Multidomain binding molecules

Abstract

The present invention relates to a multi-domain single-chain binding molecule comprising: i) a peptide-major histocompatibility complex (pMHC)-binding domain comprising a first variable region-constant region concatenation (VC1) and a second variable region-constant region concatenation (VC2); 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 and Fc2 regions dimerize to form an Fc domain. These binding molecules can be used to treat diseases such as cancer and infectious diseases.

Description

[Technical Field]

[0001] [Sequence table] This application contains a Sequence Listing, which is incorporated herein by reference in its entirety. The XML copy, created on August 2, 2023, is named P206512WO ST.26 Sequence Listing.xml and is 55320 bytes in size. [Background technology]

[0002] Many protein-based therapeutics, including antibody fragments and fusion proteins, are rapidly cleared from the body after administration. Their short circulatory half-lives are typically due to their small size, which allows for efficient clearance via renal filtration, and a lack of protection against intracellular degradation. In such cases, frequent administration or long infusion times are required to maintain effective drug concentrations over long periods of time. Several strategies have been employed to extend circulatory half-life and improve dosing. These strategies include increasing the hydrodynamic radius of proteins by attaching flexible hydrophilic molecules such as carbohydrates or PEG (polyethylene glycol), and exploiting recirculation via the fetal Fc receptor (FcRn) by attaching antibody Fc domains or serum albumin (Non-Patent Document 1).

[0003] Strategies that exploit FcRn-mediated recycling are particularly attractive because they pose a lower risk of inducing immunogenicity in vivo and can achieve long half-lives. For example, the half-life of a BiTE™-format T cell-engaging bispecific antibody has been reported to exceed 200 hours after attachment of an Fc domain (Non-Patent Document 2). Similarly, a TriTac™-format bispecific antibody incorporating an albumin-binding domain has been reported to have a half-life of more than 4 days (Non-Patent Document 3).

[0004] Fusion proteins containing a soluble T cell receptor (TCR) fused to an anti-CD3 antibody fragment represent a relatively new category of immune cell (e.g., T cell)-engaging bispecific fusion proteins with in vivo half-lives ranging from 6 to 8 hours (Non-Patent Document 4, Non-Patent Document 5). This half-life is much shorter than that of conventional monoclonal antibodies, which typically have half-lives ranging from 260 to 720 hours (Non-Patent Document 6). Furthermore, TCR-anti-CD3 fusion proteins have demonstrated advantageous therapeutic properties, including picomolar potency (Non-Patent Document 7). Therefore, there is a need to identify suitable approaches 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 without affecting other therapeutic properties, thereby reducing dosing frequency and maintaining effective concentrations over extended periods.

[0005] Unlike conventional antibodies, TCRs are designed to recognize short peptides (peptide-HLA) derived from intracellular antigens and presented on the cell surface by human leukocyte antigens (HLA). Effective immune synapse formation between peptide-HLA complexes on antigen-presenting cells and corresponding receptors on immune cells, such as T cells, relies on carefully orchestrated interactions that can be disrupted, for example, by increasing the intermembrane distance (Non-Patent Document 8, Non-Patent Document 9). Fusion techniques to extend the half-life of TCR-containing proteins, such as the attachment of antibody Fc domains or serum albumin, are therefore extremely challenging due to the risk of disrupting the geometry of the interactions required for TCR binding. Similar challenges apply to fusion proteins containing antibodies that bind to peptide-HLA complexes, known as TCR-like or TCR-mimetic antibodies.

[0006] Patent Document 1 describes a technique for extending the half-life of a TCR-anti-CD3 fusion protein by fusing it with an immunoglobulin Fc domain or an albumin-binding domain. However, such multi-domain binding molecules are large and complex proteins with countless possible formats, i.e., the possible combinations of the position and orientation of each domain (and each region within each domain) in one or more polypeptide chains. The position and orientation of each domain (and its region) within the molecule, as well as the number of polypeptide chains present, can affect the characteristics of the binding molecule, such as activity, half-life, and manufacturability. Therefore, there remains a need to identify suitable formats for such multi-domain binding molecules. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2020 / 157211 [Non-patent literature]

[0008] [Non-Patent Document 1] Konnteman, Curr Opin Biotechnol. 2011 Dec;22(6):868-76 [Non-patent document 2] Lorenczewski, et al., Blood 2017. 130(Suppl 1), 2815 [Non-patent document 3] Wesche et al., Cancer Res 2018;78(13 Suppl):Abstract nr 3814 [Non-patent document 4] Sato et al., 2018 J Clin Onc 2018 36, no. 15, suppl 9521-9521 [Non-Patent Document 5] Middleton et al., J Clin Onc 2016 34, no. 15, suppl 3016-3016 [Non-patent document 6] Ovacik & Lin, 2018 Clin Transl Sci, 11:540 [Non-Patent Document 7] Lowe et al. 2019 Cancer treatment reviews, vol. 77 35-43 [Non-patent document 8] Choudhuri et al., 2005 Nature Jul 28;436(7050):578-82 [Non-Patent Document 9] Holland et al J Clin Invest. 2020;130(5):2673-2688 Summary of the Invention

[0009] The present invention generally relates to multi-domain binding molecules. Specifically, the present invention relates to multi-domain binding molecules comprising: i) a peptide-major histocompatibility complex (pMHC)-binding domain comprising a first variable region and constant region concatenated together (VC1) and a second variable region and constant region concatenated together (VC2); ii) an immune cell engaging (ICE) domain (e.g., 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 and Fc2 regions dimerize to form an Fc domain. These binding molecules can be used to treat diseases such as cancer, infectious diseases, and autoimmune diseases.

[0010] Description of the Invention The present inventors have tested over 35 different formats (i.e., the orientation and location of each domain within the polypeptide) of a multidomain binding molecule containing a pMHC-binding domain, a T cell-engaging immune effector domain, and a half-life-extending domain. In doing so, the inventors found that in many formats, fusion of a TCR-anti-CD3 fusion protein to an Fc domain resulted in a significant loss of in vitro potency. However, the inventors surprisingly identified formats of the molecule that can be expressed as a single polypeptide chain, significantly extending the half-life and retaining the high potency of the original molecule. Example 8 further demonstrates that the identified formats function advantageously with TCRs that bind to a variety of targets.

[0011] In a first aspect, i) a peptide-major histocompatibility complex (pMHC) binding domain comprising a first variable region and constant region linkage (VC1) and a second variable region and constant region linkage (VC2), wherein VC1 and VC2 dimerize to form a pMHC binding domain; ii) an immune cell engaging (ICE) domain; 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; A multi-domain single chain binding molecule comprising: the ICE domain is linked to the N-terminus of VC1, VC1 is linked via its C-terminus to the N-terminus of the FC1 region, the FC1 region is linked via its C-terminus to the N-terminus of VC2, and VC2 is linked via its C-terminus to the N-terminus of the FC2 region; and Multi-domain single-chain binding molecules are provided in which the pMHC binding domain and the T cell engaging immune effector domain are capable of binding to pMHC complexes and immune cells, respectively.

[0012] In one embodiment, i) a peptide-major histocompatibility complex (pMHC) binding domain comprising a first variable region and constant region linkage (VC1) and a second variable region and constant region linkage (VC2), wherein VC1 and VC2 dimerize to form a pMHC binding domain; 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); 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; A multi-domain single chain binding molecule comprising: the T cell engaging immune effector domain is linked to the N-terminus of VC1, VC1 is linked via its C-terminus to the N-terminus of the FC1 region, the FC1 region is linked via its C-terminus to the N-terminus of VC2, and VC2 is linked via its C-terminus to the N-terminus of the FC2 region; and Multi-domain single-chain binding molecules are provided in which the pMHC binding domain and the T cell engaging immune effector domain are capable of binding to pMHC complexes and T cells, respectively.

[0013] In another embodiment, i) a soluble TCR comprising a first variable region and constant region concatenation (VC1) and a second variable region and constant region concatenation (VC2), wherein VC1 comprises the variable and constant regions of TCR beta having the amino acid sequence set forth in SEQ ID NO: 16, or a sequence which is at least 90%, at least 95%, or at least 98% identical thereto, and VC2 comprises the variable and constant regions of TCR alpha having the amino acid sequence set forth in SEQ ID NO: 14, or a sequence which is at least 90%, at least 95%, or at least 98% identical thereto; ii) an anti-CD3 scFv comprising an antibody light chain variable region (TCE-VL) having the amino acid sequence set forth 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 set forth in SEQ ID NO: 32, or a sequence that is at least 90%, at least 95%, or at least 98% identical thereto; iii) a half-life extending domain comprising a first IgG Fc region (FC1) having the amino acid sequence set forth in SEQ ID NO: 42, or a sequence at least 90%, at least 95%, or at least 98% identical thereto, and a second IgG Fc region (FC2) having the amino acid sequence set forth in SEQ ID NO: 43, or a sequence 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; A multi-domain single chain binding molecule comprising: the T cell engaging immune effector domain is linked to the N-terminus of VC1, VC1 is linked via its C-terminus to the N-terminus of the FC1 region, the FC1 region is linked via its C-terminus to the N-terminus of VC2, and VC2 is linked via its C-terminus to the N-terminus of the FC2 region; and Multi-domain single-chain binding molecules are provided in which the pMHC binding domain and the T cell engaging immune effector domain are capable of binding to pMHC complexes and T cells, respectively.

[0014] In another embodiment, a multi-domain single-chain binding molecule is provided that comprises the amino acid sequence set forth in SEQ ID NO:45.

[0015] In a still further aspect, there is provided a nucleic acid encoding a multi-domain binding molecule. Also provided is an expression vector comprising a nucleic acid of this aspect. Additionally, there is provided a host cell comprising a nucleic acid or vector of this aspect.

[0016] Also provided in a further aspect is a method of making a multi-domain binding molecule, comprising maintaining the host cell described above under conditions optimal for expression of the nucleic acid, and isolating the multi-domain binding molecule.

[0017] In a further aspect, a pharmaceutical composition comprising the multi-domain binding molecule is provided.

[0018] The multi-domain binding molecule, nucleic acid, vector, host cell, or pharmaceutical composition of any of the above aspects can be used in the treatment of diseases such as cancer, infectious diseases, and autoimmune diseases. Accordingly, in a further aspect, there is also provided a multi-domain binding molecule, nucleic acid, vector, host cell, or pharmaceutical composition for use as a medicament. In a still further aspect, there is provided a method of treatment comprising administering a multi-domain binding molecule, nucleic acid, vector, host cell, or pharmaceutical composition to a patient in need of treatment.

[0019] Peptide-major histocompatibility complex (pMHC) binding domain As used herein, a "pMHC-binding domain" is a protein domain capable of binding to a peptide-MHC complex. A first variable and constant region combination (VC1) and a second variable and constant region combination (VC2) dimerize to form a pMHC-binding domain. In this context, "VC1" refers to the region of the pMHC-binding domain sequence that includes the first variable and constant region combination, and "VC2" refers to the region that includes the second variable and constant region combination. The pMHC-binding site is located within the variable regions of VC1 and VC2. Suitable variable and constant region sequences include the variable and constant regions of a TCR or antibody. As used herein, the terms "MHC" and "HLA" are used interchangeably.

[0020] The pMHC binding domain may comprise at least a portion of a TCR alpha chain and a TCR beta chain. For example, the variable regions of VC1 and VC2 may be TCR variable regions. VC1 may comprise either a TCR alpha variable region or a TCR beta variable region, and VC2 may comprise the other of a TCR alpha variable region and a TCR beta variable region. For example, (i) VC1 may comprise either (a) the variable and constant regions of TCR alpha, or (b) the variable and constant regions of TCR beta, and (ii) VC2 may comprise the other of (a) or (b). Preferably, VC1 comprises the variable and constant regions of TCR β, and VC2 comprises the variable and constant regions of TCR α.

[0021] The pMHC-binding domain may be a T cell receptor (TCR), such as a soluble TCR comprising the variable and constant regions of the TCR. The TCR sequences defined herein are described with reference to the IMGT nomenclature, which is widely known and available to those skilled in the field of TCRs. See, for example, 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 Supplement 1: Supplement 100; and Lefranc, (2003), Leukemia 17(1): 260-266. Briefly, a TCR consists of two disulfide-bonded chains. Each chain (α and β) is generally considered to have two extracellular regions: a variable region and a constant region. A short junction region connects the variable and constant regions and is typically considered part of the α variable region. Additionally, the β chain usually contains a short diversity region adjacent to the junction region, which is also typically considered part of the β variable region. The variable region of each chain of a typical TCR is located at the N-terminus and contains three complementarity-determining regions (CDRs) embedded in framework sequences. The CDRs contain the recognition site for peptide-MHC binding.

[0022] Alternatively, the pMHC-binding domain may comprise an antibody variable region. The VC1 and VC2 variable regions may be antibody heavy chain variable regions or antibody light chain variable regions. For example, VC1 may comprise either a heavy chain antibody variable region or a light chain antibody variable region, and VC2 may comprise the other of a heavy chain antibody variable region or a light chain antibody variable region. In this regard, the pMHC-binding domain may be a TCR-like antibody, also known as a "TCR mimetic antibody" (TCRm-Ab). For example, the pMHC-binding domain may comprise the variable region of a TCR-like antibody. Antibodies do not naturally recognize pMHC complexes. However, it is known that antibodies with specificity for pMHC can be engineered, 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.

[0023] The pMHC-binding domain may comprise at least one immunoglobulin constant region. For example, the constant regions in VC1 and VC2 may be immunoglobulin constant regions. The constant regions may correspond to the constant regions from the TCR α chain or TCR β chain (TRAC or TRBC, respectively). Alternatively, the constant region of the pMHC-binding domain may be the constant region from an antibody light chain or antibody heavy chain (CL, CH1, CH2, CH3, or CH4). The constant region may be full-length or truncated. The TCR constant region may be truncated to remove the transmembrane domain and cytoplasmic tail. When the constant region is truncated, preferably only the membrane-associated and cytoplasmic portions are removed from the C-terminus. When the pMHC-binding domain comprises a TCR α chain sequence or a TCR β chain sequence, VC1 and VC2 may comprise a TCR variable region and a TCR constant region, respectively. Preferably, VC1 and VC2 do not comprise the transmembrane or cytoplasmic domain, i.e., the pMHC-binding domain is preferably soluble. Additional mutations may be introduced into the amino acid sequence of the constant region relative to the native constant region. The constant region may also contain residues, either naturally occurring or introduced, that allow dimerization, for example, by disulfide bonding between two cysteine ​​residues.

[0024] When present, the TCR portion of the molecules of the invention may be an αβ heterodimer. The α-β heterodimeric TCR portion of the molecules of the invention may comprise an α chain TRAC constant region sequence and / or a β chain TRBC1 constant region sequence or a TRBC2 constant region sequence. As noted above, the constant regions may be in soluble form (i.e., without transmembrane or cytoplasmic domains). One or both of the constant regions may 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 an N to K variant at position 4 of TRAC (Bragado et al. International Immunology. 1994 Feb;6(2):223-30).

[0025] The α-chain constant region sequence and the β-chain constant region sequence can be modified by truncation or substitution to eliminate the native disulfide bond between Cys4 in exon 2 of TRAC and Cys2 in exon 2 of TRBC1 or TRBC2. The α-chain constant region sequence and / or the β-chain constant region sequence can have a disulfide bond introduced between residues of the respective constant domains, as described, for example, in WO 2003 / 020763, WO 2004 / 033685, and WO 2006 / 000830, and, for example, U.S. Pat. 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 constant regions may be modified by substitution of a cysteine ​​residue at Thr48 of TRAC and Ser57 of TRBC1 or TRBC2, where the cysteine ​​forms a disulfide bond between the α and β constant regions of the TCR. TRBC1 or TRBC2 may further comprise a cysteine ​​to alanine mutation at position 75 of the constant domain and an asparagine to aspartic acid mutation at position 89 of the constant domain. One or both of the extracellular constant regions present in the αβ heterodimer may be truncated at one or more C-termini, for example, 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 may be truncated by 8 amino acids.

[0026] The amino acid sequences of the variable and constant regions of VC1 and VC2 may correspond to those found in nature or may contain one or more mutations compared 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 incorporated to improve stability and manufacturability. The VC1 and VC2 sequences can be derived from human sequences.

[0027] The VC1 and VC2 sequences contain one or more engineered cysteine ​​residues in the constant regions, allowing for the formation of a non-native disulfide bond between VC1 and VC2. Suitable locations for introducing disulfide bonds between residues in the respective constant regions are described in WO 2003 / 020763 and WO 2004 / 033685. Single-chain TCRs are also described in WO 2004 / 033685, WO 98 / 39482, WO 01 / 62908, Weidanz et al. (1998) J Immunol Methods 221 (1-2): 59-76, Hoo et al. (1992) Proc Natl Acad Sci USA 89(10): 4759-4763, and Schodin (1996) Mol Immunol 33(9): 819-829.

[0028] VC1 may comprise one of the TCR alpha variable region or the TCR beta variable region, and VC2 may comprise the other of the TCR alpha variable region and the TCR beta variable region. (i) the TCR alpha variable region comprises the CDRs of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5 as CDR1, CDR2, and CDR3, respectively; and (ii) The TCR β variable region comprises the CDRs of SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11 as CDR1, CDR2, and CDR3, respectively.

[0029] Alternatively, the TCR alpha and TCR beta CDR sequences may each optionally have one, two, three, or four amino acid substitutions relative to the sequences above.

[0030] The TCR alpha variable region may comprise CDRs as CDR1, CDR2 and CDR3, respectively, that are at least 90%, at least 95%, at least 98%, or at least 99% identical to the sequences of SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, and / or the TCR beta variable region may comprise CDRs as CDR1, CDR2 and CDR3, respectively, that are at least 90%, at least 95%, at least 98%, or at least 99% identical to the sequences of SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11.

[0031] The TCR alpha variable region may comprise CDRs corresponding to the sequences of SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, and may 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, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:28, and / or the TCR beta variable region may comprise CDRs corresponding to the sequences of SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, and may 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, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:30.

[0032] The TCR alpha variable region may be at least 80% identical to the sequence of SEQ ID NO: 2, and the TCR beta variable region may be at least 80% identical to the sequence of SEQ ID NO: 8. The TCR alpha variable region may be at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 2, and the TCR beta variable region may be at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 8. Preferably, the TCR alpha variable region has the sequence set forth in SEQ ID NO: 2, and the TCR beta variable region has the sequence set forth in SEQ ID NO: 8.

[0033] VC1 may comprise one of the TCR alpha constant region or the TCR beta constant region, and VC2 may comprise the other of the TCR alpha constant region and the TCR beta constant region. The TCR alpha constant region may be at least 80% identical to the sequence of SEQ ID NO: 15, and the TCR beta constant region may be at least 80% identical to the sequence of SEQ ID NO: 19. The TCR alpha 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 TCR beta 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 TCR alpha constant region has the sequence set forth in SEQ ID NO: 15, and the TCR beta constant region has the sequence set forth in SEQ ID NO: 19.

[0034] VC1 may comprise one of the variable and constant regions of TCR α or the variable and constant regions of TCR β, and VC2 may comprise the other of the variable and constant regions of TCR α and TCR β. The variable and constant regions of TCR α may comprise or consist of an amino acid sequence at least 80% identical to the sequence of SEQ ID NO: 14, and the variable and constant regions of TCR β may comprise or consist of an amino acid sequence at least 80% identical to the sequence of SEQ ID NO: 16. The variable and constant regions of TCR α may comprise or consist of an amino acid sequence at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 14, and the variable and constant regions of TCR β may comprise or consist of an amino acid sequence at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 16. Preferably, the variable and constant regions of TCR α comprise or consist solely of the amino acid sequence set forth in SEQ ID NO: 14, and the variable and constant regions of TCR β comprise or consist solely of the amino acid sequence set forth in SEQ ID NO: 16.

[0035] Those skilled in the art will appreciate that the multi-domain binding molecule formats of the present invention may be applied to other TCR sequences as well. For example, amino acid sequences of other suitable TCR chains are set forth in WO 2011 / 001152, WO 2017 / 109496, WO 2017 / 175006, and WO 2018 / 234319, as well as, for example, 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).

[0036] As is well known in the art, protein molecules can undergo post-translational modifications. Glycosylation is one such modification, which involves the covalent attachment of oligosaccharide moieties to specific amino acids within TCR or antibody chains. For example, asparagine residues or serine / threonine residues are well-known sites for oligosaccharide attachment. The glycosylation state of a particular protein depends on several factors, including the protein sequence, protein conformation, and the availability of certain enzymes. Furthermore, the glycosylation state (i.e., the type of oligosaccharide, the covalent linkages, and the total number of attachments) can affect protein function. Therefore, controlling glycosylation is often desirable when producing recombinant proteins. Controlled glycosylation has been used to improve antibody-based therapeutics (Jefferis et al., (2009) Nat Rev Drug Discov Mar;8(3):226-34.). Glycosylation can be controlled, for example, 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 native human proteins (Sinclair and Elliott, (2005) Pharm Sci. Aug; 94(8):1626-35). Alternatively, glycosylation can lead to inconsistent manufacturing, which is undesirable for therapeutic molecules. Residues with a high risk of glycosylation, such as asparagine, can be substituted with alternative amino acids, such as glutamine.

[0037] VC1 and / or VC2 may contain one or more amino acid substitutions that eliminate one or more glycosylation sites. A substitution in this context is with respect to the native (e.g., wild-type) or unmodified sequence. For example, (i) VC1 or VC2 may comprise a variable region and a constant region of TCR alpha comprising one or more amino acid substitutions at positions selected from the group consisting of N24, N148, N182, and N193, numbered according to SEQ ID NO: 14; and / or (ii) The other of VC1 and VC2 may comprise a variable and constant region of TCR β comprising an amino acid substitution at position N184, numbered according to SEQ ID NO: 16. The substitution may be an Asn to Gln (i.e., N to Q). Preferably, the variable and constant regions of TCR α comprise N24Q, N148Q, N182Q, and N193Q substitutions, numbered according to SEQ ID NO: 14, and the variable and constant regions of TCR β comprise an N184Q substitution, numbered according to SEQ ID NO: 16.

[0038] The pMHC-binding domain may not be completely aglycosylated, 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). A single glycosylation site may be present in the VC1 or VC2 variable region. A single glycosylation site may be present at position N18 of the TCR β variable region, numbered according to SEQ ID NO: 16. Advantageously, the inventors have determined that multi-domain binding proteins with this single glycosylation site retain affinity for peptide-MHC binding and potency in target cell killing, as well as have superior manufacturability (e.g., protein production yield, resistance to heat stress and aggregation) compared to other glycosylated and / or aglycosylated variants.

[0039] The pMHC-binding domain binds to MHC complexed with a peptide antigen. The peptide antigen may be a disease-associated antigen. The pMHC-binding domain can bind to a tumor-associated antigen peptide complexed with MHC. For example, the peptide antigen may be a peptide derived from GP100, NYESO, MAGEA4, or PRAME, as described in WO 2011 / 001152, WO 2017 / 109496, WO 2017 / 175006, and WO 2018 / 234319. The tumor-associated antigen may be PIWIL1. For example, the pMHC-binding domain binds to SLSNRLYYL (SEQ ID NO: 56) HLA-A. * The tumor-associated antigen may be PRAME. Preferably, the pMHC binding domain is SLLQHLIGL (SEQ ID NO: 1) HLA-A * Binds to the 02 complex.

[0040] immune cell engaging domain As used herein, an "immune cell engaging domain" is a protein domain that can bind to a target on an immune cell and / or modify an immune response, e.g., promote or suppress an immune response such as T cell activation. Immune cell engaging domains are also referred to herein as "ICE" domains.

[0041] 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 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 a heavy chain antibody variable region (e.g., VHH).

[0042] The immune cell engaging domain may be a T cell engaging immune effector domain. As used herein, a "T cell engaging immune effector domain" is a protein domain that can bind to a target on a T cell and promote an immune response. The T cell engaging immune effector domain may 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 a T cell engaging immune effector domain. "TCE-VL" and "TCE-VH" may also be referred to herein as "TCEVL" and "TCEVH."

[0043] 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 CD3, e.g., specifically bind to CD3 (i.e., the T cell engaging immune effector domain can be a CD3-binding protein). The T cell engaging immune 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 through interaction 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).

[0044] Alternatively, the immune cell engaging domain may be an immunosuppressive factor. As used herein, the term "immunosuppressive factor" refers to any molecule, e.g., a protein, that can inhibit an immune response, such as inhibiting T cell activation. The immunosuppressive factor can bind to a target (e.g., an antigen). For example, the immunosuppressive factor may be an immune checkpoint agonist, i.e., a molecule that induces immune checkpoint signaling. The immunosuppressive factor may include an antigen-binding portion that can bind to an antigen. The antigen of the immunosuppressive factor may be located on an immune cell, such as a T cell. The binding molecule may include an antibody or an antigen-binding fragment thereof; for example, the antibody may be a single-domain antibody, such as the variable region of a heavy chain antibody. Alternatively, the antibody may be a single-chain variable fragment (scFv), or an antibody-like scaffold of similar size, or any other binding protein that inhibits T cells through the induction of immune checkpoint signaling. Such immunosuppressive factors are described below.

[0045] The immune cell engaging domain may comprise an antigen-binding portion capable of binding to an antigen. The antigen of the immune cell engaging domain may be located on an immune cell, such as a T cell. The binding molecule may 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. "Native" or "conventional" in this context refers to an antibody having domains and domain arrangements identical to those of antibodies found in nature and comprising antibody-derived CDR and FR sequences. In natural / conventional four-chain antibodies, e.g., human antibodies, 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 binding recognition and specificity to the antigen. Conventional antibody binding sites are primarily composed of residues from the "antibody complementarity-determining regions" (CDRs) or hypervariable regions. In some cases, residues from non-hypervariable regions or framework regions (FR) may influence the overall domain structure and thus the binding site. CDR refers to the amino acid sequence that defines both the binding affinity and specificity of a natural antibody binding site. The light chain and heavy chain of a conventional four-chain antibody each have three CDRs, designated CDR1-L, CDR2-L, CDR3-L, and CDR1-H, CDR2-H, CDR3-H, respectively. Thus, the antigen-binding site of a conventional four-chain antibody contains six CDRs, including the CDR sets from each of VH and VL.

[0046] "Engineered" antibody formats include functional antibody fragments, single-chain antibodies, single-domain antibodies, and chimeric, humanized, bispecific, or multispecific antibodies. Engineered antibody formats further include constructs in which a TCR-derived CDR, optionally including three, two, or one additional N-terminal and / or C-terminal framework residues, or an entire variable domain from a TCR is 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, particularly the antigen-binding or variable region of a full-length antibody, that retains the ability to bind to a target antigen. Examples of functional antibody "fragments" include Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, and diabodies. For example, a binding molecule of the present invention may comprise an scFv.

[0047] The antibody may also be a single-domain antibody, such as a heavy-chain antibody variable region. 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 immune cell engaging domain may comprise, for example, a VHH (i.e., a heavy-chain antibody variable domain). As known in the art, the antigen-binding site of a single-domain antibody, such as a VHH, may comprise three CDRs (as opposed to six in conventional four-chain antibodies). The term "antigen-binding portion of an antibody" as used herein encompasses such binding sites. Alternatively or additionally, the binding molecule may comprise a Fab fragment or an Fv fragment. The term "Fab" ("fragment antigen-binding") refers to an antigen-binding fragment of an antibody, which comprises the antibody light chain (VL-CL) and the variable CH1 domain of the antibody heavy chain (VH-CH1). A Fab fragment typically has a molecular weight of about 50,000 daltons. An Fv fragment is the N-terminal portion of an antibody Fab fragment, and consists of one variable portion of a light chain (VL) and one variable portion of a heavy chain (VH).

[0048] The immune cell engaging domain may comprise an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL), which associate to form an antigen-binding moiety capable of binding to an antigen. Thus, the antigen-binding moiety may comprise a VH and a VL. For example, the immune cell engaging domain may comprise an scFv comprising a VH and a VL.

[0049] Other suitable antigen-binding moieties include heavy chain antibodies (hcAb), single domain antibodies (sdAb), minibodies (Tramontano et al (1994) J. Mol. Recognition 7, 9-24), camelid heavy chain antibody variable domains (VHH), variable domains of novel antigen receptors (VNAR), affibodies (Nygren PA (2008) FEBS J. 275, 2668-2676), alphabodies (see WO 2010 / 066740), designed ankyrin repeat domains (DARPins) (Stumpp et al (2008) Drug Discovery Today 13, 695-701), anticalins (Skerra et al (2008) FEBS J. 275, 2677-2683), knottins (Kolmar et al (2008) FEBS J. 275, 2684-2690), and engineered CH2 domains (nanobiodies, see Dimitrov DS (2009) mAbs 1, 26-28).

[0050] The antigen-binding portion may be or comprise a heavy chain variable domain comprising, consisting of, or consisting 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 (which retains the antigen-binding site). The antigen-binding portion may be a heavy chain antibody. The antigen-binding portion may be a heavy chain variable domain sequence of an antibody derived from a conventional four-chain antibody, such as, but not limited to, a VH sequence derived from a human antibody. Preferably, the antigen-binding portion is or comprises a variable domain of a heavy chain antibody (e.g., a camelid antibody), such as a VHH (also referred to herein as a "VHH domain"). Preferably, the antigen-binding portion is a VHH.

[0051] As described herein, an immune cell engaging domain may comprise an antigen-binding moiety (e.g., an antibody antigen-binding moiety) that binds to an antigen located on an immune cell. In the context of the present invention, an "immune cell" may refer to, for example, a T cell or a B cell. In particular, the antigen of the antigen-binding moiety may be a T cell surface antigen.

[0052] The immune cell engaging domain may be a single-chain variable fragment (scFv). A "single-chain Fv," also abbreviated as "sFv" or "scFv," is an antibody fragment comprising a VH antibody domain and a VL antibody domain connected in a single polypeptide chain. The scFv polypeptide may further comprise a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. For a review of scFvs, see Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Rosenburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994).

[0053] The immune cell engaging domain may be a CD3 effector. Examples of CD3 effectors include, but are not limited to, anti-CD3 antibodies or antibody fragments, particularly anti-CD3 scFvs or antibody-like scaffolds. The immune cell engaging domain may be a T cell engaging immune effector domain, which may be an anti-CD3 scFv. Additional immune effectors include, but are not limited to, antibodies (including fragments, derivatives, and variants thereof) that bind to antigens on T cells. Such antigens include CD28, 4-1bb (CD137), or CD16, or any molecule that exerts an effect on the immune synapse. A particularly preferred immune effector is an anti-CD3 antibody, or a functional fragment or variant of the above-mentioned anti-CD3 antibody. 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.

[0054] Preferably, the immune cell engaging domain comprises: (i) a VL region comprising the CDRs of SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35 as CDR1, CDR2, and CDR3, respectively; (ii) a VH region comprising the CDRs of SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38 as CDR1, CDR2, and CDR3, respectively; is a T cell engaging immune effector domain comprising

[0055] Alternatively, the immune cell engaging domain may be (i) a VL region comprising the CDRs of SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35 as CDR1, CDR2, and CDR3, respectively; (ii) a VH region comprising the CDRs of SEQ ID NO: 48, SEQ ID NO: 37, and SEQ ID NO: 38 as CDR1, CDR2, and CDR3, respectively;

[0039] The T cell engaging immune effector domain may be

[0056] The above VL and VH CDR sequences may each optionally have one, two, three, or four amino acid substitutions relative to the above sequences.

[0057] The TCE-VL may comprise CDRs as CDR1, CDR2, and CDR3, respectively, that are at least 90%, at least 95%, at least 98%, or at least 99% identical to the sequences of SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35, and / or the TCE-VH may comprise CDRs as CDR1, CDR2, and CDR3, respectively, that are at least 90%, at least 95%, at least 98%, or at least 99% identical to the sequences of SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38.

[0058] Alternatively, TCE-VL may comprise CDRs as CDR1, CDR2, and CDR3, respectively, that are at least 90%, at least 95%, at least 98%, or at least 99% identical to the sequences of SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35, and / or TCE-VH may comprise CDRs as CDR1, CDR2, and CDR3, respectively, that are at least 90%, at least 95%, at least 98%, or at least 99% identical to the sequences of SEQ ID NO: 48, SEQ ID NO: 37, and SEQ ID NO: 38.

[0059] The TCE-VL may comprise or consist of an amino acid sequence at least 80% identical to the sequence of SEQ ID NO: 31, and the TCE-VH may comprise or consist of an amino acid sequence at least 80% identical to the sequence of SEQ ID NO: 32. The TCE-VL may comprise or consist of an amino acid sequence at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 31, and the TCE-VH may comprise or consist of an amino acid sequence at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 32. Preferably, the TCE-VL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 31, and the TCE-VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 32.

[0060] Alternatively, the TCE-VL may comprise or consist of an amino acid sequence at least 80% identical to the sequence of SEQ ID NO: 31, and the TCE-VH may comprise or consist of an amino acid sequence at least 80% identical to the sequence of SEQ ID NO: 41. The TCE-VL may comprise or consist of an amino acid sequence at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 31, and the TCE-VH may comprise or consist of an amino acid sequence at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 41. For example, the TCE-VL may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 31, and the TCE-VH may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 41.

[0061] As described above, the immune cell engaging domain or T cell engaging immune effector domain may be an scFv. The immune cell engaging domain or T cell engaging immune effector domain may be an scFv comprising or consisting of an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 17 or SEQ ID NO: 40. The scFv 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: 17 or SEQ ID NO: 40. Preferably, the scFv comprises or consists of the amino acid sequence set forth in SEQ ID NO: 17. Alternatively, the scFv may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 40.

[0062] Alternatively, the immune cell engaging domain may be an immunosuppressant. For example, targets of the immunosuppressant include PD-1 (programmed cell 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-lymphocyte and T-lymphocyte attenuating factor, 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), TIGI It may be an immune checkpoint molecule such as T (T cell immunoreceptor with Ig and ITIM domains), LAG3 (lymphocyte activation gene 3), NOX2 (nicotinamide adenine dinucleotide phosphate NADPH oxidase isoform 2), TIM-3 (T cell immunoglobulin domain and mucin domain 3), VISTA (V domain Ig suppressor of T cell activation), SIGLEC7 (sialic acid-binding immunoglobulin-type lectin 7, also known as CD328), and SIGLEC9 (sialic acid-binding immunoglobulin-type lectin 9, also known as CD329).

[0063] In this regard, the immunosuppressive factor may be an agonist of one or more of the above-mentioned immune checkpoint molecules. Thus, the immunosuppressive factor may be an immune checkpoint agonist (i.e., inhibit immune activation). Suitable immune checkpoint agonists, including natural ligands and antibodies, are reviewed, for example, in Paluch et al., Front Immunol, 2018, 9:2306.

[0064] Instead of comprising the antigen-binding portion of an antibody, the immunosuppressive factor may comprise one member of a receptor-ligand pair, thereby allowing the immunosuppressive factor to bind to the other member of the receptor-ligand pair. The target ligand or target receptor may be located on an immune cell. For example, the immunosuppressive factor may comprise a ligand of an immune checkpoint molecule as described above. In particular, the immunosuppressive factor may comprise a portion of PD-L1 (e.g., a soluble extracellular domain) capable of binding to PD-1. Such an immunosuppressive factor can engage immune cells by binding to PD-1 and stimulate PD-1 signaling.

[0065] Alternatively, the immunosuppressive factor may comprise an agonist antibody capable of binding to an immune checkpoint molecule and stimulating its signal transduction. For example, the immunosuppressive factor may be or comprise a PD-1 agonist antibody (e.g., a single-domain antibody). Such a PD-1 agonist preferably does not compete with PD-L1 for binding to PD-1. The PD-1 agonist may be a full-length antibody or a fragment thereof, such as an scFv antibody or Fab fragment, or a single-domain antibody. Examples of such antibodies are provided in WO 2011 / 110621, WO 2010 / 029434, and WO 2018 / 024237. Thus, the antigen of the immunosuppressive factor may be PD-1, and the antigen-binding portion of the immunosuppressive factor may be a PD-1 agonist. The antigen-binding portion of the immunosuppressive factor may comprise a single-domain antibody, optionally a VHH. For example, the immunosuppressive factor may be a PD-1 agonist VHH.

[0066] As described above, the immunosuppressive factor may be a PD-1 agonist. As used herein, the term "PD-1 agonist" refers to any molecule that can bind to PD-1 and activate PD-1 signaling, including, for example, PD-1 ligand, PD-L1, and PD-1 agonist antibodies. Activation of the PD-1 pathway downregulates immune activity, promotes peripheral immune tolerance, and prevents autoimmunity (Keir et al., Annu Rev Immunol, 26:677-704, 2008; Okazaki et al., Int Immunol, 19:813-824, 2007).

[0067] Half-life extension domain As used herein, a "half-life prolonging domain" refers to a protein domain that extends the half-life of a multi-domain binding protein relative to a multi-domain binding protein lacking the half-life prolonging domain. The half-life prolonging domain comprises a first IgG Fc region (FC1) and a second IgG Fc region (FC2), wherein the FC1 and FC2 regions dimerize to form an Fc domain. As used herein, the term "Fc region" is used to refer to a region of a single polypeptide chain that comprises at least the CH2 and CH3 domain sequences, whereas the term "Fc domain" refers to a dimer of two Fc regions (i.e., FC1 and FC2).

[0068] As used herein, the term "half-life" refers to a pharmacokinetic property of a binding molecule, which is a measure of the average duration of a binding molecule after administration. The half-life of a binding molecule can be expressed as the time required to remove 50 percent of a known amount of the binding molecule from a patient's body (or other mammal) or a specific compartment thereof, for example, as measured in serum (i.e., circulating half-life) or other tissue.

[0069] A longer half-life not only allows for a reduction in the amount of drug given to a patient, but also allows for less frequent administration. A longer half-life can be beneficial, for example, in the treatment of cancer, infectious diseases, or autoimmune diseases or conditions. Binding molecules with increased half-lives can also be generated by modifying amino acid residues identified to be involved in the interaction between Fc and the FcRn receptor. Binding molecules comprising an Fc region containing one or more modifications that enhance binding to FcRn may have a half-life that is increased by 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 comprising a native Fc region. A binding molecule comprising an Fc region containing one or more modifications that enhance binding to FcRn may have a half-life that is 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 longer, compared to a binding molecule comprising a native Fc region.

[0070] Patent document 1 describes a technique for extending the half-life of a TCR-anti-CD3 fusion protein by fusing it to an IgG Fc domain. The present inventors have surprisingly found that the multi-domain binding molecules of the present invention maintain the extended half-life provided by the Fc domain in the format disclosed in Patent document 1, but furthermore have significantly higher potency.

[0071] The Fc domain of an immunoglobulin may be any antibody Fc domain. The Fc domain is the tail region of an antibody that interacts with cell surface Fc receptors and several proteins of the complement system. An Fc domain comprises two polypeptide chains (i.e., two Fc "regions"), each of which has two or three heavy chain constant domains (termed CH2, CH3, and CH4) and, optionally, a hinge region. The two Fc region chains may be linked by one or more disulfide bonds within the hinge region. The Fc domains from immunoglobulin subclasses IgG1, IgG2, and IgG4 bind to FcRn and undergo FcRn-mediated recycling, resulting in a long circulating half-life (3 to 4 weeks) and thus an extended half-life for the multi-domain binding molecules of the present invention. The interaction of IgG with FcRn is localized in the Fc region spanning 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 may be an IgG1 Fc domain, i.e., the F1 and F2 regions may be IgG1 Fc regions. The Fc domain may be derived from a human sequence.

[0072] The FC1 region may comprise or consist of an amino acid sequence at least 80% identical to the sequence of SEQ ID NO: 42, and the FC2 region may comprise or consist of an amino acid sequence at least 80% identical to the sequence of SEQ ID NO: 43. The FC1 region may comprise or consist of an amino acid sequence at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 42, and the FC2 region may comprise or consist of an amino acid sequence 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 set forth in SEQ ID NO: 42, and the FC2 region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 43. As will be understood by those skilled in the art, the sequences set forth above for FC1 and FC2 are also suitable vice versa. For example, the FC1 region may comprise or consist solely of the amino acid sequence set forth in SEQ ID NO: 43, and the FC2 region may comprise or consist solely of the amino acid sequence set forth in SEQ ID NO: 42.

[0073] 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 desirable therapeutic properties. For example, knobs-into-hole (KiH) mutations can be engineered into the CH3 domain to promote heterodimerization. Thus, the half-life extending domain can contain one or more amino acid substitutions that promote dimerization between the FC1 and FC2 regions. Such substitutions include "knob-in-hole" substitutions. In this case, one chain (i.e., either the FC1 or FC2 region) is engineered to contain a bulky protruding residue (i.e., 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., hole). For example, a knob can be constructed by replacing a small amino acid side chain with a larger one. A hole can be constructed by replacing a large amino acid side chain with a smaller one. Without wishing to be bound by theory, it is believed that this stabilizes heterodimers between the FC1 and FC2 regions by favoring the formation of heterodimers over other species, such as homomultimers of FC1 and FC2, thereby increasing the stability and manufacturability of the multi-domain binding molecules of the present invention.

[0074] Suitable positions and substitutions for KiH mutations and other mutations that promote dimerization of Fc regions are known in the art and include those described in Merchant et al., Nat Biotechnol 16:677 (1998), Ridgway et al., Prot Engineering 9:617 (1996), and Atwell et al. J Mol Biol 270,1 (1997): 26-35. For example, the substitutions that form corresponding knobs and holes in the two Fc regions can correspond to one or more pairs shown in the following table:

[0075] [Table 1]

[0076] Substitutions in the table above are indicated by the original residue, followed by its position using the EU numbering system, then the incorporated residue (all residues are indicated by the single-letter amino acid code). Multiple substitutions are separated by colons.

[0077] The FC1 and FC2 regions may contain one or more substitutions in the table above. For example: (i) one of the FC1 and FC2 regions may comprise one or more amino acid substitutions selected from the group consisting of T366S, L368A, T394S, F405A, Y407A, Y407T, and Y407V according to the EU numbering scheme; and (ii) The other of the FC1 and FC2 regions may contain one or more amino acid substitutions selected from the group consisting of T366W, T366Y, T366W, T394W, and F405W according to the EU numbering scheme. The substitutions in (i) and (ii) are hole-forming and knob-forming substitutions, respectively. The FC1 region may contain one or more of the substitutions in (i), and the FC2 region may contain one or more of the substitutions in (ii).

[0078] for example, (i) one of the FC1 and FC2 regions may contain one or more amino acid substitutions selected from the group consisting of T366S, L368A, and Y407V according to the EU numbering scheme; and (ii) The other of the FC1 and FC2 regions may contain the amino acid substitution T366W according to the EU numbering scheme. The FC1 region may contain one or more of the substitutions in (i), and the FC2 region may contain the substitution in (ii).

[0079] Preferably, (i) one of the FC1 and FC2 regions comprises amino acid substitutions of T366S, L368A, and Y407V according to the EU numbering scheme, and (ii) the other of the FC1 and FC2 regions comprises amino acid substitutions of T366W according to the EU numbering scheme. For example, the FC1 region may comprise amino acid substitutions of T366S, L368A, and Y407V according to the EU numbering scheme, and the FC2 region may comprise amino acid substitutions of T366W according to the EU numbering scheme.

[0080] The Fc domain may also contain 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 cellular cytotoxicity (ADCC). A modification that attenuates effector function may be a modification that alters the glycosylation pattern of the Fc domain, for example, a modification that results in a deglycosylated Fc domain. Alternatively, a modification that attenuates effector function may be a modification that does not alter the glycosylation pattern of the Fc domain. A modification that attenuates effector function may 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 extending domain may comprise one or more amino acid substitutions selected from the group consisting of S228P, E233P, L234A, L235A, L235E, L235P, G236R, G237A, P238S, F241A, V264A, D265A, H268A, D270A, N297A, N297G, N297Q, E318A, K322A, L328R, P329G, P329A, A330S, A330L, P331A, and P331S according to the EU numbering scheme. Particular modifications include the N297G or N297A substitution in the Fc region of human IgG1 (EU numbering). Other suitable modifications include substitutions of L234A, L235A, and P329G (EU numbering) in the Fc region of human IgG1, which result in attenuated effector function. The Fc region in the multi-domain binding molecules of the present invention may contain a substitution at residue N297, numbered according to the EU index. For example, the substitution may be an N297G substitution or an N297A substitution. Other suitable mutations (e.g., at residue N297) are known to those skilled in the art.

[0081] An Fc variant with reduced effector function refers to an Fc variant in which effector function (e.g., activities such as CDC, ADCC, and / or FcR binding) 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 which may have other mutations). An Fc variant with reduced effector function may be an Fc variant in which all detectable effector function is abolished compared to the wild-type Fc region. Assays for measuring effector function are known in the art and are described below.

[0082] To confirm the reduction / impairment of CDC and / or ADCC activity, in vitro and / or in vivo cytotoxicity assays can be performed. For example, Fc receptor (FcR) binding assays can be performed to confirm that an Fc region or fusion protein lacks FcγR binding (and thus likely lacks ADCC activity) but retains FcRn binding ability. NK cells, the primary cells mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. 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 to assess ADCC activity of a molecule of interest are described in U.S. Pat. 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) and U.S. Pat. No. 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)).

[0083] Substitutions can be introduced into the FC1 and FC2 regions that abolish or reduce binding to Fcγ receptors, and / or increase binding to FcRn, and / or prevent Fab arm exchange, and / or remove protease sites. In this regard, the half-life prolonging domain may also comprise one or more amino acid substitutions that prevent or reduce binding to activating receptors. The half-life prolonging domain may comprise one or more amino acid substitutions that prevent or reduce binding to FcγR. For example, the FC1 and / or FC2 regions may comprise an amino acid substitution of N297G according to the EU numbering scheme. Both the FC1 and FC2 regions may comprise an amino acid substitution of N297G.

[0084] The half-life prolonging domain may comprise one or more amino acid substitutions compared to an unmodified half-life prolonging domain, wherein the one or more amino acid substitutions enhance the binding of the Fc domain to FcRn. Methods for measuring binding to FcRn are known (see, for example, 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.)). The in vivo FcRn binding and serum half-life of human FcRn high-affinity binding polypeptides can be assayed, for example, in transgenic mice or transfected human cell lines expressing human FcRn, or in primates administered with polypeptides having mutant Fc regions. WO 2004 / 42072 (Presta) describes antibody substitutions that improve or reduce FcR binding. 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) describes amino acid substitutions in the Fc region of antibodies that are suitable for enhancing FcRn binding.

[0085] The modification(s) in the Fc region (e.g., amino acid substitutions, insertions, or deletions) that enhance binding of the Fc domain to FcRn are numbered 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, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, The modification may be at one or more positions selected from the group consisting of: 5, 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. Optionally, the Fc region may comprise non-naturally occurring amino acid residues at additional and / or alternative positions known in the art.

[0086] More specifically, the Fc region comprises 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, 241H ... 41R, 243W, 243L, 243Y, 243R, 243Q, 244H, 245A, 247L, 247V, 247G, 251F, 252 Y, 254T, 255L, 256E, 256M, 262I, 262A, 262T, 262E, 263I, 263A, 263T, 263M, 2 64L, 264I, 264W, 264T, 264R, 264F, 264M, 264Y, 264E, 265G, 265N, 265Q, 265 Y, 265F, 265V, 265I, 265L, 265H, 265T, 266I, 266A, 266T, 266M, 267Q, 267L, 2 68E, 269H, 269Y, 269F, 269R, 270E, 280A, 284M, 292P, 292L, 296E, 296Q, 296 D, 296N, 296S, 296T, 296L, 2961, 296H, 269G, 297S, 297D, 297E, 298H, 298I, 298T, 298F, 299I, 299L, 299A, 299S, 299V, 299H, 299F, 299E, 305I, 313F, 31 6D, 325Q, 325L, 325I, 325D, 325E, 325A, 325T, 325V, 325H, 327G, 327W, 327N, 327L, 328S, 328M, 328D, 328E, 328N, 328Q, 328F, 328I, 328V, 328T, 328H, 32 8A, 329F, 329H, 329Q, 330K, 330G, 330T, 330C, 330L, 330Y, 330V, 330I, 330F, 330R, 330H, 331G, 331A, 331L, 331M, 331F, 331W, 331K, 331Q, 331E, 331S, 33 1V, 331I, 331C, 331Y, 331H, 331R, 331N, 331D, 331T, 332D, 332S, 332W, 332F,The Fc region may comprise at least one substitution selected from the group consisting of 332E, 332N, 332Q, 332T, 332H, 332Y, 332A, 339T, 370E, 370N, 378D, 392T, 396L, 416G, 419H, 421K, 440Y, and 434W. Optionally, the Fc region may comprise additional and / or alternative non-naturally occurring amino acid residues known in the art.

[0087] The modification(s) (e.g., amino acid substitutions, insertions, or deletions) in the Fc region that enhance binding to FcRn may be at one or more positions selected from the group consisting of 234, 235, and 331, as numbered by the EU index as set forth in Kabat. For example, the Fc region may include at least one substitution selected from the group consisting of 234F, 235F, 235Y, and 331S, as numbered by the EU index as set forth in Kabat.

[0088] The modification(s) (e.g., amino acid substitutions, insertions, or deletions) in the Fc region that enhance binding to FcRn may be at one or more positions selected from the group consisting of 239, 330, and 332, as numbered by the EU index as set forth in Kabat. For example, the Fc region may include at least one substitution selected from the group consisting of 239D, 330L, and 332E, as numbered by the EU index as set forth in Kabat.

[0089] The modification(s) in the Fc region that enhance binding to FcRn (e.g., amino acid substitutions, insertions, or deletions) may be at one or more positions selected from the group consisting of 252, 254, and 256 as numbered by the EU index as set forth in Kabat. For example, the Fc region may include at least one substitution selected from the group consisting of 252Y, 254T, and 256E as numbered by the EU index as set forth in Kabat, as described in U.S. Pat. No. 7,083,784, the contents of which are incorporated herein by reference in their entirety. The Fc region may include all of the following substitutions as numbered by the EU index as set forth in Kabat: 252Y, 254T, and 256E.

[0090] The substitutions that enhance binding to FcRn listed above are relative to the corresponding wild-type Fc region (e.g., the Fc region of human IgG1 or IgG4) and may be present in either the FC1 or FC2 portion of the Fc domain, or preferably both. In other words, the substitutions involve amino acids that are not normally present in the corresponding wild-type Fc region, e.g., the Fc region of human IgG1 or IgG4. In this regard, the term "substitution" as used herein refers to the presence of one of the listed amino acids in a polypeptide and does not necessarily require the replacement of one amino acid with another. In one embodiment, the FC1 and / or FC2 regions contain the following amino acid substitutions, numbered according to the EU numbering scheme: 252Y, 254T, and 256E.

[0091] Additionally or alternatively, for manufacturing reasons, mutations may be made to remove or replace amino acids that may be subject to post-translational modifications, such as glycosylation, as described herein. The immunoglobulin Fc may be fused to other domains (i.e., VC1 or VC2) within the molecules of the invention via linker and / or hinge sequences as described herein. Alternatively, no linker may be used.

[0092] The FC1 region may comprise or consist of an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 57, and the FC2 region may 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 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: 57, and the FC2 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: 58. The FC1 region may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 57, and the FC2 region may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 58. As will be understood by those skilled in the art, the sequences set forth above for FC1 and FC2 are also suitable vice versa. For example, the FC1 region may comprise or consist solely of the amino acid sequence set forth in SEQ ID NO: 57, and the FC2 region may comprise or consist solely of the amino acid sequence set forth in SEQ ID NO: 58.

[0093] The two Fc regions in the molecules of the invention may comprise a CH2 constant domain and a CH3 constant domain and all or part of a hinge sequence. The hinge sequence may substantially or partially correspond to a hinge region from IgG1, IgG2, IgG3, or IgG4. The hinge sequence may be an IgG1 hinge sequence, such as the amino acid sequence set forth in SEQ ID NO: 44. The hinge may comprise all or part of the core hinge domain and all or part of the lower hinge region.

[0094] Suitable half-life extended formats of the multi-domain binding molecules of the present invention are also described in the application entitled "Multi-Domain Binding Molecules," filed concurrently herewith, which claims the benefit of priority to U.S. Provisional Application No. 63 / 371,863, filed August 18, 2022, the contents of which are incorporated herein by reference.

[0095] Format and Linker As used herein, the term "format" refers to the position and orientation of each domain (and each region within each domain) and the number of polypeptide chains in a multi-domain binding molecule of the invention. A schematic diagram of an exemplary multi-domain binding molecule format is shown in FIG. 1. The immune cell engaging domain in the exemplary binding molecule shown in FIG. 1 is a T cell engaging immune effector domain comprising a VH (TCE-VH) and a VL (TCE-VL). As described earlier herein, other types of immune cell engaging domains (e.g., single domain antibodies, VHHs, etc.) are also suitable. The pMHC binding domain and immune cell engaging domain of such molecules are capable of binding to pMHC complexes and immune cells, respectively. In this regard, the pMHC binding domain and immune cell engaging domain may be capable of simultaneously binding to pMHC complexes and immune cells, respectively.

[0096] In the multi-domain binding molecule format of the present invention, the immune cell engaging domain is linked to the N-terminus of VC1, which is linked via its C-terminus to the N-terminus of the FC1 region, which is linked via its C-terminus to the N-terminus of VC2, and VC2 is linked via its C-terminus to the N-terminus of FC2. Each region is covalently linked to a single polypeptide chain. This format can be represented as N-ICE-VC1-FC1-VC2-FC2-C. The inventors have confirmed that this format of molecule has the highest activity (i.e., potency and selectivity) and production yield among over 35 different formats tested.

[0097] When the immune cell engaging domain is a T cell engaging immune effector domain comprising a VH and a VL, this format can be represented as N-(TCEVL-TCEVH or TCEVH-TCEVL)-VC1-FC1-VC2-FC2-C. The inventors have determined that molecules of this format have the highest activity (i.e., potency and selectivity) and production yield among over 35 different formats tested.

[0098] The multi-domain binding molecules of the present invention are in a single-chain format. In this context, "single-chain" is used to describe a multi-domain binding molecule that is expressed as a single polypeptide chain comprising a pMHC-binding domain, an immune cell-engaging domain, and a half-life-extending domain.

[0099] Preferably, VC1 comprises the variable and constant regions of TCRβ, and VC2 comprises the variable and constant regions of TCRα, the immune cell engaging domain is an anti-CD3 scFv, and the Fc domain is an IgG1 Fc domain.

[0100] Two or more of the ICE region, TCE-VH region, TCE-VL region, VC1 region, VC2 region, FC1 region, and / or FC2 region may be linked to each other via a linker and / or an IgG hinge sequence. The linker sequence may be flexible because it is composed primarily of amino acids such as glycine, alanine, and serine, which lack bulky side chains that may limit flexibility. Such linkers include "glycine-serine" linkers, which refer to linkers containing only or primarily glycine and serine residues, e.g., (GGGGS)n. Alternatively, a linker with greater rigidity may be desirable. An example of a more rigid linker is an α-helix-forming linker having the sequence (EAAAK)n. A usable or optimal length for the linker sequence can be easily determined. Often, the linker sequence will be less than about 15, e.g., less than 10, or between 2 and 10 amino acids in length. The linker may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length. Examples of suitable linkers that may be used in multi-domain binding molecules are known in the art and include those described in WO 2010 / 133828 and Chen et al Adv Drug Deliv Rev. 2013;65(10):1357-1369. For example, the linker(s) present in a multi-domain binding protein of the invention may have a sequence selected from the group of 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).

[0101] Suitable IgG hinge sequences are known in the art and include the exemplary IgG1 hinge sequence set forth in SEQ ID NO: 44. Other suitable IgG hinge sequences include the truncated IgG1 hinge sequence set forth in SEQ ID NO: 52, and the IgG4 hinge set forth in SEQ ID NO: 53.

[0102] The ICE domain may be linked via its C-terminus to the N-terminus of VC1. In this regard, a multi-domain binding molecule of the invention may have the following format: N-ICE-VC1-FC1-VC2-FC2-C. When the immune cell engaging domain is a T cell engaging immune effector domain comprising a VH and a VL, the TCE-VL domain may be linked via its C-terminus to the N-terminus of the TCE-VH domain, and the TCE-VH domain may be linked via its C-terminus to the N-terminus of VC1. In this regard, a multi-domain binding molecule of the invention may have the following format: N-TCEVL-TCEVH-VC1-FC1-VC2-FC2-C.

[0103] VC1 may comprise the variable and constant regions of TCR β, and VC2 may comprise the variable and constant regions of TCR α. VC1 and VC2 may therefore dimerize to form a soluble TCR. In this regard, preferably, the multi-domain binding molecule of the invention has the following format: N-ICE-TCR β-FC1-TCR α-FC2-C or N-TCEVL-TCEVH-TCR β-FC1-TCR α-FC2-C (where "TCR β" refers to the variable and constant regions of TCR β, and "TCR α" refers to the variable and constant regions of TCR α).

[0104] When present, the TCE-VL domain may be linked to the TCE-VH domain via a sequence comprising a glycine-serine linker. Preferably, the sequence linking the TCE-VL domain and the TCE-VH domain is the amino acid sequence set forth in SEQ ID NO: 39.

[0105] The ICE domain or TCE-VH region may be linked to VC1 via a sequence comprising or consisting solely of a glycine-serine linker. Preferably, the sequence linking the ICE domain or TCE-VH region to VC1 is the amino acid sequence set forth in SEQ ID NO: 18.

[0106] VC1 may be linked to the FC1 region via a sequence comprising an IgG hinge sequence, and / or VC2 may be linked to the FC2 region via a sequence comprising an IgG hinge sequence. The IgG hinge sequence may 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.

[0107] The sequence linking the VC1 and FC1 regions may further comprise a glycine-serine linker, and / or the sequence linking the VC2 and FC2 regions may further comprise a glycine-serine linker. Preferably, the glycine-serine linker has the sequence shown in SEQ ID NO: 47. Preferably, these sequences are in the following format from N-terminus to C-terminus: VC1-GS linker-IgG hinge-FC1 and VC2-GS linker-IgG hinge-FC2.

[0108] The FC1 region may be linked to VC2 via a sequence containing a glycine-serine linker. Preferably, the glycine-serine linker linking the FC1 region and the VC2 region has the sequence shown in SEQ ID NO:47.

[0109] The multi-domain binding molecules of the present invention are single polypeptide chains (see Figure 1). Multi-domain binding molecules may be soluble and / or recombinant and / or isolated. The complete amino acid sequences of two exemplary multi-domain binding molecules are shown in SEQ ID NO:45 and SEQ ID NO:46.

[0110] 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 solely of the amino acid sequence shown in SEQ ID NO: 45.

[0111] A multi-domain binding molecule may have an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 46. A 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. A multi-domain binding molecule may comprise or consist of the amino acid sequence shown in SEQ ID NO: 46.

[0112] Optionally, the above multi-domain binding molecule sequences may be further fused to one or more other polypeptide sequences.

[0113] The above sequence is SLLQHLIGL (SEQ ID NO: 1) HLA-A * The present invention relates to multi-domain binding molecules comprising TCR chains that bind to the .02 complex. One skilled in the art can adapt these sequences to different targets by replacing the TCR chains in SEQ ID NO:45 and SEQ ID NO:46 with the sequences of a different TCR of interest. Similarly, one skilled in the art can replace the anti-CD3 scFv sequence (i.e., the T cell engaging immune effector domain) in SEQ ID NO:45 or SEQ ID NO:46 with another T cell engaging immune effector domain, for example, a different anti-CD3 scFv sequence.

[0114] Preferably, a) VC1 comprises the variable and constant regions of TCRβ; b) VC2 comprises the variable and constant regions of TCR alpha; c) the immune cell engaging domain is a T cell engaging immune effector domain that is an anti-CD3 scFv; d) FC1 has the amino acid sequence set forth in SEQ ID NO: 42, or an amino acid sequence which is at least 90%, or at least 95%, or at least 98% identical thereto; and e) FC2 has the amino acid sequence set forth in SEQ ID NO: 43, or an amino acid sequence which is at least 90%, at least 95%, or at least 98% identical thereto.

[0115] The multi-domain binding molecule preferably comprises the following amino acid sequences in the following order from N-terminus to C-terminus: a) the amino acid sequence of an anti-CD3 scFv (TCE-VL and TCE-VH), optionally followed by a linker sequence as shown in SEQ ID NO: 18; b) the amino acid sequence of the variable and constant regions of TCRβ (VC1); c) a linker sequence as set forth in SEQ ID NO: 47 followed by an IgG hinge sequence as set forth in SEQ ID NO: 44; d) an Fc region (FC1) having the sequence set forth in SEQ ID NO: 42; e) a linker sequence as shown in SEQ ID NO: 47; f) the amino acid sequence of the variable and constant regions of TCRα (VC2); g) a linker sequence as set forth in SEQ ID NO: 47 followed by an IgG hinge sequence as set forth in SEQ ID NO: 44, and h) an Fc region (FC2) having the sequence set forth in SEQ ID NO: 43; Includes:

[0116] The constant region of TCR β may have the amino acid sequence shown in SEQ ID NO: 19, and / or the constant region of TCR α may have the amino acid sequence shown in SEQ ID NO: 15. The multidomain binding molecule may not contain any amino acid sequences other than the sequences in a) to h) above.

[0117] The anti-CD3 scFv may comprise or consist of the amino acid sequence shown in SEQ ID NO: 17 or the amino acid sequence shown in SEQ ID NO: 40.

[0118] Amino acid sequence Phenotypically silent variants of any molecule disclosed herein are also within the scope of the present invention. As used herein, the term "phenotypically silent variant" is understood to refer to variants that contain one or more additional amino acid changes, including substitutions, insertions, and deletions, in addition to those set forth above, and that have a phenotype similar to the corresponding molecule without said change(s). For purposes of this application, phenotype refers to binding affinity (K D and / or binding half-life) and specificity. Phenotypes for soluble multi-domain binding molecules can include binding affinity and specificity, as well as immunostimulatory potency and purification yield.

[0119] A phenotypically silent variant may contain one or more conservative substitutions and / or one or more tolerated substitutions. By tolerated substitutions is meant substitutions that are phenotypically silent, even though they are not included in the definition of conservative substitutions set forth below. Those skilled in the art will recognize that various amino acids are "conservative" because they have similar properties. One or more such amino acids of a protein, polypeptide, or peptide can often be substituted with one or more other such amino acids without eliminating the desired activity of the protein, polypeptide, or peptide.

[0120] Thus, the amino acids glycine, alanine, valine, leucine, and isoleucine (amino acids with aliphatic side chains) can often be substituted for one another. Of these possible substitutions, glycine and alanine are preferred for one another because they have relatively short side chains, and valine, leucine, and isoleucine are preferred for one another because they have larger, hydrophobic aliphatic side chains. Other amino acids that are often 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 understood that amino acid substitutions within the scope of the present invention can be made using naturally occurring or non-naturally occurring amino acids. For example, it is contemplated herein that the methyl group of alanine may be replaced with an ethyl group, and / or minor changes may be made to the peptide backbone. Whether natural or synthetic amino acids are used, it is preferred that only L-amino acids be present.

[0121] Substitutions of this nature are often referred to as "conservative" or "semi-conservative" amino acid substitutions. Accordingly, the present invention extends to the use of molecules comprising any of the above amino acid sequences, but with one or more conservative substitutions and / or one or more permissive substitutions in said sequence, such that the amino acid sequence of said molecule, or any domain or region thereof, has at least 90% identity, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a sequence disclosed herein.

[0122] "Identity," as known in the art, is the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between polypeptide sequences or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. While several methods exist for measuring the identity between two polypeptide sequences or two polynucleotide sequences, commonly used methods for determining identity are codified 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)).

[0123] Amino acid sequences can be compared using programs such as the CLUSTAL program. This program compares amino acid sequences and finds the optimal alignment by inserting spaces appropriately into either sequence. It is possible to calculate amino acid identity or similarity (identity + conservation of amino acid type) for the optimal alignment. Programs such as BLASTx align the longest stretch of similar sequences and assign a value for their goodness of fit. In this way, comparisons can be made by finding several regions of similarity, each with a different score. Both types of identity analysis are contemplated in the present invention.

[0124] The percent identity of two amino acid sequences or two nucleic acid sequences is determined by aligning the sequences for optimal comparison (e.g., gaps can be introduced in the first sequence for best alignment with this sequence) and comparing the amino acid residues or nucleotides at corresponding positions. The "best alignment" is the alignment in which the two sequences have 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).

[0125] The determination of percent identity between two sequences can be achieved using mathematical algorithms known to those skilled in the art. One 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, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the BLASTn and BLASTp programs of Altschul, et al. (1990) J. Mol. Biol. 215:403-410. The determination of percent identity between two nucleotide sequences can be performed using the BLASTn program. The determination of percent identity between two protein sequences can be performed using the BLASTp program. 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 that detects distant relationships between molecules (supra). 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, word size=3 and expectation threshold=10. Parameters can be selected to automatically adjust for short input sequences. Another example of a mathematical algorithm used 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, incorporates such an algorithm.Other algorithms for sequence analysis known in the art include ADVANCE and ADAM, described in Torellis and Robotti (1994) Comput. Appl. Biosci., 10:3-5, and FASTA, described in Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-8. Within FASTA, ktup is a control option that sets the sensitivity and speed of the search. For purposes of assessing percent identity in this disclosure, BLASTp with default parameters is used as the comparison method. Furthermore, when the stated percent identity provides a non-integer value for an amino acid (i.e., a sequence of 25 amino acids with 90% sequence identity would provide a value of "22.5"), the resulting value is truncated to the next integer, i.e., "22." Thus, in the given example, a sequence with 22 matches out of 25 amino acids would have a sequence identity within 90%.

[0126] As will be apparent to one skilled in the art, it may be possible to shorten or extend the sequences provided at the C-terminus and / or N-terminus by one, two, three, four, five, or more residues without substantially affecting the functional characteristics of the molecule, e.g., the TCR portion. The sequences provided at the C-terminus and / or N-terminus may be shortened or extended by one, two, three, four, or five residues. All such variants are encompassed by the present invention.

[0127] Mutations, including conservative and permissive substitutions, insertions, and deletions, can be introduced into the sequences shown using any suitable method, including, but not limited to, polymerase chain reaction (PCR)-based methods, restriction enzyme-based cloning, or ligation-independent cloning (LIC) procedures. These methods are detailed in many standard molecular biology textbooks. For further details regarding polymerase chain reaction (PCR) and restriction enzyme-based cloning, see Sambrook & Russell (2001) "Molecular Cloning - A Laboratory Manual" (3rd ed.), CSHL Press. Further information regarding ligation-independent cloning (LIC) procedures can be found in Rashtchian, (1995) Curr Opin Biotechnol 6(1): 30-6. The protein sequences shown herein can be obtained from recombinant expression, solid-phase synthesis, or any other suitable method known in the art.

[0128] Evaluation of binding properties and activity of multi-domain binding molecules Binding affinity (equilibrium constant K D ) and binding half-life (T 1 / 2 Methods for determining the binding affinity (denoted as K ) are known to those skilled in the art. Binding affinity and binding half-life can be determined using surface plasmon resonance (SPR) or biolayer interferometry (BLI), for example using a BIAcore or Octet instrument, respectively. A doubling of affinity is expressed as K D It will be understood that T 1 / 2 is the dissociation rate constant (k off ) is calculated by dividing by T 1 / 2 When doubled, k off is halved. K for TCR D value and k offValues ​​are typically measured for soluble forms of TCRs, i.e., truncated forms of TCRs such that cytoplasmic and transmembrane domain residues are removed. To account for variations between independent measurements, particularly interactions with dissociation times greater than 20 hours, the binding affinity and / or binding half-life of a given protein can be measured several times, e.g., three or more times, using the same assay protocol, and the results averaged. To compare binding data between two samples (i.e., two different proteins and / or two preparations of the same protein), it is preferable to perform measurements using the same assay conditions (e.g., temperature). Measurement methods described for TCRs can also be applied to the multi-domain binding molecules described herein.

[0129] Certain multidomain binding molecules of the present invention can generate highly potent T cell responses in vitro against antigen-positive cells, particularly cells that present low levels of antigens typical of cancer cells (i.e., on the order of 5-100 antigens per cell, e.g., 50 antigens per cell) (Bossi et al., (2013) Oncoimmunol. 1;2(11):e26840; Purbhoo et al., (2006). J Immunol 176(12):7308-7316.). Such TCRs may be suitable for incorporation into the multidomain binding molecules described herein. The measured T cell response may be the release of T cell activation markers such as interferon-γ or granzyme B, or other measures of T cell activation, such as target cell killing or T cell proliferation. A highly potent response would be in the nM-pM range, e.g., an EC of 500 nM or less, preferably 1 nM or less, or 500 pM or less. 50 It may have a value.

[0130] Alternatively, certain binding molecules of the invention bind to CD8 + Cell killing and / or CD4 +This can result in a very strong anti-inflammatory response, such as inhibition of inflammation. Such binding molecules may exist in soluble form and may contain immune cell-engaging domains that are immunosuppressive factors, such as PD-1 agonists, or interleukins or cytokines, such as IL-2, IL-4, IL-10, or IL-13. The measured anti-inflammatory response may be a CD8 + Cell killing and / or CD4 + Inflammation inhibition and / or CD8 + The anti-inflammatory response may be an inhibition of the T cell signaling pathway. Suitable methods for assessing the anti-inflammatory response are known in the art, including the Jurkat NFAT cell reporter assay. Preferably, a highly potent response is in the pM range, i.e., an IC of 1000 pM or less. 50 Preferably, the maximal inhibition obtained in the reporter assay is greater than 50%, for example 80% or greater.

[0131] The molecules encompassed by the present invention may have an improved half-life. Methods for determining whether a protein has an improved half-life will be clear to those skilled in the art. For example, the protein's ability to bind to fetal Fc receptor (FcRn) is evaluated. In this regard, increased binding affinity to FcRn increases the serum half-life of the protein (see, for example, Kim et al. Eur J Immunol., 24:2429, 1994).

[0132] The half-life of a protein 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 intravenously injected into mice, and its plasma concentration is measured periodically over 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 can be measured periodically using ELISA. The resulting clearance curve should be biphasic, i.e., have an α-phase and a β-phase. To determine the in vivo half-life of a protein, the clearance rate in the β-phase is calculated and compared to the clearance rate of the wild-type or unmodified protein.

[0133] Nucleic acids, vectors, and host cells The present invention provides nucleic acids encoding the multi-domain binding molecules of the present invention. The nucleic acid may be cDNA. The nucleic acid may be mRNA. The nucleic acid may be non-naturally occurring and / or purified and / or engineered. The nucleic acid sequence may be codon-optimized depending on the expression system used. As known to those skilled in the art, expression systems may include bacterial cells such as E. coli, or yeast cells, or mammalian cells, or insect cells, or the expression system may be a cell-free expression system.

[0134] The present invention also provides constructs in the form of plasmids, vectors, transcription cassettes, or expression cassettes comprising at least one nucleic acid as described above. The present invention also provides recombinant host cells comprising one or more of the constructs described above. As noted above, nucleic acids encoding specific binding molecules of the present invention constitute an aspect of the present invention, as do methods for producing specific binding molecules of the present invention comprising expression from nucleic acids encoding specific binding molecules of the present invention. Expression can be conveniently achieved by culturing recombinant host cells containing the nucleic acid under appropriate conditions. Following production by expression, the specific binding molecules can be isolated and / or purified using any suitable technique and then used as appropriate.

[0135] Systems for cloning and expression of polypeptides in a wide variety of host cells are well known. Suitable host cells include bacteria, mammalian cells, yeast, and baculovirus systems. Mammalian cell lines available in the art for expressing heterologous polypeptides 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. The expression of antibodies and antibody fragments in prokaryotic cells such as E. coli is well established in the art. For a review, see, e.g., Plueckthun, Bio / Technology 9:545-551 (1991). Expression in eukaryotic cells in culture is also available to those skilled in the art as an option for producing specific binding molecules; for recent reviews, see, e.g., Reff, Curr. Opinion Biotech. 4:573-576 (1993); Trill et al., Curr. Opinion Biotech. 6:553-560 (1995).

[0136] Suitable vectors can be chosen or constructed containing appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes, and other sequences, as appropriate. The vector may be any suitable vector known in the art, including plasmids or viral vectors (e.g., phage or phagemid), as appropriate. For further details, see, e.g., Sambrook et al., "Molecular Cloning: A Laboratory Manual," 2nd ed., Cold Spring Harbor Laboratory Press (1989). For example, many known techniques and protocols for preparing nucleic acid constructs, mutagenesis, sequencing, introducing DNA into cells, and manipulating nucleic acids in gene expression and protein analysis are described in detail in Ausubel et al., eds., "Short Protocols in Molecular Biology," 2nd ed., John Wiley & Sons (1992).

[0137] The present invention also provides host cells containing the nucleic acids disclosed herein. Furthermore, the present invention provides methods comprising introducing such nucleic acids into host cells. Any available technique can be used for introduction. For eukaryotic cells, suitable techniques include calcium phosphate transfection, DEAE-dextran, electroporation, liposome-mediated transfection, and transduction using retroviruses or other viruses, such as vaccinia, or, in the case of insect cells, baculovirus. For bacterial cells, suitable techniques include calcium chloride transformation, electroporation, and transfection using bacteriophages. Following introduction, expression from the nucleic acid can be caused or permitted, for example, by culturing the host cells under conditions for gene expression.

[0138] Suitable host cells for cloning or expressing the polynucleotides and / or vectors of the present invention are known in the art. Suitable host cells for expressing (glycosylated) proteins are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures can also be used as hosts. See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe the PLANTIBODIES™ technology for producing antibodies in transgenic plants). Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to growth in suspension may be useful. Other examples of useful mammalian host cell lines are SV40-transformed monkey kidney CV1 line (COS-7), human embryonic kidney line (e.g., 293 or 293T cells described in Graham, FL et al., J. Gen Virol. 36 (1977) 59-74), baby hamster kidney cells (BHK), mouse Sertoli cells (TM4 cells described in Mather, JP, Biol. Reprod. 23 (1980) 243-252), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor (MMT 060562), TRI cells (e.g., Mather, JP et al., Annals NY 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, BKC (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268. Host cells can be eukaryotic cells, such as Chinese hamster ovary (CHO) cells or lymphoid cells (e.g., Y0, NS0, Sp20 cells).

[0139] The nucleic acids of the invention may be integrated into the genome (e.g., chromosome) of the host cell. Integration can be facilitated by the inclusion of sequences that facilitate recombination with the genome, according to standard techniques.

[0140] Methods for producing multi-domain binding molecules Further provided herein is a method of making a multi-domain binding molecule as described herein, comprising maintaining a host cell of the invention under conditions optimal for expression of a nucleic acid or expression vector of the invention, and isolating the multi-domain binding molecule.

[0141] Methods for producing recombinant proteins are well known in the art. After cloning a nucleic acid encoding a protein into an expression construct or expression vector, these can be transfected into host cells that do not otherwise produce the protein, such as E. coli cells, yeast cells, insect cells, or mammalian cells such as monkey COS cells, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, or myeloma cells. Exemplary mammalian cells used to express proteins are CHO cells, myeloma cells, or HEK cells. Molecular cloning techniques to achieve these goals are known in the art and are described, for example, in Ausubel et al. (editors), 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). A wide variety of cloning and in vitro amplification techniques are suitable for constructing recombinant nucleic acids. Methods for producing recombinant antibodies are also known in the art. See, e.g., U.S. Patent No. 4,816,567 or U.S. Patent No. 5,530,101.

[0142] Nucleic acids can be operably linked to a promoter and inserted into an expression construct or expression vector for further cloning (DNA amplification) or expression in a cell-free system or in cells. As used herein, the term "promoter" should be interpreted in its broadest context and includes the transcriptional regulatory sequences of a genomic gene, including the TATA box or initiator element required for accurate transcription initiation, with or without additional regulatory elements (e.g., upstream activation sequences, transcription factor binding sites, enhancers, and silencers) that alter expression of the nucleic acid, for example, in response to developmental and / or external stimuli or in a tissue-specific manner. In this context, the term "promoter" is also used to describe a recombinant, synthetic, or fusion nucleic acid, or derivative, that confers, activates, or enhances expression of an operably linked nucleic acid. Exemplary promoters may include additional copies of one or more specific regulatory elements that further enhance expression and / or alter the spatial and / or temporal expression of the nucleic acid. As used herein, the term "operably linked" means that the promoter is positioned relative to the nucleic acid such that expression of the nucleic acid is controlled by the promoter.

[0143] Many vectors for intracellular expression are commercially available. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, a protein-coding sequence (e.g., a sequence obtained from the information provided herein), an enhancer element, a promoter, and a transcription termination sequence. Those skilled in the art will be aware of sequences suitable 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, alpha factor leader, or acid phosphatase leader), or mammalian secretion signals (e.g., herpes simplex gD signal).

[0144] Exemplary promoters active in mammalian cells include the cytomegalovirus immediate early promoter (CMV-IE), the human elongation factor 1α 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 comprising the CMV enhancer / β-actin promoter or immunoglobulin promoter, or active fragments thereof. Examples of useful mammalian host cell lines are the SV40-transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651), the human embryonic kidney line (293 cells or 293 cells subcloned for growth in suspension culture), baby hamster kidney cells (BHK, ATCC CCL 10), or Chinese hamster ovary cells (CHO).

[0145] Exemplary promoters suitable for expression in yeast cells, such as yeast cells selected from the group including Pichia pastoris, Saccharomyces cerevisiae, and S. pombe, include, but are not limited to, the ADH1 promoter, the GAL1 promoter, the GALA promoter, the CUP1 promoter, the PH05 promoter, the nmt promoter, the RPR1 promoter, or the TEF1 promoter.

[0146] Host cells used to produce proteins 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), RPM1-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing mammalian cells. Media for culturing the other cell types discussed herein are known in the art.

[0147] Methods for isolating proteins are known in the art. If the protein is secreted into the culture medium, the supernatant from such expression systems can first be concentrated using a commercially available protein concentration filter, such as an Amicon ultrafiltration unit or a Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF can be included in any of the above steps to inhibit proteolysis, and antibiotics can be included to prevent the growth of adventitious contaminants. Alternatively or additionally, the supernatant can be filtered and / or separated from the cells expressing the protein, for example, using continuous centrifugation.

[0148] 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 chromatography), or any combination of the above.

[0149] These methods are known in the art and are described, for example, in WO 99 / 57134 or in Antibodies: A Laboratory Manual, Ed Harlow and David Lane (editors), Cold Spring Harbor Laboratory, (1988). Those skilled in the art will also recognize that proteins 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 proteins are then purified using methods known in the art, such as affinity purification. For example, proteins containing hexa-His tags are purified by contacting a sample containing the protein with nickel-nitrilotriacetic acid (Ni-NTA), which specifically binds to the hexa-His tag immobilized on a solid or semi-solid support, washing the sample to remove unbound proteins, and subsequently eluting the bound proteins. Alternatively or additionally, a ligand or antibody that binds to the tag is used in affinity purification methods.

[0150] The molecules of the present invention may be suitable for high-yield purification. The yield may be determined based on the amount of material retained during the purification process (i.e., the amount of correctly folded material obtained at the end of the purification process relative to the amount of solubilized material obtained before refolding) and / or the yield may be based on the amount of correctly folded material obtained at the end of the purification process relative to the original culture volume. High yield means a yield of more than 1%, or more than 5%, or higher. High yield means a yield of more than 1 mg / ml, or more than 3 mg / ml, or more than 5 mg / ml, or higher.

[0151] Pharmaceutical compositions and medical methods For administration to a patient, the molecules, nucleic acids, expression vectors, or cells of the invention may be provided as part of a pharmaceutical composition together with one or more pharmaceutically acceptable carriers or excipients. The pharmaceutical composition may be in any suitable form (e.g., depending on the desired method of administration to a patient). The pharmaceutical composition may be provided in unit dosage form, generally in a hermetically sealed container, and may be provided as part of a kit. Such a kit will usually (but not necessarily) include instructions for use. Such a kit may include a plurality of the unit dosage forms described above.

[0152] Pharmaceutical compositions may be adapted for administration by any suitable route, such as parenteral (including subcutaneous, intramuscular, intrathecal, or intravenous), enteral (including oral or rectal), inhalation, or intranasal. Such compositions may be prepared by any method known in the art of pharmacy, for example, by mixing the active ingredient with the carrier(s) or excipient(s) under sterile conditions. Methods for preparing proteins in a form suitable for administration to a subject (e.g., pharmaceutical compositions) are known in the art and include, for example, those described in Remington's Pharmaceutical Sciences (18th ed., Mack Publishing Co., Easton, PA, 1990) and the US Pharmacopeia: National Formulary (Mack Publishing Company, Easton, PA, 1984).

[0153] Pharmaceutical compositions typically comprise a solution of the multidomain binding molecules of the present invention (or the nucleic acids, cells, or vectors of the present invention) dissolved in a pharmaceutically acceptable carrier, such as an aqueous carrier. A variety of aqueous carriers can be used, such as buffered saline. Pharmaceutical compositions may contain pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, toxicity adjusting agents, and the like, as needed to approximate physiological conditions, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, and the like. The concentration of the molecules of the present invention in these formulations can vary widely and will be selected primarily based on fluid volume, viscosity, body weight, and the like, depending on the particular mode of administration selected and the patient's needs. Exemplary carriers include water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles, such as mixed oils and ethyl oleate, may also be used. Liposomes may also be used as carriers. The vehicle may contain minor amounts of additives that enhance isotonicity and chemical stability, such as buffers and preservatives.

[0154] The molecules of the present invention may have an ideal safety profile for use as a therapeutic agent. An ideal safety profile means that in addition to exhibiting good specificity, the molecules of the present invention can pass further preclinical safety tests. Examples of such tests include whole blood assays to confirm that cytokine release in the presence of whole blood is minimal, thus reducing the risk of potential cytokine release syndrome in vivo, and alloreactivity tests to confirm that the molecules are unlikely to recognize alternative HLA types.

[0155] The dosage of the molecules of the invention can vary between wide limits, depending on the disease or disorder being treated, the age and condition of the individual being treated, etc. Ultimately, the physician will determine the appropriate dosage to be used.

[0156] The multidomain binding molecules, pharmaceutical compositions, vectors, nucleic acids, and cells of the invention may be provided in substantially pure form, e.g., 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.

[0157] The multi-domain binding molecules of the present invention may also be associated with a therapeutic agent. Therapeutic agents that may be associated with the molecules of the present invention include immunomodulators and immune effectors, radioactive compounds, enzymes (e.g., perforin), or chemotherapeutic agents (e.g., cisplatin). To ensure that the toxic effect is exerted at the desired location, the toxin may be in a liposome linked to the multi-domain binding molecules described herein, thereby allowing the compound to be released slowly. This prevents harmful effects during transport within the body and ensures that the toxin has its maximum effect after the multi-domain binding molecules described herein bind to the relevant antigen-presenting cells.

[0158] Examples of suitable therapeutic agents include, but are not limited to: Small molecule cytotoxic agents, i.e., compounds capable of killing mammalian cells with a molecular weight of less than 700 daltons, may contain toxic metals that may have cytotoxic effects. Furthermore, these small molecule cytotoxic agents should be understood to include prodrugs, i.e., compounds that break down or are converted under physiological conditions to release a cytotoxic agent. Examples of such cytotoxic agents include cisplatin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodium photofrin II, temozolomide, topotecan, trimetreate glucuronate, auristatin E, vincristine, and doxorubicin. Peptide cytotoxins, i.e., proteins or fragments thereof that have the ability to kill mammalian cells, such as ricin, diphtheria toxin, Pseudomonas exotoxin A, DNase, and RNase. Radionuclides, i.e., unstable isotopes of elements that decay with the simultaneous emission of one or more alpha or beta particles or gamma rays, such as iodine-131, rhenium-186, indium-111, yttrium-90, bismuth-210 and bismuth-213, actinium-225, and astatine-213. Chelating agents can be used to facilitate the association of these radionuclides with the multi-domain binding molecule. Immunostimulators, i.e., immune effector molecules that stimulate the immune response, such as cytokines such as IL-2 and IFN-γ. Superantigens and their mutants. TCR-HLA fusions, for example fusions to peptide-HLA complexes, where the peptide is derived from a common human pathogen such as Epstein-Barr virus (EBV). Chemokines such as IL-8, platelet factor 4, melanoma growth stimulating protein, etc. Antibodies or fragments thereof, including anti-T cell antibodies or anti-NK cell determinant antibodies (e.g., anti-CD3, anti-CD28, or anti-CD16). An antibody or fragment thereof that binds to a molecule located at the immune synapse. Alternative protein scaffolds with antibody-like binding properties. Complement activator. Heterologous protein domains, allologous protein domains, viral / bacterial protein domains, viral / bacterial peptides.

[0159] The multidomain binding molecules, nucleic acids, vectors, pharmaceutical compositions, and cells of the present invention can be used to treat diseases such as cancer, particularly cancers associated with expression of tumor-associated antigens, such as GP100, NYESO, MAGEA4, or PRAME, as described in International Publication Nos. WO 2011 / 001152, WO 2017 / 109496, WO 2017 / 175006, and WO 2018 / 234319, and corresponding 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.

[0160] The cancer to be treated may be a cancer associated with PRAME expression. "Associated with PRAME expression" means that the cancer contains cancer cells that express PRAME. In this regard, the cancer may be a PRAME-positive cancer. The cancer may be known to be associated with PRAME expression, and therefore PRAME expression may not be assessed. Alternatively, PRAME expression can be assessed using any method known in the art, including, for example, histological methods. However, the present invention is not intended to be limited to the treatment of 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 Hodgkin's lymphoma. For example, the cancer associated with PRAME expression may be melanoma. The melanoma may be uveal melanoma or cutaneous melanoma. The lung cancer may 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, and the ovarian cancer can be epithelial ovarian cancer, such as high-grade serous ovarian cancer.

[0161] The multi-domain binding molecules, nucleic acids, vectors, pharmaceutical compositions, and cells of the present invention can be used to treat infectious diseases. Infectious diseases can be caused by bacterial, viral, fungal, or parasitic pathogens. Any infectious disease caused by a pathogen that results in antigen-presenting cells displaying MHC bound to pathogen-derived peptides may be suitable for treatment with the multi-domain binding molecules of the present invention. The multi-domain binding molecules of the present invention are particularly well suited for infectious diseases in which antigen-presenting cells display pathogen peptides at levels lower than optimal for the innate immune system to eliminate the infection without additional treatment. The infectious disease can be a chronic infectious disease. Exemplary infectious diseases include hepatitis B virus (HBV) infection and human immunodeficiency virus (HIV) infection.

[0162] The multidomain binding molecules of the present invention can be used in methods for treating autoimmune diseases, such as type 1 diabetes. Given the potential for serious adverse events associated with systemic immunosuppression, organ-specific immunosuppression, rather than systemic immunosuppression, may be a beneficial therapeutic route. In autoimmunity, growing evidence suggests that impairment of the PD-1 pathway plays an important role in the pathogenesis of the disease. Genetic polymorphisms in PD-1, PD-L1, and PD-L2 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. Therefore, activation of PD-1 on autoreactive lymphocytes may serve as 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 and tissue specificity) that can be administered less frequently.

[0163] The present invention also provides the following: a multi-domain binding molecule, nucleic acid, vector, pharmaceutical composition or cell of the invention for use in medicine, preferably for use in a method for the treatment of cancer or tumors or infectious diseases; a multi-domain binding molecule, nucleic acid, vector, pharmaceutical composition or cell of the invention for use in medicine, preferably for use in a method for the treatment of cancer or tumors or autoimmune diseases or infectious diseases; Use of a multidomain binding molecule, nucleic acid, vector, pharmaceutical composition, or cell of the invention in the manufacture of a medicament for treating cancer or tumors or infectious diseases; Use of a multidomain binding molecule, nucleic acid, vector, pharmaceutical composition, or cell of the invention in the manufacture of a medicament for treating cancer or a tumor or an autoimmune disease or an infectious disease; a method for treating cancer or tumor or infectious disease in a patient, the method comprising administering to the patient a multi-domain binding molecule, nucleic acid, vector, pharmaceutical composition or cell of the invention; A method for treating cancer or a tumor or an autoimmune disease or an infectious disease in a patient, the method comprising administering to the patient a multi-domain binding molecule, nucleic acid, vector, pharmaceutical composition, or cell of the invention; and An injectable formulation for administration to a human subject comprising a multi-domain binding molecule, nucleic acid, vector, pharmaceutical composition, or cell of the invention.

[0164] The method of treatment may further comprise administering, separately, in combination, or sequentially, additional anti-tumor agents, examples of which are known in the art and may include immune activators and / or T cell modulators.

[0165] Kits and manufactured products In another aspect, kits or articles of manufacture are provided containing materials useful for the treatment and / or prevention of the above diseases.

[0166] The kit may include (a) a container containing a molecule, nucleic acid, vector, or cell of the invention, optionally in a pharmaceutically acceptable carrier or diluent, and (b) a package insert containing instructions for treating a disease (e.g., cancer, an immune disease, or an autoimmune disease) in a subject. The kit may further include (c) at least one additional therapeutically active compound or drug.

[0167] 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 comprising the molecule, nucleic acid, vector, or cell of the invention and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is the molecule, nucleic acid, vector, or cell of the invention. The label or package insert indicates that the composition is used for treating a subject to be treated, e.g., a subject suffering from or predisposed to developing a disease described herein, and provides specific guidance regarding dosage and dosing intervals of the composition and other medications. The kit may further include an additional container containing a pharmaceutically acceptable diluent buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and / or dextrose solution. The kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0168] Optionally, the kit further comprises a container containing an effective amount of a second medicament, wherein the molecule, nucleic acid, vector, or cell of the invention is the first medicament, and the kit further comprises instructions in the package insert for treating a subject with the second medicament.

[0169] Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description, and fall within the scope of the appended claims. Preferred features of each aspect of the invention are applicable to each of the other aspects mutatis mutandis. All documents referred to herein are incorporated by reference to the fullest extent permitted by law. [Brief explanation of the drawings]

[0170] [Figure 1]1A and 1B are schematic diagrams of exemplary multi-domain single-chain binding molecules of the invention, where Figure 1a shows a representation of the domain arrangement from N-terminus to C-terminus, and Figure 1b shows a hypothetical representation of the folded structure of the molecule. [Figure 2] Figure 2 shows the results of an ELISpot assay using IFNγ as a readout for T cell activation. As a comparison, using the same TCR, multi-domain molecules were constructed using the format previously disclosed in WO 2007 / 024990 and tested alongside the single chain format shown in Figure 1. In Figure 2, a schematic representation of each format is placed to show the corresponding data points. [Figure 3] FIG. 10 depicts graphs of surface plasmon resonance experiments assessing the binding of mol093v9 and mol093v11 to pHLA, CD3, and FcRn, respectively. [Figure 4] Figure 1 shows the pharmacokinetic profile evaluated in Tg32 SCID mice. Mice were dosed at 1 mg / Kg by IV bolus and serial blood samples were collected over a 21-day period. Samples were detected in serum by electrochemiluminescence immunoassay. The graph shows serum concentrations over time for four individual mice. [Figure 5] FIG. 10 is a graph showing the results of an ELISPot assay in which T cell activation of mol093v9 and mol093v11 was evaluated in vitro. [Figure 6] Figure 6 depicts a graph showing the results of an ELISPot assay directly comparing T cell activation of mol093v9 with an alternative molecule (WO 2018 / 234319) that targets the same PRAME peptide but does not contain a half-life-extending Fc domain. Figure 6 shows that both molecules drive similarly potent T cell responses. [Figure 7] FIG. 10 depicts a graph demonstrating real-time killing of antigen-positive cells in the presence of mol093v9 and mol093v11 as determined using the xCELLigence platform. [Figure 8]

[0023] Figure 8 depicts a graph showing the results of a T cell killing assay directly comparing mol093v9 to an alternative molecule (WO 2018 / 234319) that targets the same PRAME peptide but does not contain a half-life extended Fc domain. Figure 8 shows that mol093v9 demonstrates comparable killing data to the non-HLE version of the molecule ("Mol001"). [Figure 9] Figure 1 shows data from an ELISPOT T cell activation assay obtained using two normal cell lots (cardiac cells (HCM27) and lung epithelial cells (HSAEpiC9)) for one PBMC effector donor. Minimal T cell activation against normal cells was observed at concentrations of mol093v9 and mol093v11 below 1.1 nM of fusion molecule. [Figure 10] Figure 10 depicts a graph showing the results of an ELISPOT T cell activation assay directly comparing the reactivity of normal cells to mol093v9 with an alternative molecule (WO 2018 / 234319) that targets the same PRAME peptide but does not contain a half-life-extending Fc domain. Figure 10 shows that both molecules similarly lack reactivity with normal cells from skin (melanocytes) and kidney (renal proximal tubules). [Figure 11] FIG. 1 depicts a graph showing T cell activation as measured by IFNγ release for the TCR anti-CD3 fusion molecule a40b23U28-mol93 in the presence of antigen-positive and antigen-negative cancer cell lines. [Figure 12] FIG. 10 depicts a graph showing T cell activation as measured by IFNγ release for the half-life extended TCR anti-CD3 fusion molecules a40b23U28-mol93 and a40b23U28-mol14 in the presence of antigen-positive cells. DETAILED DESCRIPTION OF THE INVENTION

[0171] Array Description SEQ ID NO: 1 HLA-A * 02 Restrictive peptide: SLLQHLIGL.

[0172] SEQ ID NO: 2: Amino acid sequence of the alpha chain variable domain of an exemplary TCR. The CDRs (CDR1, CDR2, and CDR3) are underlined and are designated SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively, and the framework regions (FR1, FR2, FR3, and FR4) are italicized and are designated SEQ ID NO: 27, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 28, respectively. This sequence contains the N24Q mutation (double underlined), which removes the N-linked glycosylation site. TIFF2025528212000003.tif14170

[0173] SEQ ID NO: 8: Amino acid sequence of the TCR β chain variable domain of an exemplary TCR. The CDRs (CDR1, CDR2, and CDR3) are underlined and are designated SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively, and the framework regions (FR1, FR2, FR3, and FR4) are italicized and are designated SEQ ID NO: 29, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 30, respectively. TIFF2025528212000004.tif15170

[0174] SEQ ID NO: 14: Amino acid sequence of the TCR alpha chain of an exemplary TCR. The CDRs (CDR1, CDR2, and CDR3) are underlined and are designated SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively; the framework regions (FR1, FR2, FR3, and FR4) are italicized and are designated SEQ ID NO: 27, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 28, respectively. The constant region is shown in bold and is designated SEQ ID NO: 15. Within the constant region, the non-native cysteine ​​residue introduced to create an interchain disulfide bond is double underlined (constant region position 48). This sequence also contains the following substitutions (double underlined): N24Q, N148Q, N182Q, and N193Q, which remove each N-linked glycosylation site. TIFF2025528212000005.tif29170

[0175] SEQ ID NO: 16: Amino acid sequence of the TCR β chain of an exemplary TCR. The CDRs (CDR1, CDR2, and CDR3) are underlined and are designated SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively; the framework regions (FR1, FR2, FR3, and FR4) are italicized and are designated SEQ ID NO: 29, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 30, respectively. The constant region is shown in bold (not underlined) and is designated SEQ ID NO: 19. Within the constant region, the non-native cysteine ​​residue introduced to create an interchain disulfide bond is shaded (constant region position 57). This sequence also includes the N184Q substitution (double underlined), which removes an N-linked glycosylation site. TIFF2025528212000006.tif29170

[0176] SEQ ID NO: 17. An exemplary anti-CD3 scFv (T cell-engaging immune effector domain), referred to herein as "U0." The light chain variable domain (VL) is in italics and is designated SEQ ID NO: 31. The light chain CDRs (CDR1, CDR2, and CDR3) are underlined and are designated SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35. The heavy chain variable domain (VH) is shown in bold and is designated SEQ ID NO: 32. The heavy chain CDRs (CDR1, CDR2, and CDR3) are underlined and are designated SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38. The glycine-serine linker connecting VL and VH is shown in plain text and is designated SEQ ID NO: 39. TIFF2025528212000007.tif37170

[0177] SEQ ID NO:40: Another exemplary anti-CD3 scFv (T cell-engaging immune effector domain), referred to herein as "U28." This sequence is the same as SEQ ID NO:17 above, except for two substitutions (T164A and I201F) that are double-underlined. The light chain variable domain (VL) is in italics and is designated SEQ ID NO:31. The light chain CDRs (CDR1, CDR2, and CDR3) are underlined and are designated SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35. The heavy chain variable domain (VH) is shown in bold and is designated SEQ ID NO:41. The heavy chain CDRs (CDR1, CDR2, and CDR3) are underlined and are designated SEQ ID NO:48, SEQ ID NO:37, and SEQ ID NO:38. The glycine-serine linker connecting VL and VH is shown in plain text and is designated SEQ ID NO:39. TIFF2025528212000008.tif36170

[0178] SEQ ID NO: 54 Unmodified human IgG1 Fc region (CH2 and CH3 domains) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0179] SEQ ID NO: 42: Exemplary IgG1 Fc region sequence. This sequence has four double-underlined substitutions compared to the unmodified IgG1 Fc sequence above (SEQ ID NO: 54). These are the N297G substitution, which inhibits binding to FcγR, and the T366S, L368A, and Y407V substitutions (forming a hole) that promote dimerization with another Fc region (e.g., SEQ ID NO: 43) containing the T366W substitution (forming a knob). The numbering of the substitutions in this sequence follows the EU numbering scheme. TIFF2025528212000009.tif30170

[0180] SEQ ID NO: 43: Another exemplary IgG1 Fc region sequence. This sequence has two double-underlined substitutions compared to the unmodified IgG1 Fc sequence above (SEQ ID NO: 54). These are the N297G substitution, which inhibits binding to FcγR, and the T366W substitution (a knob-forming substitution), which promotes dimerization with another Fc region (e.g., SEQ ID NO: 42) containing the T366S, L368A, and Y407V substitutions (a hole-forming substitution). The numbering of the substitutions in this sequence follows the EU numbering scheme. TIFF2025528212000010.tif29170

[0181] An exemplary IgG1 Fc region sequence is shown below: SEQ ID NO: 57. This sequence has seven substitutions (bold) compared to the unmodified IgG1 Fc sequence above (SEQ ID NO: 54). These are the N297G substitution, which inhibits binding to FcγR; the T366S, L368A, and Y407V substitutions (hole-forming substitutions) that promote dimerization with another Fc region (e.g., SEQ ID NO: 58) containing the T366W substitution (knob-forming substitution); and the M252Y, S254T, and T256E substitutions that enhance binding to FcRn. The numbering of the substitutions in this sequence follows the EU numbering scheme. TIFF2025528212000011.tif29170

[0182] A further exemplary IgG1 Fc region sequence is shown below: SEQ ID NO: 58. This sequence has five substitutions (bold) compared to the unmodified IgG1 Fc sequence above (SEQ ID NO: 54). These are the N297G, T366S, L368A, and Y407V substitutions (hole-forming substitutions) that inhibit binding to FcγR; the T366W substitution (knob-forming substitution) that promotes dimerization with another Fc region (e.g., SEQ ID NO: 57); and the M252Y, S254T, and T256E substitutions that enhance binding to FcRn. The numbering of the substitutions in this sequence follows the EU numbering scheme. TIFF2025528212000012.tif28170

[0183] SEQ ID NO: 44 Exemplary IgG1 hinge sequence (comprising a C to S substitution at position 5 numbered according to SEQ ID NO: 44 compared to the native human IgG1 sequence): EPKSSDKTHTCPPCP

[0184] SEQ ID NO: 52 Truncated IgG1 hinge sequence: DKTHTCPPCP

[0185] SEQ ID NO: 53 IgG4 hinge sequence: ESKYGPPCPSCP

[0186] SEQ ID NO:45: Complete amino acid sequence of an exemplary multi-domain single-chain binding molecule designated "mol093v11." The T cell-engaging immune effector domain (underlined) is the anti-CD3 scFv sequence ("U0") shown in SEQ ID NO:17. The pMHC-binding domain is double underlined and comprises the TCR β chain sequence (in this case "VC1") shown in SEQ ID NO:16 (double underline, regular letters) and the TCR α chain sequence (in this case "VC2") shown in SEQ ID NO:14 (double underline, bold letters). The half-life extending domain is an Fc domain that is a dimer formed between the Fc region sequence (in this case the FC1 region) shown in SEQ ID NO:42 (italics) and the Fc region sequence (in this case the FC2 region) shown in SEQ ID NO:43 (italics and bold). TIFF2025528212000013.tif145170

[0187] SEQ ID NO:46: Complete amino acid sequence of an exemplary multi-domain single-chain binding molecule designated "mol093v9." The T cell-engaging immune effector domain (underlined) is the anti-CD3 scFv sequence ("U28") shown in SEQ ID NO:40. The pMHC-binding domain is double underlined and comprises the TCR β chain sequence (in this case "VC1") shown in SEQ ID NO:16 (double underline, regular letters) and the TCR α chain sequence (in this case "VC2") shown in SEQ ID NO:14 (double underline, bold letters). The half-life extending domain is an Fc domain that is a dimer formed between the Fc region sequence (in this case the FC1 region) shown in SEQ ID NO:42 (italics) and the Fc region sequence (in this case the FC2 region) shown in SEQ ID NO:43 (italics and bold). TIFF2025528212000014.tif145170

[0188] SEQ ID NO:55: Complete amino acid sequence of an exemplary multi-domain single-chain binding molecule designated "a40b23U28-mol93." The T cell-engaging immune effector domain (underlined) is the anti-CD3 scFv sequence ("U28") set forth 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 comprises the TCR β chain sequence (in this case "VC1") (double underline, regular letters) and the TCR α chain sequence (in this case "VC2") (double underline, bold letters). The half-life extending domain is an Fc domain that is a dimer formed between the Fc region sequence set forth in SEQ ID NO:42 (in this case the FC1 region) (italics) and the Fc region sequence set forth in SEQ ID NO:43 (in this case the FC2 region) (italics and bold). TIFF2025528212000015.tif143170

[0189] SEQ ID NO: 56 Amino acid sequence of the PIWIL1 peptide bound by a40b23U28-mol93 (when complexed with MHC). SLSNRLYYL

[0190] Additional linker sequences: 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). [Example]

[0191] The present invention will be more fully understood by reference to the following examples. However, these examples should not be construed as limiting the scope of the present invention. The examples and embodiments described herein are for illustrative purposes only, and it is understood that various modifications or changes therein will be suggested to those skilled in the art and are included within the scope of this application and the scope of the appended claims.

[0192] Example 1 - Multi-domain molecules with improved potency Multidomain molecules comprising a TCR-anti-CD3 fusion protein and incorporating a half-life-extending Fc domain have previously been described and shown to be functional in U.S. Patent No. 5,627,999. However, such molecules were subsequently found to have a significantly reduced ability to activate T cells in vitro compared to non-Fc-fused versions of the molecule and were therefore deemed suboptimal for therapeutic use. Further engineering was undertaken to identify novel molecular formats with improved therapeutic properties.

[0193] We constructed a multidomain molecule in which each functional domain is arranged on a single polypeptide chain. Figure 1a shows a schematic representation of the domain arrangement, and Figure 1b shows a hypothetical representation of the folded structure of the molecule.

[0194] In a first example, the TCR domain of the multi-domain single chain molecule is linked to an HLA-A domain derived from PRAME, as previously described (WO 2018 / 234319). * The molecule was designed to recognize the 02-restricted peptide SLLQHLIGL (SEQ ID NO: 1). The ability of this molecule to drive T cell activation in the presence of antigen-positive cancer cells was investigated using an ELISpot assay, using IFNγ as a readout for T cell activation. As a comparison, the same TCR was used to construct a multi-domain molecule using the format previously disclosed in U.S. Patent No. 5,629,493 and tested alongside the single-chain format shown in FIG. 1.

[0195] The data presented in Figure 2 demonstrate that the single chain molecule is able to drive significantly improved T cell responses against antigen-positive cancer cells than previously disclosed formats.

[0196] Additionally, 37 molecular formats with alternative domain arrangements were generated and tested for in vitro efficacy. None of these formats performed better than the molecules shown in Figure 1. Little or no response was observed in 17 of these formats, low-level responses were observed in 15 formats, and moderate-level responses were observed in 2 formats. The remaining 3 formats demonstrated increased cross-reactivity against antigen-negative cell lines in addition to reduced potency against antigen-positive cells.

[0197] Example 2 - Preparation of single-chain multi-domain molecules targeting PRAME Two multidomain molecules (designated mol093v9 and mol093v11) were prepared using the format shown in Figure 1. The TCR regions of both molecules were cloned into the HLA-A domain derived from PRAME. * The two molecules were designed to recognize the CD3-restricted peptide SLLQHLIGL. 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 shown in SEQ ID NO:46 and SEQ ID NO:45, respectively.

[0198] Expression Mol093v9 and mol093v11 were expressed in Cho cells using Thermo's ExpiCHO™ transient expression protocol. Briefly, 6×10 cells were cultured. 6 Transfection was performed after dilution to a concentration of 100 μg. Cells were harvested 14 days after transfection and the temperature was shifted to 32°C on day 1 after transfection. Feed additions were performed on days 1 and 5 after transfection. Clarification was performed by two consecutive centrifugation steps at 300 × g and 17,500 × g. The resulting supernatant was passed through 0.45 μm and 0.2 μm membrane filters.

[0199] purification The clarified supernatant was purified using Protein A, followed by a size-exclusion chromatography step. A 15 cm bed height MabSelect Extra Protein A resin column was prepared. 50 column volumes of the supernatant were loaded onto the column and eluted using pH 3.0 sodium citrate buffer. The eluted product was collected over three column volumes, filtered through a 0.2 μm membrane filter, and neutralized by adding 2 M Tris. The Protein A eluate was concentrated to at least 2 mg / mL using tangential flow filtration (Pellicon™ XL50 with Ultracel™ 30 kDa membrane) and then loaded onto HiLoad 26 / 600 Superdex SEC resin. 5% of the column volume was loaded onto the column. The product was eluted in phosphate-citrate buffer, and the relevant fraction was filtered through a 0.22 μm membrane filter.

[0200] yield The concentration of the purified material was determined by absorbance at 280 nm using a Nanodrop spectrophotometer.

[0201] The calculated yield per liter of supernatant was 9.6 mg / L for mol093v9 and 17 mg / L for mol093v11.

[0202] stability The stability of the molecule was assessed by SEC UPLC for 14 days after freeze / thaw cycles and / or under conditions of i) heat stress and ii) agitation. The results are shown in the table immediately below, which indicates the purity of the mol093v11 monomer under the conditions indicated. In both cases, the purity of the monomer was deemed acceptable.

[0203] [Table 2]

[0204] Example 3 - Binding affinity and kinetics of multi-domain molecules targeting PRAME To verify that the TCR and anti-CD3 portions of the molecules bound 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).

[0205] method The chip used was from the Biotin CAPture Kit series S (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 guanidine hydrochloride (8 M) (GuHCl) and sodium hydroxide (1 M) (NaOH) in a 3 + 1 ratio. The flow rate was 20 μL / min, with a contact time of 120 s. The chip was activated with biotin CAPture reagent (diluted 1:16 in PBS + P20). The flow rate was 2 μL / min for 300 s. The amount captured was 1600 response units (RU). Biotinylated pHLA was injected at 10 μg / mL for 120 s at a flow rate of 10 μL / min.

[0206] For single-cycle kinetic analysis, serial dilutions of mol093v9 and mol093v11 were injected at a flow rate of 60 μL / min (maximum concentration = 15 nM), with 200 s of dissociation between injections and 7200 s of dissociation for the fifth injection.

[0207] Subsequently, CD3(γε) was injected at a concentration of 300 nM and a flow rate of 10 μL / min for 60 seconds.

[0208] To capture FcRn, flow cells were primed with PBS + P20 (0.005%), pH 6.0. Biotinylated FcRn was injected at 5 μg / mL for 120 seconds at a flow rate of 2 μL / min. After FcRn injection, 5 μM biotin was injected into all flow cells at 10 μL / min for 120 seconds. The amount of FcRn was 450 RU.

[0209] Mol093v9 or mol093v11 were injected at 15 nM at a flow rate of 10 μL / min for 300 seconds and allowed to dissociate for 600 seconds. The responses were 152 RU and 163 RU for mol093v9 and mol093v11, respectively.

[0210] Kinetic parameters were calculated using the manufacturer's software. The dissociation phase was fitted to a single exponential decay equation, which allowed for the calculation of the half-life. The equilibrium constant K D k off / k on It was calculated from.

[0211] result Mol093v9 and mol093v11 exhibited picomolar affinity for the pHLA complex, along with high levels of CD3 activity and FcRn binding. The data are shown in Figure 3, and the binding parameters are summarized in the table immediately below.

[0212] [Table 3]

[0213] Example 4 - Pharmacokinetics Pharmacokinetic properties were evaluated in Tg32 SCID mice. Test articles were administered via IV bolus at 1 mg / kg to four mice per compound, and blood samples were collected serially over a 21-day period. Samples were detected in serum by electrochemiluminescence immunoassay using biotinylated PRAME peptide-HLA capture and sulfo-tagged anti-scFv antibody detection. Figure 4 shows serum concentrations over time for four individual mice. PK parameters were extracted by noncompartmental analysis.

[0214] result Mol93v9 terminal phase 1 / 2 was calculated to be 9 days. The results of the non-compartmental analysis are shown in the table immediately below.

[0215] [Table 4]

[0216] The results of the two-compartment modeling using NONMEM are shown in the table immediately below.

[0217] [Table 5]

[0218] Example 5 - In vitro T cell activation Mol093v9 and Mol093v11 were identified as SLLQHLIGL-HLA-A * CD3 on cells presenting the O2 complex + Their ability to mediate potent and specific activation of T cells was assessed. Interferon-γ (IFN-γ) release was used as a readout for T cell activation.

[0219] method Assays were performed using a human IFN-γ ELISPOT kit (BD Biosciences) according to the manufacturer's instructions. Briefly, target cells were cultured at 1 × 10 per ml in assay medium (RPMI 1640 containing 10% heat-inactivated FBS and 1% penicillin-streptomycin-L-glutamine). 6 Cells were prepared at a density of 50,000 cells per well and plated at 50,000 cells per well in a volume of 50 μl. Peripheral blood mononuclear cells (PBMCs) isolated from fresh donor blood were used as effector cells and plated at an approximately 1:1 ratio with target cells in a volume of 50 μl (the exact number of PBMCs used in each experiment is donor-dependent and can be adjusted to generate responses within a range appropriate for the assay). Fusion molecules were titrated incrementally from 10 nM to obtain the indicated final concentrations (spanning the expected clinically relevant range) and added to wells in a volume of 50 μl.

[0220] Plates were prepared according to the manufacturer's instructions. Target cells, effector cells, and fusion molecules were added to the relevant wells and adjusted to a final volume of 200 μl with assay medium. All reactions were performed in triplicate. Control wells omitting the fusion molecules were also prepared. Plates were then incubated overnight (37°C / 5% CO2). The next day, plates were washed three times with wash buffer (prepared in deionized water using a 1x PBS bag containing 0.05% Tween-20). Primary detection antibody was then added to each well in a volume of 50 μl. Plates were incubated for 2 hours at room temperature and then washed three times again. Secondary 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 step. 15 minutes before use, one drop (20 μl) of AEC chromogen was added per 1 ml of AEC substrate, mixed, and 50 μl was added to each well. The color development of the spots was monitored periodically, and the plates were washed with tap water to stop the color reaction. After the plates were dried at room temperature for at least 2 hours, spot counting was performed using a CTL analyzer equipped with Immunospot software (Cellular Technology Limited).

[0221] In this example, the following cell lines were used as target cells: Antigen positive: Mel624-human melanoma cell line NCI-H1755-Non-small cell lung cancer (NSCLC) cell line OV56-ovarian serous carcinoma cell line THP-1-acute monocytic leukemia cell line NCI-H1703-Lung squamous cell carcinoma cell line COV318-Ovarian serous carcinoma cell line Antigen negative: TY-KNU-Ovarian serous adenocarcinoma (HLA-A * 02 negative; PRAME negative) NCI-H1693-Non-small cell lung cancer (NSCLC) cell line (HLA-A * 02 positive; PRAME negative).

[0222] result Mol093v9 and mol093v11 were shown to potently activate T cells in the presence of various antigen-positive cancer cells. 50 Values ​​were calculated from data obtained using PBMCs from two separate donors. T cell activation EC for both donors 50 Values ​​are shown in the table immediately below. Figure 5 shows data obtained from donor 1. Limited responses were observed in antigen-negative cell lines.

[0223] [Table 6]

[0224] Comparison Data The T cell activation driven by mol093v9 was directly compared to alternative molecules that target the same PRAME peptide but do not contain a half-life-extending Fc domain. Such molecules are described in WO 2018 / 234319 and U.S. Pat. No. 11,427,624 (the contents of each are incorporated herein by reference). ELISPot assays were performed as described above. Figure 6 shows that both molecules drive similarly potent T cell responses.

[0225] Example 6 - T cell killing Mol093v9 and Mol093v11 were evaluated for their ability to mediate potent and specific killing of antigen-positive cancer cells.

[0226] method Assays were performed using either the xCELLigence platform with the appropriate 96-well plate for impedance reading (xCELLigence E-plate 96 PET, part number 300600900) or the Incucyte live-cell imaging platform with the CellPlayer 96-well Caspase-3 / 7 Apoptosis Assay Kit (Essen BioScience, catalog number 4440) according to the manufacturer's instructions. Target cells were plated at their optimal density (the number of targets added per well varied for each cell line and was previously titrated to determine optimal conditions) and incubated overnight to allow them to adhere. Test molecules were prepared at various concentrations, and 50 μl of each was added to the relevant wells to achieve final concentrations between 100 fM and 10 nM. Effector cells were used at a 10:1 effector-to-target cell ratio and plated in 50 μl. Control samples containing either effector or target cells alone were also prepared, along with fusion-free controls. For the xCELLigence platform, the final volume in the plate was adjusted to 200 μl using assay medium. The percentage of cell lysis was determined using a normalized cell index (impedance measurement). For the Incucyte platform, NucView assay reagent was adjusted to 30 μM and 25 μl was added to every well, bringing the final volume to 150 μl (yielding a final concentration of 5 μM). The number of apoptotic cells in each image was determined and measured by 1 mm 2 The number of apoptotic cells per 1000 cells was recorded. In all cases, assays were performed in triplicate and measurements were taken every 2 hours for 96 hours.

[0227] result The data shown in Figure 7 demonstrates real-time killing of antigen-positive cells in the presence of mol093v9 and mol093v11 as determined using the xCELLigence platform. 50 The values ​​are shown in the table immediately below and were in the low pM range. Limited killing of antigen-negative cell lines was detected.

[0228] [Table 7]

[0229] Comparison Data We directly compared T cell activation driven by mol093v9 with an alternative molecule that targets the same PRAME peptide but does not contain a half-life-extending Fc domain. Such molecules are described in WO 2018 / 234319. Killing assays were performed using the Incucyte platform as described above. Figure 8 shows that both molecules drive similarly potent killing responses.

[0230] Example 7 - Minimal reactivity to high-risk normal tissues To demonstrate the specificity of mol093v9 and mol093v11, further studies were performed using the same ELISPOT assay as above and a panel of normal cells derived from healthy human tissues as targets, including heart, lung, kidney, and skin.

[0231] The TCR-anti-CD3 fusion molecule was tested at six different concentrations, ranging from 50 pM to 10 nM, against target normal cell lots co-cultured with PBMCs from healthy donors. 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.

[0232] result Figure 9 shows data obtained using two normal cell lots (cardiac cells (HCM27) and lung epithelial cells (HSAEpiC9)) for one PBMC effector donor. Minimal T cell activation against normal cells was observed at concentrations of mol093v9 and mol093v11 below 1.1 nM of the fusion molecule.

[0233] Comparison Data Normal cell reactivity to mol093v9 was directly compared to an alternative molecule that targets the same PRAME peptide but does not contain a half-life-extending Fc domain. Such a molecule is described in WO 2018 / 234319. Figure 10 shows that both molecules similarly lack reactivity with normal cells from skin (melanocytes) and kidney (renal proximal tubules).

[0234] Example 8 - Multi-domain single-chain binding molecules targeting the PIWIL1 peptide-MHC complex exhibit potent and specific T cell activation In contrast to the above-described mol093v9 and mol093v11, which bind to the PRAME peptide-MHC complex, we designed an additional multi-domain single-chain binding molecule containing a pMHC-binding domain that targets the PIWIL1 peptide (SLSNRLYYL, SEQ ID NO: 56)-MHC complex. The full sequence of the resulting molecule, designated "a40b23U28-mol93," is shown in SEQ ID NO: 55. The sequence of a40b23U28-mol93 is identical to mol093v9 except for the TCR α and TCR β variable domains. The TCR α and TCR β variable domains of a40b23U28-mol93 correspond to SEQ ID NO: 28 ("a40") and SEQ ID NO: 36 ("b23"), respectively, in UK Patent Application No. 2300226.4.

[0235] a40b23U28-mol93 and SLSNRLYYL (SEQ ID NO: 56)-HLA-A * Binding to the 02 complex was determined using SPR as described in Example 3 above. Binding affinity (K D ) is 50 pM, and the binding half-life (t 1 / 2 ) was 11.8 hours. These results were comparable to the equivalent TCR-anti-CD3 fusion molecule without the half-life extending (i.e., Fc) domain.

[0236] The ability of a40b23U28-mol93 to stimulate T cell activation was determined by measuring IFNγ secretion 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) cells were used as antigen-positive target cells. NCI-H1755 cells were used as antigen-negative cells. Data were plotted using PRISM software, and EC values ​​were calculated from the curves. 50 values ​​were calculated.

[0237] Figure 11 shows that a40b23U28-mol93 has EC2s in the low pM range against two antigen-positive cell lines. 50 values ​​(42.1 pM for KATO-III and 164.0 pM for CL11), indicating little to no response in the presence of antigen-negative cells (at concentrations of a40b23U28-mol93 less than 1 nM). Equivalent TCR-anti-CD3 fusion molecules lacking the half-life-extending (i.e., Fc) domain exhibited EC values ​​of 12.9 pM and 52.7 pM for KATO-III and CL11 cells, respectively. 50 had.

[0238] These data demonstrate that the multi-domain single-chain binding molecules of the invention, comprising a pMHC-binding domain that targets the PIWIL1 peptide-MHC complex, retain the same high affinity and potency as comparable TCR-anti-CD3 fusion molecules lacking the half-life extending (i.e., Fc) domain, and retain specificity for antigen-positive cells.

[0239] In further experiments, a40b23U28-mol93 was compared with an alternative multidomain molecule format called a40b23U28-mol14. a40b23U28-mol14 has the same amino acid sequences of the individual domains as a40b23U28-mol93, except that it is arranged in a two-chain format, as shown in Figure 12. In this two-chain format, the first chain (the left chain in Figure 12) contains, from N- to C-terminus, the TCR α chain variable domain, the TCR α chain constant domain, and the Fc region. The second chain (the right chain in Figure 12) contains, from N- to C-terminus, the anti-CD3 scFv, the TCR β chain variable domain, the TCR β chain constant domain, and the Fc region. T cell activation against the KATO III cell line was compared between the two molecules. As shown in Figure 12, both molecules drive T cell activation, but Mol93 exhibits a more potent response than Mol14.

[0240] The results described in this example demonstrate that TCR variable domains that bind to PIWIL1 peptide-MHC complexes 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 PRAME peptide-MHC or PIWIL1 peptide-MHC), the multi-domain single chain format was effective in extending half-life (by providing an Fc domain) without significantly affecting affinity for the target or potency of T cell activation.

Claims

1. i) comprising a linkage between a first variable region and a constant region (VC1) and a linkage between a second variable region and a constant region (VC2), wherein VC1 comprises the variable region and constant region of TCRβ, and VC2 comprises the variable region and constant region of TCRα, and VC1 and VC2 dimerize to form a pMHC-binding domain, ii) A T-cell engaging immunoeffector domain comprising an antibody light chain variable region (TCE-VL) and an antibody heavy chain variable region (TCE-VH), iii) A half-life extension 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, A multi-domain single-chain binding molecule, including The T cell-engaging immune effector domain is ligated to the N-terminus of VC1, VC1 is ligated to the N-terminus of the FC1 region via its C-terminus, the FC1 region is ligated to the N-terminus of VC2 via its C-terminus, and VC2 is ligated to the N-terminus of the FC2 region via its C-terminus, and The pMHC-binding domain and the T-cell-engaging immune effector domain are multi-domain single-chain binding molecules that can bind to the pMHC complex and T cells, respectively.

2. The T cell-engaging immune effector is ScFv, the multi-domain single-chain binding molecule according to claim 1.

3. The multiple-domain single-chain binding molecule according to claim 1 or 2, wherein the TCE-VL region is linked to the N-terminus of the TCE-VH region via its C-terminus, and the TCE-VH region is linked to the N-terminus of VC1 via its C-terminus.

4. The multi-domain single-chain binding molecule according to claim 1 or 2, wherein the T cell-engaging immune effector domain is a CD3 effector domain that activates T cells through interaction with CD3 and / or the TCR / CD3 complex.

5. The pMHC-binding domain binds to a tumor-associated antigen peptide that forms a complex with MHC, as described in claim 1 or 2.

6. The TCE-VH region, TCE-VL region, VC1 region, VC2 region, FC1 region, and two or more FC2 regions are linked to each other via linkers and / or IgG hinge arrays. The linker(s) may have a sequence selected from the group consisting of GGGGS (SEQ ID NO: 18), GGGSG (SEQ ID NO: 20), GGSGG (SEQ ID NO: 21), GSGGG (SEQ ID NO: 22), GSGGGGP (SEQ ID NO: 23), GGEPS (SEQ ID NO: 24), GGEGGGP (SEQ ID NO: 25), GGEGGGSEGGGGS (SEQ ID NO: 26), GGGGSGGGG (SEQ ID NO: 47), GGGGSGGGGGSGGGGGSGGGGGSGGGS (SEQ ID NO: 39), GGGGSGGGGGSGGGGGS (SEQ ID NO: 49), EAAAK (SEQ ID NO: 50), and EAAAKEAAAAKEAAAAK (SEQ ID NO: 51). A multi-domain single-chain binding molecule according to claim 1 or 2.

7. VC1 is connected to the FC1 region via an array including an IgG hinge array, and / or VC2 is connected to the FC2 region via an array including an IgG hinge array. The multi-domain binding single-chain molecule according to claim 1 or 2, wherein the IgG hinge sequence may be at least 80% identical to sequence number 44.

8. The half-life extension domain comprises one or more amino acid substitutions that promote dimerization between the FC1 region and the FC2 region, optionally, (a) (i) One of the FC1 region and the FC2 region contains one or more amino acid substitutions selected from the group consisting of T366S, L368A, T394S, F405A, Y407A, Y407T, and Y407V according to the EU numbering scheme, (ii) The other of the FC1 region and the FC2 region contains one or more amino acid substitutions selected from the group consisting of T366W, T366Y, T366W, T394W, and F405W according to the EU numbering scheme, or (b) (i) One of the FC1 region and the FC2 region contains one or more amino acid substitutions selected from the group consisting of T366S, L368A, and Y407V according to the EU numbering scheme, (i) The other of the FC1 region and the FC2 region includes the amino acid substitution T366W according to the EU numbering scheme, A single-chain molecule capable of binding multiple domains according to claim 1 or 2.

9. The aforementioned half-life extension domain includes one or more amino acid substitutions selected from the group consisting of S228P, E233P, L234A, L235A, L235E, L235P, G236R, G237A, P238S, F241A, V264A, D265A, H268A, D270A, N297A, N297G, N297Q, E318A, K322A, L328R, P329G, P329A, A330S, A330L, P331A, and P331S according to the EU numbering scheme, and optionally includes, The FC1 region and / or the FC2 region are: (i) containing amino acid substitutions of N297G according to the EU numbering scheme; or (ii) Including amino acid substitutions of M252Y, S254T, and T256E, which are numbered according to the EU numbering scheme, A single-chain molecule capable of binding multiple domains according to claim 1 or 2.

10. The aforementioned T cell-engaging immune effector domain is (a) (i) A VL region containing the CDRs of sequence numbers 33, 34, and 35 as CDR1, CDR2, and CDR3, respectively, and (ii) A VH region containing the CDRs of sequence numbers 36, 37, and 38 as CDR1, CDR2, and CDR3, respectively, and / or (b) The VL region is at least 80% identical to the sequence of sequence number 31, and the VH region is at least 80% identical to the sequence of sequence number 32. A multi-domain binding single-chain molecule according to claim 1 or 2, comprising:

11. From the N-terminus to the C-terminus, the amino acid sequence is as follows: a) A sequence in which the amino acid sequence of anti-CD3 scFv is followed by the linker sequence shown in SEQ ID NO: 18, b) Amino acid sequences of the variable and constant regions of TCRβ, c) A sequence in which the linker sequence shown in sequence number 47 is followed by the IgG hinge sequence shown in sequence number 44. d) Fc region having the sequence shown in sequence number 42, e) Linker sequence shown in Sequence ID No. 47, f) Amino acid sequences of the variable and constant regions of TCRα, g) A sequence in which the linker sequence shown in sequence number 47 is followed by the IgG hinge sequence shown in sequence number 44, and h) Fc region having the sequence shown in sequence number 43, A multi-domain binding single-chain molecule according to claim 1 or 2, comprising:

12. A nucleic acid encoding a multi-domain binding single-chain molecule as described in claim 1.

13. An expression vector comprising the nucleic acid described in claim 12.

14. A host cell comprising the nucleic acid described in claim 12 or the expression vector described in claim 13.

15. A method for producing a multi-domain binding single-chain molecule according to claim 1 or 2, comprising: maintaining a host cell according to claim 14 under conditions optimal for the expression of the nucleic acid according to claim 12 or the expression vector according to claim 13; and isolating the multi-domain binding single-chain molecule.

16. A pharmaceutical composition comprising a multi-domain binding single-chain molecule according to claim 1 or 2, a nucleic acid according to claim 12, an expression vector according to claim 13, or a host cell according to claim 14.

17. A pharmaceutical composition according to claim 16, to be used as a pharmaceutical.

18. Used in the treatment of cancer, autoimmune diseases, or infectious diseases. The pharmaceutical composition according to claim 16, wherein the cancer may be related to PRAME expression.

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