steps at 300 xg and 17,500 xg. The resulting supernatant was passed through 0.45 μm and 0.2 μm membrane filters.

[0307] Purify

[0308] Purify the clarified supernatant by protein A and size exclusion chromatography steps. Prepare a 15 cm bed height MabSelect Extra protein A resin column. Load 50 column volumes of the supernatant onto the column and elute using a sodium citrate buffer at pH 3.0. After three column volumes had been collected, the eluate was neutralized by adding 2 M Tris and filtered through a 0.2 μm membrane filter. The protein A eluate was concentrated to at least 2 mg / mL using tangential flow filtration (using a 30 kDa membrane of XL50), and then loaded onto a HiLoad 26 / 600 Superdex SEC resin. The column was loaded with 5% of the column volume. The product was eluted into a phosphate citrate buffer, and the relevant fractions were filtered through a 0.22 μm membrane filter.

[0309] Yield

[0310] Measure the concentration of the purified material using a Nanodrop spectrophotometer by absorbance at 280 nm.

[0311] The calculated yield per liter of supernatant was 9.6 mg / L for mol093v9 and 17 mg / L for mol093v11.

[0312] Stability

[0313] Molecular stability was evaluated by SEC UPLC after freeze - thaw cycles, and / or after 14 days under i) thermal stress and ii) agitation conditions. The results are provided in the table immediately below, which shows the mol093v11 monomer purity under the indicated conditions. In each case, the monomer purity was considered acceptable.

[0314]

[0315] Example 3 - Binding affinity and kinetics of multi-domain molecules targeting PRAME

[0316] To verify that the TCR and anti - CD3 moieties of the molecule bind to their respective target molecules, single - cycle kinetics was performed by surface plasmon resonance (SPR) on a T200 BIAcore, followed by a single injection of CD3(γε).

[0317] Method

[0318] The chip used was from the Serie - S Biotin Capture Kit (Cytiva). The running buffer was phosphate - buffered saline (PBS) pH 7.2 containing 0.005% P20. The chip was regenerated by three consecutive injections of a solution of 8M guanidine hydrochloride (GuHCl) and 1M sodium hydroxide (NaOH) in a ratio of 3 + 1. The flow rate was 20 μL / min and the contact time was 120 sec. The chip was activated with a biotin capture reagent diluted 16:1 with PBS + P20. The flow rate was 2 μL / min for 300 sec. The capture level was 1600 response units (RU). The biotinylated pHLA was injected at 10 μg / mL, flow rate 10 μL / min for 120 sec.

[0319] For single - cycle kinetic analysis, serial dilutions of mol093v9 and mol093v11 were injected at a flow rate of 60 μL / min (highest concentration = 15 nM), with dissociation for 200 sec between each injection and 7200 sec for the 5th injection.

[0320] Subsequently, CD3(γε) was injected at a concentration of 300 nM, flow rate 10 μL / min for 60 sec.

[0321] For FcRn capture, initially PBS + P20 0.005% pH 6.0 was added to the flow cell. The biotinylated FcRn was injected at 5 μg / mL, flow rate 2 μL / min for 120 sec. After the FcRn injection, biotin was injected at 5 μM on all flow cells, flow rate 10 μL / min for 120 sec. The amount of FcRn was 450 RU.

[0322] Inject Mol093v9 or mol093v11 at 15 nM with a flow rate of 10 μL / min for 300 seconds and dissociate for 600 seconds. The responses for mol093v9 and mol093v11 are 152 RU and 163 RU, respectively.

[0323] Use the manufacturer's software to calculate kinetic parameters. Fit the dissociation phase to a single exponential decay equation to enable calculation of the half-life. From k off / k on Calculate the equilibrium constant K D 。

[0324] Results

[0325] Mol093v9 and mol093v11 exhibit picomolar affinity for the pHLA complex, high levels of CD3 activity, and binding to FcRn. The data are as Figure 3 shown and the binding parameters are summarized in the table immediately below.

[0326]

[0327] Example 4 - Pharmacokinetics

[0328] Pharmacokinetic properties were evaluated in Tg32 SCID mice. The test article was administered by IV bolus at 1 mg / Kg, 4 mice per compound, and blood samples were collected continuously over a 21-day period. Samples were detected in serum by electrochemiluminescence immunoassay, captured with biotinylated PRAME peptide-HLA and detected using a sulfo-labeled anti-scFv antibody. Figure 4 Serum concentrations over time for 4 individual mice are shown. PK parameters were extracted by non-compartmental analysis.

[0329] Results

[0330] The terminal t1 / 2 of Mol93v9 was calculated to be 9 days. The results of non-compartmental analysis are shown in the table immediately below.

[0331]

[0332] The results of 2-compartment simulation using NONMEM are shown in the table immediately below.

[0333]

[0334] Example 5 - In vitro T cell activation

[0335] Evaluate the ability of Mol093v9 and Mol093v11 to mediate potent and specific activation of CD3+ T cells against cells presenting the SLLQHLIGL-HLA-A*02 complex. Interferon-γ (IFN-γ) release was used as a readout for T cell activation.

[0336] Methods

[0337] Assays were performed using a human IFN-γ ELISPOT kit (BD Biosciences) according to the manufacturer's instructions. Briefly, target cells were prepared at a density of 1 x 10 6 / ml in assay medium (containing 10% heat-inactivated FBS and 1% penicillin-streptomycin-L-glutamine) and plated at 50,000 cells / well in a 50 μl volume. Peripheral blood mononuclear cells (PBMCs) isolated from fresh donor blood were used as effector cells and plated in a 50 μl volume with target cells at an approximate ratio of 1:1 (the exact number of PBMCs used per experiment depends on the donor and can be adjusted to produce a response within the appropriate assay range). The fusion molecules were titrated down from 10 nM to obtain the indicated final concentrations (across the expected clinically relevant range) and added to the wells in a 50 μl volume.

[0338] Plates were prepared according to the manufacturer's instructions. Target cells, effector cells, and fusion molecules were added to the relevant wells and the final volume was made up to 200 μl with assay medium. All reactions were repeated in triplicate. Control wells were also prepared by omitting the fusion molecule. The plates were then incubated overnight (37 °C / 5% CO 2 2). The next day, the plates were washed three times with buffer (1x PBS sachet, containing 0.05% Tween-20, prepared in deionized water). Then the first detection antibody was added to each well in a 50 μl volume. The plates were incubated at room temperature for 2 hours and then washed three times again. The second detection was performed by adding 50 μl of diluted streptavidin-HRP to each well, incubating at room temperature for 1 hour, and repeating the washing steps. Immediately before use, one drop (20 μl) of AEC chromophore was added to 1 ml of AEC substrate and mixed, and 50 μl was added to each well. The development of the spots was monitored regularly and the plates were washed with tap water to terminate the color development reaction. The plates were allowed to dry at room temperature for at least 2 hours and then the spots were counted using a CTL analyzer (Cellular Technology Limited) equipped with Immunospot software.

[0339] In this example, the following cell lines were used as target cells:

[0340] Antigen positive:

[0341] Mel624 - Human melanoma cell line

[0342] NCI - H1755 - Non - small cell lung cancer (NSCLC) cell line

[0343] OV56 - Ovarian serous carcinoma cell line

[0344] THP - 1 - Acute monocytic leukemia cell line

[0345] NCI - H1703 - Lung squamous cell carcinoma cell line

[0346] COV318 - Ovarian serous carcinoma cell line

[0347] Negative antigen:

[0348] TY - KNU - Ovarian serous adenocarcinoma (HLA - A*02 - ve; PRAME - ve)

[0349] NCI - H1693 - Non - small cell lung cancer (NSCLC) cell line (HLA - A*02 + ve; PRAME - ve)

[0350] Results

[0351] In the presence of various antigen - positive cancer cells, Mol093v9 and mol093v11 demonstrated efficient activation of T cells. The EC 50 values were calculated based on the data and obtained using PBMC from two independent donors. The EC 50 values for T - cell activation of the two donors are shown in the table immediately below. Figure 5 Data obtained from donor 1 are shown. Limited responses were observed in antigen - negative cell lines.

[0352]

[0353] Comparative data

[0354] Directly compare T - cell activation driven by mol093v9 and an alternative molecule that targets the same PRAME peptide but does not contain a half - life - extended Fc domain. Such molecules are described in WO 2018 / 234319 and U.S. Patent No. 11,427,624, the content of each of which is incorporated herein by reference. ELISPot assays were performed as described above. Figure 6 Showed that the two molecules drive similar efficient T - cell responses.

[0355] Example 6 - T cell killing

[0356] Evaluate the ability of Mol093v9 and Mol093v11 to mediate efficient and specific killing of antigen - positive cancer cells.

[0357] Method

[0358] Use the xCELLigence platform with a 96-well plate suitable for impedance reading (xCELLigence E-Plate 96PET part number 300600900), or the Incucyte live cell imaging platform using the CellPlayer 96-well Caspase-3 / 7 apoptosis assay kit (EssenBioScience, catalog number 4440), and perform the assay according to the manufacturer's instructions. Seed the target cells at their respective optimal densities (the number of target cells added per well is different for each cell line and has been titrated previously to determine the optimal conditions) and incubate overnight to allow them to adhere. Prepare test molecules at various concentrations and add 50 μl per well to the relevant wells such that the final concentration is between 100 fM and 10 nM. Effector cells are used at an effector-target ratio of 10:1 and seeded at 50 μl. Control samples without the fusion protein are also prepared, as well as samples containing only effector cells or only target cells. For the xCELLigence platform, adjust the final volume in the plate to 200 μl using the assay medium. Determine the percentage of cell lysis using the normalized cell index (impedance measurement). For the Incucyte platform, prepare the NucView assay reagent at 30 μM and add 25 μl to each well, and bring the final volume to 150 μl (resulting in a 5 μM final concentration). Determine the number of apoptotic cells in each image and record the apoptotic cells per mm2. In all cases, the assay is repeated three times, every 2 hours for 96 hours.

[0359] Results

[0360] Figure 7 The data in shows the real-time killing of antigen-positive cells in the presence of mol093v9 and mol093v11 as determined using the xCELLigence platform. EC 50 values are shown in the table immediately below and are in the low pM range. Limited killing was detected in the antigen-negative cell lines.

[0361]

[0362] Comparative data

[0363] Directly compare the T cell activation driven by mol093v9 and an alternative molecule targeting the same PRAME peptide but not containing the half-life extended Fc domain. Such molecules are described in WO 2018 / 234319. As described above, perform the killing assay using the Incucyte platform. Figure 8 Shows that these two molecules drive similar highly efficient killing responses.

[0364] Example 7 - Minimal reactivity against high-risk normal tissues

[0365] To determine the specificity of mol093v9 and mol093v11, the same ELISPOT method as described above and a set of normal cells derived from healthy human tissues were used as targets for further testing. The normal tissues included heart, lung cancer, kidney, and skin.

[0366] For batches of target normal cells co-cultured with PBMCs from healthy donors, the TCR-anti-CD3 fusion molecule was tested at 6 different concentrations ranging from 50 pM to 10 nM. Control measurements were performed using samples without the fusion molecule and samples in which the normal cells were replaced with NCI-H1755 (antigen-positive) cells.

[0367] Results

[0368] Figure 9 Data are shown for two normal cell batches (heart cells (HCM27) and lung epithelial cells (HSAEpiC9)) using one PBMC effector donor. Minimal T cell activation against normal cells was observed for mol093v9 and mol093v11 concentrations up to and including 1.1 nM of the fusion molecule.

[0369] Comparative data

[0370] Direct comparison of normal cell reactivity against mol093v9 and an alternative molecule that targets the same PRAME peptide but does not contain the half-life extended Fc domain. Such molecules are described in WO 2018 / 234319. Figure 10 It is shown that both molecules show a similar lack of reactivity against normal cells from skin (melanocytes) and kidney (renal proximal tubules).

[0371] Example 8 - Multi-domain single-chain binding molecules targeting PIWIL1 peptide-MHC complexes show efficient and specific T cell activation

[0372] Another multi-domain single-chain binding molecule was designed to contain a pMHC-binding domain targeting the PIWIL1 peptide (SLSNRLYYL, SEQ ID NO:56)-MHC complex, rather than mol093v9 and mol093v11 that bind to the PRAME peptide-MHC complex as described above. The full-length sequence of the resulting molecule (designated "a40b23U28-mol93") is provided in SEQ ID NO:55. Except for the TCRα and TCRβ variable domains, the sequence of a40b23U28-mol93 is identical to mol093v9. The TCRα variable domain and TCRβ variable domain of a40b23U28-mol93 correspond to SEQ ID NOs:28 ("a40") and 36 ("b23") in GB application number 2300226.4, respectively.

[0373] As described in Example 3 above, the binding of a40b23U28-mol93 to the SLSNRLYYL (SEQ ID NO:56)-HLA-A*02 complex was measured using SPR. The binding affinity (K D ) was 50 nM, and the binding half-life (t1 / 2) was 11.8 hours. These results are comparable to those of an equivalent TCR-anti-CD3 fusion molecule without a half-life extension (i.e., Fc) domain.

[0374] The ability of a40b23U28-mol93 to drive T cell activation was measured by IFNγ secretion assay using an ELISpot assay. The assay was performed using a human IFN-γ ELISPOT kit (BD Biosciences) according to the manufacturer's instructions. Peripheral blood mononuclear cells (PBMCs) isolated from fresh donor blood were used as effector cells. In this assay, KATOIII (gastric cancer) and CL11 (colon cancer) were used as antigen-positive target cells. NCI-H1755 was used as an antigen-negative cell. The data were plotted using PRISM software, and the EC 50 values were calculated from the curves.

[0375] Figure 11 a40b23U28-mol93 produced EC 50 values in the low pM range against the two antigen-positive cell lines (EC 50 value for KATO-III was 42.1 pM and EC 50 value for CL11 was 164.0 pM), and produced little to no response in the presence of antigen-negative cells (at a40b23U28-mol93 concentrations less than 1 nM). Equivalent TCR-anti-CD3 fusion molecules without a half-life extension (i.e., Fc) domain had EC50 12.9 pM and 52.7 pM, respectively.

[0376] These data confirm that the multi-domain single-chain binding molecules of the invention comprising the pMHC binding domain of a targeted PIWIL1 peptide-MHC complex retain a high affinity and potency comparable to that of an equivalent TCR-anti-CD3 fusion molecule without a half-life extension (i.e., Fc) domain, and retain specificity for antigen-positive cells.

[0377] In a further experiment, a40b23U28-mol93 was compared to an alternative multi-domain molecular form designated a40b23U28-mol14. a40b23U28-mol14 has the same individual domain amino acid sequences as a40b23U28-mol93, except arranged in a double-chain form as described in Figure 12 . In this double-chain form, the first chain (the left-hand chain in Figure 12 ) contains the variable domain of the TCRα chain, the constant domain of the TCRα chain, and an Fc region in the N-terminal to C-terminal direction. The second chain (the right-hand chain in Figure 12 ) contains an anti-CD3 scFv, the variable domain of the TCRβ chain, the constant domain of the TCRβ chain, and an Fc region in the N-terminal to C-terminal direction. T cell activation against the KATOIII cell line was compared between the two molecules. As shown in Figure 12 , both molecules drive T cell activation; however, Mol93 drives a more potent response than Mol14.

[0378] The results described in this example confirm that the variable domains of a TCR that bind a PIWIL1 peptide-MHC complex can be incorporated into the multi-domain single-chain binding molecules of the invention. In other words, regardless of the specific TCR sequence used (i.e., targeting a PRAME peptide-MHC or a PIWIL1 peptide-MHC), the multi-domain single-chain format effectively extends the half-life (by providing an Fc domain) without significantly affecting the affinity for the target or the potency of T cell activation.

Claims

1. A multi-domain single-chain binding molecule, comprising: i) a peptide-major histocompatibility complex (pMHC) binding domain, which comprises a first variable region (VC1) linked to a constant region and a second variable region (VC2) linked to a constant region, wherein VC1 and VC2 dimerize to form the pMHC binding domain; ii) an immune cell engaging (ICE) domain; and iii) a half-life extending domain, which comprises a first IgG Fc region (FC1) and a second IgG Fc (FC2), wherein the FC1 region and the FC2 region dimerize to form an Fc domain; wherein the ICE domain is linked to the N-terminus of VC1, VC1 is linked through its C-terminus to the N-terminus of the FC1 region, the FC1 region is linked through its C-terminus to the N-terminus of VC2, and VC2 is linked through its C-terminus to the N-terminus of the FC2 region; and wherein the pMHC binding domain and the T cell engaging immune effector domain are capable of binding to a pMHC complex and an immune cell, respectively.

2. The multi-domain binding molecule according to claim 1, wherein the immune cell engaging domain comprises: i) an antibody light chain variable region (ICE-VL) and an antibody heavy chain variable region (ICE-VH); or ii) a single domain antibody (ICE-SD).

3. The multi-domain binding molecule according to claim 1 or claim 2, wherein the immune cell is a T cell and the immune cell engaging domain is selected from: i) a T cell binding immune effector domain; or ii) a T cell binding immunosuppressor.

4. A multi-domain single-chain binding molecule, comprising: i) a peptide-major histocompatibility complex (pMHC) binding domain, which comprises a first variable region (VC1) linked to a constant region and a second variable region (VC2) linked to a constant region, wherein VC1 and VC2 dimerize to form the pMHC binding domain; ii) a T cell engaging immune effector domain, which comprises an antibody light chain variable region (TCE-VL) and an antibody heavy chain variable region (TCE-VH); and iii) a half-life extending domain, which comprises a first IgG Fc region (FC1) and a second IgG Fc region (FC2), wherein the FC1 region and the FC2 region dimerize to form an Fc domain; wherein the T cell engaging immune effector domain is linked to the N-terminus of VC1, VC1 is linked through its C-terminus to the N-terminus of the FC1 region, the FC1 region is linked through its C-terminus to the N-terminus of VC2, and VC2 is linked through its C-terminus to the N-terminus of the FC2 region; and wherein the pMHC binding domain and the T cell engaging immune effector domain are capable of binding to a pMHC complex and a T cell, respectively.

5. The multi-domain binding molecule according to claim 1, wherein the T cell engaging immune effector is a ScFv.

6. The multi-domain binding molecule according to any one of the preceding claims, wherein (i) VC1 comprises (a) the variable and constant regions of TCRα or (b) the variable and constant regions of TCRβ, and (ii) VC2 comprises the other of (a) and (b).

7. The multi-domain binding molecule according to claim 6, wherein VC1 comprises the variable and constant regions of TCRβ and VC2 comprises the variable and constant regions of TCRα.

8. The multi-domain binding molecule according to any one of claims 1 to 5, wherein (i) VC1 comprises (a) the variable region of a heavy chain antibody or (b) the variable region of a light chain antibody, and (ii) VC2 comprises the other of (a) and (b).

9. The multi-domain binding molecule according to claim 4 or any one of claims 5 to 8 dependent on claim 4, wherein the TCE-VL region is linked to the N-terminus of the TCE-VH region by its C-terminus and the TCE-VH region is linked to the N-terminus of VC1 by its C-terminus.

10. The multi-domain binding molecule according to any one of the preceding claims, wherein the immune cell engaging domain or the T cell engaging immune effector domain is a CD3 effector domain that activates T cells by interaction with CD3 and / or the TCR / CD3 complex.

11. The multi-domain binding molecule according to any one of the preceding claims, wherein the immune cell engaging domain or the T cell engaging immune effector domain is an anti-CD3 scFv.

12. The multi-domain binding molecule according to any one of the preceding claims, wherein the pMHC binding domain binds a tumor-associated antigen peptide complexed with MHC.

13. The multi-domain binding molecule according to any one of the preceding claims, wherein two or more of the ICE, TCE-VH, TCE-VL, VC1, VC2, FC1 and FC2 regions are linked to each other by a linker and / or an IgG hinge sequence.

14. The multi-domain binding molecule according to claim 13, wherein one or more of said linkers have a sequence selected from the following: GGGGS (SEQ ID NO:18), GGGSG (SEQ ID NO:20), GGSGG (SEQ ID NO:21), GSGGG (SEQ ID NO:22), GSGGGP (SEQ ID NO:23), GGEPS (SEQ ID NO:24), GGEGGGP (SEQ ID NO:25), GGEGGGSEGGGS (SEQ ID NO:26), GGGGSGGGGSGGGGSGGGGSGGGS (SEQ ID NO:39), GGGGSGGGGSGGGGS (SEQ ID NO:49), EAAAK (SEQ ID NO:50) and EAAAKEAAAKEAAAK (SEQ ID NO:51).

15. The multi-domain binding molecule according to any one of the preceding claims, wherein VC1 is linked to the FC1 region by a sequence comprising an IgG hinge sequence and / or VC2 is linked to the FC2 region by a sequence comprising an IgG hinge sequence.

16. The multi-domain binding molecule according to claim 15, wherein the IgG hinge sequence is at least 80% identical to SEQ ID NO:

44.

17. The multi-domain binding molecule according to claim 15 or claim 16, wherein the sequence linking VC1 to the FC1 region further comprises a glycine-serine linker, and / or the sequence linking VC2 to the FC2 region further comprises a glycine-serine linker.

18. The multi-domain binding molecule according to claim 17, wherein the glycine-serine linker has the sequence provided in SEQ ID NO:

47.

19. The multi-domain binding molecule according to claim 4 or any one of claims 5 to 18 dependent on claim 4, wherein the TCE-VL region is linked to the TCE-VH region by a sequence comprising a glycine-serine linker, optionally wherein the sequence is provided in SEQ ID NO:

39.

20. The multi-domain binding molecule according to claim 4 or any one of claims 5 to 19 dependent on claim 4, wherein the TCE-VH region is linked to VC1 by a sequence comprising a glycine-serine linker, optionally wherein the sequence is provided in SEQ ID NO:

18.

21. The multi-domain binding molecule according to any one of the preceding claims, wherein the FC1 region is linked to VC2 by a sequence comprising a glycine-serine linker, optionally wherein the sequence is provided in SEQ ID NO:

47.

22. The multi-domain binding molecule according to any one of the preceding claims, wherein the half-life extending domain comprises one or more amino acid substitutions that promote dimerization of the FC1 region and the FC2 region.

23. The multi-domain binding molecule according to claim 22, wherein: (i) one of the FC1 region and the FC2 region comprises one or more amino acid substitutions selected from: T366S, L368A, T394S, F405A, Y407A, Y407T, and Y407V, according to the EU numbering scheme; and (ii) the other of the FC1 region and the FC2 region comprises one or more amino acid substitutions selected from: T366W, T366Y, T366W, T394W, and F405W, according to the EU numbering scheme.

24. The multi-domain binding molecule according to claim 22, wherein: (i) one of the FC1 region and the FC2 region comprises one or more amino acid substitutions selected from: T366S, L368A, and Y407V, according to the EU numbering scheme; and (i) the other of the FC1 region and the FC2 region comprises the T366W amino acid substitution, according to the EU numbering scheme.

25. The multi-domain binding molecule according to any one of the preceding claims, wherein the half-life extending domain comprises one or more amino acid substitutions that attenuate the effector function of the Fc domain.

26. The multi-domain binding molecule according to claim 25, wherein the half-life extending domain comprises one or more amino acid substitutions selected from the following: S228P, E233P, L234A, L235A, L235E, L235P, G236R, G237A, P238S, F241A, V264A, D265A, H268A, D270A, N297A, N297G, N297Q, E318A, K322A, L328R, P329G, P329A, A330S, A330L, P331A, and P331S, according to the EU numbering scheme.

27. The multi-domain binding molecule according to claim 25 or claim 26, wherein the half-life extending domain comprises one or more amino acid substitutions that prevent or reduce binding to FcγR.

28. The multi-domain binding molecule according to claim 27, wherein the FC1 region and / or the FC2 region comprises the N297G amino acid substitution, according to the EU numbering scheme.

29. The multi-domain binding molecule according to any one of the preceding claims, wherein the half-life extending domain comprises one or more amino acid substitutions that promote binding to FcRn.

30. The multi-domain binding molecule according to claim 29, wherein the FC1 region and / or the FC2 region comprises the M252Y, S254T, and T256E amino acid substitutions, numbered according to the EU numbering scheme.

31. The multi-domain binding molecule according to claim 4 or any one of claims 5 to 30 dependent on claim 4, wherein the T cell engaging immune effector domain comprises: (i) a VL region that comprises CDRs of SEQ ID No: 33, 34, and 35 as CDR1, CDR2, and CDR3, respectively; and (ii) a VH region that comprises CDRs of SEQ ID No: 36, 37, and 38 as CDR1, CDR2, and CDR3, respectively.

32. The multi-domain binding molecule according to claim 4 or any one of claims 5 to 31 dependent on claim 4, wherein the T cell engaging immune effector domain comprises a VL region that is at least 80% identical to the sequence of SEQ ID NO: 31 and a VH region that is at least 80% identical to the sequence of SEQ ID NO:

32.

33. The multi-domain binding molecule according to any one of the preceding claims, wherein the FC1 region and the FC2 region are IgG1 Fc regions.

34. The multi-domain binding molecule according to any one of the preceding claims, wherein: i) the FC1 region comprises an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 42 and the FC2 region comprises an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 43; or ii) The FC1 region contains an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO:57 and the FC2 region contains an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO:

58.

35. The multi-domain binding molecule according to any one of the preceding claims, which comprises the following amino acid sequences in the order from the N-terminus to the C-terminus: a) The amino acid sequence of an anti-CD3 scFv, optionally followed by the linker sequence provided in SEQ ID NO:18; b) The amino acid sequence of the TCRβ variable and constant regions; c) The linker sequence provided in SEQ ID NO:47, followed by the IgG hinge sequence provided in SEQ ID NO:44; d) An Fc region having the sequence provided in SEQ ID NO:42; e) The linker sequence provided in SEQ ID NO:47; f) The amino acid sequence of the TCRα variable and constant regions; g) The linker sequence provided in SEQ ID NO:47, followed by the IgG hinge sequence provided in SEQ ID NO:44; and h) An Fc region having the sequence provided in SEQ ID NO:

43.

36. A nucleic acid encoding the multi-domain binding molecule according to any one of the preceding claims.

37. An expression vector comprising the nucleic acid according to claim 36.

38. A host cell comprising the nucleic acid according to claim 36 or the expression vector according to claim 37.

39. A method for preparing the multi-domain binding molecule according to any one of claims 1 to 35, which comprises maintaining the host cell according to claim 38 under optimal conditions for expressing the nucleic acid according to claim 36 or the expression vector according to claim 37, and isolating the multi-domain binding molecule.

40. A pharmaceutical composition comprising the multi-domain binding molecule according to any one of claims 1 to 35, the nucleic acid according to claim 36, the expression vector according to claim 37 or the host cell according to claim 38.

41. The multi-domain binding molecule according to any one of claims 1 to 35, the nucleic acid according to claim 36, the expression vector according to claim 37, the host cell according to claim 38 or the pharmaceutical composition according to claim 40, for use as a medicament.

42. A method of treatment comprising administering to a patient in need thereof the binding molecule according to any one of claims 1 to 35, the nucleic acid according to claim 36, the expression vector according to claim 37, the host cell according to claim 38 or the pharmaceutical composition according to claim 40.

43. The multi-domain binding molecule according to any one of claims 1 to 35, the nucleic acid according to claim 36, the expression vector according to claim 37, the host cell according to claim 38 or the pharmaceutical composition according to claim 40, for use in the treatment of cancer, an autoimmune disease or an infectious disease.

44. The multi-domain binding molecule, nucleic acid, expression vector, host cell or pharmaceutical composition according to claim 43, wherein the cancer is associated with PRAME expression.

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