Multidomain binding molecules

Abstract

The present invention relates to multi-domain binding molecules. The molecule comprises: i) a peptide-major histocompatibility complex (pMHC)-binding domain comprising a concatenation of a first variable region and a constant region (VC1) and a concatenation of a second variable region and a constant region (VC2), wherein VC1 and VC2 dimerize to form a pMHC-binding domain; ii) an immune cell engaging domain comprising a concatenation of an antibody light chain variable domain (TCE-VL) and an antibody heavy chain variable domain (TCE-VH); and iii) a half-life extending domain comprising a portion of a first IgG Fc region (FC1) and a portion of a second IgG1 Fc region (FC2), wherein the multi-domain binding molecule comprises a first polypeptide chain in which the immune cell engaging domain is linked to the N-terminus of VC1, a second polypeptide chain in which VC2 is linked to the N-terminus of FC1 via its C-terminus, and a third polypeptide chain comprising FC2, and wherein the pMHC-binding domain and the immune cell engaging domain are capable of binding to pMHC complexes and T cells, respectively. The binding molecules can be used to treat diseases such as cancer, autoimmune diseases, 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 8, 2023, is named P200739WO ST26 Sequence Listing.xml and is 79120 bytes in size.

[0002] The present invention generally relates to multi-domain binding molecules. In particular, the present invention relates to a multi-domain binding molecule 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), 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 portion (FC1) and a second IgG1 Fc region portion (FC2), wherein the multi-domain binding molecule comprises a first polypeptide chain in which the ICE domain is linked to the N-terminus of VC1, a second polypeptide chain in which VC2 is linked to the N-terminus of FC1 via its C-terminus, and a third polypeptide chain comprising FC2, wherein the pMHC-binding domain and the ICE domain are capable of binding to pMHC complexes and immune cells, respectively. The binding molecules can be used to treat diseases such as cancer, autoimmune diseases, and infectious diseases. [Background technology]

[0003] 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).

[0004] 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).

[0005] 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). Therefore, there is a need to identify suitable approaches for extending the half-life of TCR-immune cell engaging domain proteins, such as TCR-anti-CD3 fusion proteins, and other TCR-containing proteins. 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-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.

[0006] 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.

[0007] 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]

[0008] [Patent Document 1] International Publication No. 2020 / 157211 [Non-patent literature]

[0009] [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

[0010] Description of the Invention As shown in Example 1, the inventors tested over 40 different formats (i.e., the orientation and location of each domain within the polypeptide) of a multidomain binding molecule comprising a pMHC-binding domain, an immune cell engaging (ICE) domain (which may be an immunoactivator), 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 decrease in in vitro potency. However, the inventors surprisingly identified formats (shown in Figures 1 and 10 herein) of such molecules that retain a high degree of potency and specificity and preserve the therapeutic window of the original molecule. Examples 11-13 further demonstrate that the identified formats are advantageous for molecules comprising fusions of various (pMHC)-binding domains with various ICE domains. The identified formats function advantageously with TCRs that bind to various targets and with immune cell engaging domains that are both immunoactivators and immunosuppressants.

[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 portion of an IgG Fc region (FC1) and a second portion of an IgG1 Fc region (FC2); A multi-domain binding molecule comprising: The multi-domain binding molecule may be a first polypeptide chain in which an ICE domain is linked to the N-terminus of VC1; a second polypeptide chain in which VC2 is linked through its C-terminus to the N-terminus of FC1; a third polypeptide chain comprising FC2; and and Multi-domain binding molecules are provided in which the pMHC binding domain and the ICE 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 a concatenation of an antibody light chain variable domain (TCE-VL) and an antibody heavy chain variable domain (TCE-VH); iii) a half-life extending domain comprising a first portion of an IgG Fc region (FC1) and a second portion of an IgG1 Fc region (FC2); A multi-domain binding molecule comprising: The multi-domain binding molecule may be a first polypeptide chain in which a T cell engaging immune effector domain is linked to the N-terminus of VC1; a second polypeptide chain in which VC2 is linked through its C-terminus to the N-terminus of FC1; a third polypeptide chain comprising FC2; and and Multi-domain 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] Advantageously, the binding molecules of the invention have an extended in vitro half-life while maintaining a similar therapeutic window and potent response as comparable Fc-free molecules. In a still further aspect, there is provided one or more nucleic acids encoding one or more of the polypeptide chains of a multi-domain binding molecule according to the first aspect of the invention. Also provided is an expression vector comprising the nucleic acid(s) of this aspect. Additionally, there is provided a host cell comprising the nucleic acid or vector of this aspect.

[0014] Also provided in a further aspect is a method of making a multi-domain binding molecule according to the first aspect, comprising maintaining a host cell as described above under conditions optimal for expression of the nucleic acid, and isolating the multi-domain binding molecule.

[0015] In a further aspect, there is provided a pharmaceutical composition comprising a multi-domain binding molecule according to the first aspect.

[0016] 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, autoimmune diseases, and infectious 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.

[0017] 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.

[0018] 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.

[0019] 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 only two disulfide-bonded chains. Each chain (alpha and beta) 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 alpha variable region. In addition, the beta chain usually contains a short diversity region adjacent to the junction region, which is also typically considered part of the beta 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.

[0020] 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.

[0021] The pMHC-binding domain may comprise a constant region. For example, VC1 and VC2 may each comprise a constant region. The constant region may correspond to the constant region from the TCR α chain or TCR β chain (TRAC or TRBC, respectively). Alternatively, the constant region of the pMHC-binding domain may be a constant region from an antibody light chain or antibody heavy chain (e.g., which may comprise one or more or all of the CL, CH1, CH2, CH3, or CH4 domains). 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.

[0022] When present, the TCR portion of the molecules of the invention may be an αβ heterodimer. The alpha-beta heterodimeric TCR portion of the molecules of the invention may comprise an alpha chain TRAC constant region sequence and / or a beta 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).

[0023] The alpha and beta chain constant region sequences 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 alpha and / or beta chain constant region sequences can have disulfide bonds introduced between residues of the respective constant domains, as described, for example, in WO 2003 / 020763, WO 2004 / 033685, and WO 2006 / 000830. The alpha and beta constant regions can be modified by substitution of a cysteine residue at Thr48 in TRAC and Ser57 in TRBC1 or TRBC2, where the cysteine forms a disulfide bond between the alpha and beta 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 alpha chain extracellular constant region may be truncated by 8 amino acids.

[0024] 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.

[0025] 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, where VC1 and VC2 comprise TCR constant regions, and suitable positions for introducing a disulfide bond between residues in each constant region are described in WO 2003 / 020763 and WO 2004 / 033685. Single-chain TCRs are further 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, Schodin (1996) Mol Immunol 33(9): 819-829.

[0026] 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: 2, SEQ ID NO: 3, and SEQ ID NO: 4 as CDR1, CDR2, and CDR3, respectively; and (ii) The TCR β variable region comprises the CDRs of SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10 as CDR1, CDR2, and CDR3, respectively.

[0027] 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.

[0028] 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: 2, SEQ ID NO: 3 and SEQ ID NO: 4, 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: 8, SEQ ID NO: 9 and SEQ ID NO: 10.

[0029] The TCR alpha variable region may be at least 80% identical to the sequence of SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and the TCR beta variable region may be at least 80% identical to the sequence of SEQ ID NO:11 or SEQ ID NO:12. 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:5, SEQ ID NO:6, or SEQ ID NO:7, 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:11 or SEQ ID NO:12. Preferably, the TCR alpha variable region has the sequence set forth in SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and the TCR beta variable region has the sequence set forth in SEQ ID NO:11 or SEQ ID NO:12. The TCR alpha variable region may have the sequence set forth in SEQ ID NO:5, and the TCR beta variable region may have the sequence set forth in SEQ ID NO:11. Alternatively, the TCR alpha variable region may have the sequence set forth in SEQ ID NO:6, and the TCR beta variable region may have the sequence set forth in SEQ ID NO:11. Alternatively, the TCR alpha variable region may have the sequence shown in SEQ ID NO:7 and the TCR beta variable region may have the sequence shown in SEQ ID NO:12.

[0030] 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: 29, and the TCR beta constant region may be at least 80% identical to the sequence of SEQ ID NO: 31. 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: 29, 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: 31. Preferably, the TCR alpha constant region has the sequence set forth in SEQ ID NO: 29, and the TCR beta constant region has the sequence set forth in SEQ ID NO: 31.

[0031] Alternatively, the TCR alpha constant region may be at least 80% identical to the sequence of SEQ ID NO: 30, and the TCR beta constant region may be at least 80% identical to the sequence of SEQ ID NO: 32. 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: 30, 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: 32. Preferably, the TCR alpha constant region has the sequence set forth in SEQ ID NO: 30, and the TCR beta constant region has the sequence set forth in SEQ ID NO: 32.

[0032] 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: 24, SEQ ID NO: 25, or SEQ ID NO: 26, 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: 27 or SEQ ID NO: 28. 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: 24, SEQ ID NO: 25, or SEQ ID NO: 26, 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: 27 or SEQ ID NO: 28. Preferably, the variable and constant regions of TCR alpha comprise or consist solely of the amino acid sequences set forth in SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26, and the variable and constant regions of TCR beta comprise or consist solely of the amino acid sequences set forth in SEQ ID NO: 27 or SEQ ID NO: 28.

[0033] The variable and constant regions of TCR alpha may have the sequence set forth in SEQ ID NO: 24, and the variable and constant regions of TCR beta may have the sequence set forth in SEQ ID NO: 27. Alternatively, the variable and constant regions of TCR alpha may have the sequence set forth in SEQ ID NO: 25, and the variable and constant regions of TCR beta may have the sequence set forth in SEQ ID NO: 27. Alternatively, the variable and constant regions of TCR alpha may have the sequence set forth in SEQ ID NO: 26, and the variable and constant regions of TCR beta may have the sequence set forth in SEQ ID NO: 28.

[0034] 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 2015 / 092362, WO 2011 / 001152, WO 2017 / 109496, WO 2017 / 175006, and WO 2018 / 234319, as well as, for example, U.S. Patent Application Publication No. 2016 / 0318988, U.S. Patent No. 8,519,100, U.S. Patent No. 11,639,374, U.S. Patent No. 11,505,590, and U.S. Patent No. 11,427,624 (the contents of each of which are incorporated herein by reference).

[0035] The multi-domain binding molecules of the present invention comprise: (a) a first polypeptide chain comprising, from N-terminus to C-terminus, in the following order: an amino acid sequence of an immune cell engaging domain (e.g., an scFv domain or a VHH domain), optionally followed by a linker sequence as set forth in SEQ ID NO: 34 and amino acid sequences of the variable and constant regions of TCR β; (b) a second polypeptide chain comprising, in the following order from N-terminus to C-terminus, the amino acid sequences of the variable and constant regions of TCRα, optionally followed by a truncated hIgG1 hinge sequence as set forth in SEQ ID NO: 33 and an Fc region having the sequence as set forth in SEQ ID NO: 50; (c) a third polypeptide chain comprising an Fc region having the sequence set forth in SEQ ID NO: 52; may have

[0036] In a preferred embodiment, the multi-domain binding molecule of the invention comprises: (a) a first polypeptide chain comprising, in the following order from N-terminus to C-terminus, the amino acid sequence of an scFv, optionally followed by a linker sequence set forth in SEQ ID NO: 34 and the amino acid sequences of the variable and constant regions of a TCR β; (b) a second polypeptide chain comprising, in the following order from N-terminus to C-terminus, the amino acid sequences of the variable and constant regions of TCRα, optionally followed by a truncated hIgG1 hinge sequence as set forth in SEQ ID NO: 33 and an Fc region having the sequence as set forth in SEQ ID NO: 50; (c) a third polypeptide chain comprising an Fc region having the sequence set forth in SEQ ID NO: 52; may have

[0037] Preferably, the scFv is an anti-CD3 scFv.

[0038] In an alternative embodiment, the multi-domain binding molecule of the invention comprises: (a) a first polypeptide chain comprising, in the following order from N-terminus to C-terminus, the amino acid sequence of a VHH, optionally followed by a linker sequence as set forth in SEQ ID NO: 34, and the amino acid sequences of the variable and constant regions of a TCR β; (b) a second polypeptide chain comprising, in the following order from N-terminus to C-terminus, the amino acid sequences of the variable and constant regions of TCRα, optionally followed by a truncated hIgG1 hinge sequence as set forth in SEQ ID NO: 33 and an Fc region having the sequence as set forth in SEQ ID NO: 50; (c) a third polypeptide chain comprising an Fc region having the sequence set forth in SEQ ID NO: 52; may have

[0039] Preferably, the VHH is a PD-1 agonist VHH.

[0040] In an alternative embodiment, a multi-domain binding molecule of the invention may comprise an Fc domain having a first Fc region comprising the sequence of SEQ ID NO:77 and a second Fc region comprising the sequence of SEQ ID NO:76.

[0041] Thus, in any of the above-described embodiments, the Fc region having the sequence shown in SEQ ID NO: 50 can be replaced with an Fc region having the sequence shown in SEQ ID NO: 77, and the Fc region having the sequence shown in SEQ ID NO: 52 can be replaced with an Fc region having the sequence shown in SEQ ID NO: 76.

[0042] The TCR α and β chains can dimerize to form a peptide-major histocompatibility complex (pMHC) binding domain.

[0043] The anti-CD3 scFv may comprise or consist of the amino acid sequence shown in SEQ ID NO:62 or the amino acid sequence shown in SEQ ID NO:63.

[0044] The TCR β constant region may have the amino acid sequence set forth in SEQ ID NO:31 and / or the TCR α constant region may have the amino acid sequence set forth in SEQ ID NO:29.

[0045] The multi-domain binding molecule may not contain any amino acid sequences other than the sequences in a) to c) above.

[0046] The TCR α and β chains can dimerize to form a peptide-major histocompatibility complex (pMHC) binding domain.

[0047] 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.

[0048] VC1 and / or VC2 may comprise one or more amino acid substitutions compared to unmodified V1 and / or VC2, wherein the one or more amino acid substitutions eliminate one or more glycosylation sites. A substitution in this context refers 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 N18, N24, N146, N180, and N191, numbered according to SEQ ID NO: 25; and / or (ii) The other of VC1 or VC2 may comprise a variable and constant region of a TCR β comprising one or more amino acid substitutions at positions selected from the group consisting of N84 and N186, numbered according to SEQ ID NO: 27. The substitution may be an Asn to Gln (i.e., an N to Q) substitution. The variable and constant region of a TCR α may comprise an N18Q, an N24Q, an N146Q, an N180Q, and an N191Q substitution, numbered according to SEQ ID NO: 25, and the variable and constant region of a TCR β comprises an N84Q and an N186Q substitution, numbered according to SEQ ID NO: 27.

[0049] 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 alpha variable region, numbered according to SEQ ID NO: 25.

[0050] 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 MAGEA4. Preferably, the pMHC-binding domain binds to GVYDGREHTV (SEQ ID NO: 1) HLA-A. * Binds to the O2 complex.

[0051] In an alternative embodiment, the peptide antigen may be a peptide derived from a beta cell antigen, such as preproinsulin (PPI). 15~24 The (SEQ ID NO: 78) peptide is one such peptide derived from the signal sequence of human PPI (Skowera, et al. 2008 J Clin Invest. 118:3390-402 and WO 2009 / 004315). This peptide binds to HLA-A * HLA-A is housed in the 02 molecule and presented on the surface of insulin-producing beta cells. * The O2 complex provides a human beta cell-specific marker that can be recognized by TCR. High expression of this PPI peptide can be detected on the surface of beta cells regardless of disease stage. Thus, PPI-targeting therapeutics may be effective at an earlier stage of disease compared with existing immunotherapies.

[0052] Immune Cell Engaging Domain As used herein, an "immune cell engaging domain" is a protein domain that can alter an immune response, for example, by promoting or suppressing an immune response, such as T cell activation.

[0053] In some embodiments, the immune cell engaging domain comprises an antibody light chain variable region (TCE-VL) and an antibody heavy chain variable region (TCE-VH). As used herein, "TCE-VL" and "TCE-VH" refer to the light chain variable region and heavy chain variable region of the immune cell engaging domain (ICE), respectively. "TCE-VL" and "TCE-VH" may also be referred to herein as "TCEVL" and "TCEVH." Thus, the immune cell engaging domain may comprise an antigen-binding site. The immune cell engaging domain is also referred to herein as an "ICE" domain.

[0054] In some embodiments, the immune cell engaging domain may be a T cell engaging immune effector domain that 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 may be a CD3 effector domain. The T cell engaging immune effector domain may bind to CD3, e.g., specifically bind to CD3 (i.e., the T cell engaging immune effector domain may be a CD3 binding protein). The T cell engaging immune effector may 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 may also be a single domain antibody, such as the variable region of a heavy chain antibody.

[0055] Alternatively, the binding molecules of the present invention may comprise immunosuppressive factors. 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 comprise 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 comprise 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.

[0056] 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).

[0057] 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.

[0058] "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.

[0059] 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 only a single antibody variable domain (e.g., a heavy-chain variable domain). Thus, an immune cell inhibitor may, for example, comprise 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). As used herein, the term "antigen-binding portion of an antibody" 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 approximately 50,000 daltons. An Fv fragment is the N-terminal portion of an antibody Fab fragment, and consists of only the variable portion of one light chain (VL) and one heavy chain (VH).

[0060] 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.

[0061] 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).

[0062] 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.

[0063] 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.

[0064] 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 immune cells, such as T cells. Such antigens include CD28, 4-1bb (CD137), or CD16, or any molecule that exerts an effect on the immune synapse. Particularly preferred immune effectors are anti-CD3 antibodies, or functional fragments or variants of the above-mentioned anti-CD3 antibodies. As used herein, the term "antibody" encompasses such fragments and variants. Examples of anti-CD3 antibodies include, but are not limited to, OKT3, UCHT-1, BMA-031, and 12F6. Antibody fragments and variants / analogs suitable for use in the compositions and methods described herein include minibodies, Fab fragments, F(ab')2 fragments, dsFv, and scFv fragments.

[0065] Preferably, the immune cell engaging domain comprises: (i) a VL region comprising the CDRs of SEQ ID NO: 68, SEQ ID NO: 69, and SEQ ID NO: 70 as CDR1, CDR2, and CDR3, respectively; (ii) a VH region comprising the CDRs of SEQ ID NO: 71, SEQ ID NO: 72, and SEQ ID NO: 73 as CDR1, CDR2, and CDR3, respectively; Includes:

[0066] Alternatively, the immune cell engaging domain may be (i) a VL region comprising the CDRs of SEQ ID NO: 68, SEQ ID NO: 69, and SEQ ID NO: 70 as CDR1, CDR2, and CDR3, respectively; (ii) a VH region comprising the CDRs of SEQ ID NO: 75, SEQ ID NO: 72, and SEQ ID NO: 73 as CDR1, CDR2, and CDR3, respectively; may include:

[0067] The above VL and VH CDR sequences may each optionally have one, two, three, or four amino acid substitutions relative to the above sequences.

[0068] 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: 68, SEQ ID NO: 69, and SEQ ID NO: 70, 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: 71, SEQ ID NO: 72, and SEQ ID NO: 73.

[0069] 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: 68, SEQ ID NO: 69, and SEQ ID NO: 70, 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: 75, SEQ ID NO: 72, and SEQ ID NO: 73.

[0070] The TCE-VL may comprise or consist of an amino acid sequence at least 80% identical to the sequence of SEQ ID NO: 66, and the TCE-VH may comprise or consist of an amino acid sequence at least 80% identical to the sequence of SEQ ID NO: 67. 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: 66, 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: 67. Preferably, the TCE-VL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 66, and the TCE-VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 67.

[0071] Alternatively, the TCE-VL may comprise or consist of an amino acid sequence at least 80% identical to the sequence of SEQ ID NO: 66, and the TCE-VH may comprise or consist of an amino acid sequence at least 80% identical to the sequence of SEQ ID NO: 74. 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: 66, 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: 74. For example, the TCE-VL may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 66, and the TCE-VH may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 74.

[0072] As described above, the immune cell engaging domain may be an scFv. The immune cell engaging 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: 62 or SEQ ID NO: 63. 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: 62 or SEQ ID NO: 63. Preferably, the scFv comprises or consists of the amino acid sequence set forth in SEQ ID NO: 63. Alternatively, the scFv may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 62.

[0073] Alternatively, the target (i.e., antigen) of the immune cell engaging domain may be an immunosuppressive factor. For example, the target may be 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- 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), TIGIT (I 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).

[0074] 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.

[0075] 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.

[0076] Alternatively, the immunosuppressive factor may comprise an agonist antibody that binds to an immune checkpoint molecule and preferably stimulates its signal transduction. For example, the immunosuppressive factor may be or comprise a PD-1 agonist antibody (e.g., a single-domain antibody). Preferably, such a PD-1 agonist does not compete with PD-L1 for binding to PD-1. The PD-1 agonist 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. Preferably, the immunosuppressive factor is a PD-1 agonist VHH.

[0077] Preferably, the immunosuppressant is 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).

[0078] 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).

[0079] 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.

[0080] 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 proteins 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 proteins comprising an Fc region with one or more modifications that enhance binding to FcRn can 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 proteins comprising a native Fc region. A binding protein 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 protein comprising a native Fc region.

[0081] 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.

[0082] 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.

[0083] The FC1 region may comprise or consist of an amino acid sequence at least 80% identical to the sequence of SEQ ID NO: 50, and the FC2 region may comprise or consist of an amino acid sequence at least 80% identical to the sequence of SEQ ID NO: 49. 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: 50, 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: 49. Preferably, the FC1 region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 50, and the FC2 region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 49. 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: 49, and the FC2 region may comprise or consist solely of the amino acid sequence set forth in SEQ ID NO: 50.

[0084] 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.

[0085] 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:

[0086] TIFF2025526897000002.tif57170

[0087] 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.

[0088] 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).

[0089] 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).

[0090] 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 T366W according to the EU numbering scheme, and the FC2 region may comprise amino acid substitutions of T366S, L368A, and Y407V according to the EU numbering scheme.

[0091] 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.

[0092] 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.

[0093] 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)).

[0094] 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.

[0095] 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 binding 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.

[0096] The modification(s) in the Fc region that promote binding to FcRn (e.g., amino acid substitutions, insertions, or deletions) are numbered by the EU index as set forth in Kabat, 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, The modification may be at one or more positions selected from the group consisting of: 66, 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.

[0097] 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.

[0098] 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.

[0099] 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.

[0100] 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.

[0101] The above-listed substitutions that promote binding to FcRn are relative to the corresponding wild-type Fc region (e.g., the Fc region of human IgG1 or IgG4) and may be present in one or preferably both of the FC1 and FC2 portions of the Fc region. 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.

[0102] In one embodiment, the FC1 and / or FC2 regions comprise the following amino acid substitutions (numbered according to the EU numbering scheme): M252Y / S254T / T256E.

[0103] 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.

[0104] 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 correspond substantially or partially 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: 33. The hinge may comprise all or part of the core hinge domain and all or part of the lower hinge region.

[0105] Format and Linker As used herein, the term "format" relates 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 representation of an exemplary multi-domain binding molecule format is shown in Figure 1. The pMHC-binding domain and immune cell engaging (ICE) domain of such a molecule are capable of binding to a pMHC complex and a T cell, respectively. In this regard, the pMHC-binding domain and the immune cell engaging (ICE) domain may be capable of simultaneously binding to a pMHC complex and a T cell, respectively.

[0106] A format of the multi-domain binding molecule of the invention comprises three polypeptide chains: a first polypeptide chain in which a T cell engaging immune effector domain is linked to the N-terminus of VC1; a second polypeptide chain in which VC2 is linked via its C-terminus to the N-terminus of FC1; and a third polypeptide chain comprising FC2.

[0107] This format can be represented as N-(TCEVL-TCEVH or TCEVH-TCEVL)-VC1, N-VC2-FC1-C, N-FC2-C. The inventors have determined that this format of the molecule has the highest activity (i.e., potency and selectivity) of over 40 different formats tested.

[0108] The multi-domain binding molecules of the invention are in a three-chain format. In this context, "three-chain" is used to describe a multi-domain binding molecule that is expressed as three separate polypeptide chains that associate with each other to form a single, three-dimensionally folded structure comprising a pMHC-binding domain, an immune cell engaging (ICE) domain, and a half-life extending domain.

[0109] 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 (ICE) is an anti-CD3 scFv, and the Fc domain is an IgG1 Fc domain.

[0110] The TCE-VH region, TCE-VL region, VC1 region, VC2 region, FC1 region, and 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 primarily composed of amino acids such as glycine, alanine, and serine, which lack bulky side chains that may limit flexibility. Examples of 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 alpha-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:34), GGGSG (SEQ ID NO:35), GGSGG (SEQ ID NO:36), GSGGG (SEQ ID NO:37), GSGGGP (SEQ ID NO:38), GGEPS (SEQ ID NO:39), GGEGGGP (SEQ ID NO:40), GGEGGGSEGGGS (SEQ ID NO:41), and GGGSGGGG (SEQ ID NO:42).Further suitable linkers include GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 43), EAAAK (SEQ ID NO: 44), and EAAAKEAAAKEAAAK (SEQ ID NO: 45), GGGGSGGGGSGGGGSGGGGSGGGS (SEQ ID NO: 46).

[0111] Suitable IgG hinge sequences are known in the art and include the exemplary truncated IgG1 hinge sequence set forth in SEQ ID NO: 33. Other suitable IgG hinge sequences include the complete IgG1 hinge sequence set forth in SEQ ID NO: 47, and the IgG4 hinge set forth in SEQ ID NO: 48.

[0112] 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, the first chain of a multi-domain binding molecule of the invention may have the following format: N-TCEVL-TCEVH-VC1-C.

[0113] VC1 may comprise the variable and constant regions of TCRβ, and VC2 may comprise the variable and constant regions of TCRα. VC1 and VC2 may therefore dimerize to form a soluble TCR. In this regard, the second chain of a multidomain binding molecule of the invention has the following format: TCRβ-FC1-C (where "TCRβ" refers to the variable and constant regions of TCRβ, and "TCRα" refers to the variable and constant regions of TCRα).

[0114] The TCE-VL region may be linked to the TCE-VH region via a sequence containing a glycine-serine linker. Preferably, the sequence linking the TCE-VL region and the TCE-VH region is the amino acid sequence shown in SEQ ID NO: 46.

[0115] The 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 TCE-VH region and VC1 is the amino acid sequence shown in SEQ ID NO: 34.

[0116] VC2 may be linked to the FC1 region via a sequence comprising an IgG hinge sequence. The IgG hinge sequence may be at least 80% identical to SEQ ID NO: 33. Preferably, the IgG hinge sequence is at least 90%, at least 95%, at least 98%, or 100% identical to SEQ ID NO: 33.

[0117] The sequence connecting the VC2 and FC1 regions may further comprise a glycine-serine linker. Preferably, the glycine-serine linker has the sequence shown in SEQ ID NO: 42. Preferably, these sequences follow the format from N-terminus to C-terminus: VC2-GS linker-IgG hinge-FC1.

[0118] 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:42.

[0119] The multi-domain binding molecules of the present invention comprise three polypeptide chains. Multi-domain binding molecules may be soluble and / or recombinant and / or isolated. Exemplary multi-domain binding molecules include: (a) SEQ ID NO: 52, SEQ ID NO: 54, and SEQ ID NO: 56; (b) SEQ ID NO: 52, SEQ ID NO: 58, and SEQ ID NO: 56, or (c) SEQ ID NO: 52, SEQ ID NO: 60, and SEQ ID NO: 61; It may contain an array of:

[0120] The first chain of the multi-domain binding molecule may have an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 56. The first chain of 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: 56. Preferably, the first chain of the multi-domain binding molecule comprises or consists of the amino acid sequence set forth in SEQ ID NO: 56.

[0121] Alternatively, the first chain of the multi-domain binding molecule may have an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 61. The first chain of 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: 61. For example, the first chain of the multi-domain binding molecule comprises or consists of the amino acid sequence set forth in SEQ ID NO: 61.

[0122] The second chain of the multi-domain binding molecule may have an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 54. The second chain of 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: 54. For example, the second chain of the multi-domain binding molecule comprises or consists of the amino acid sequence set forth in SEQ ID NO: 54.

[0123] Alternatively, the second chain of the multi-domain binding molecule may have an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 58. The second chain of 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: 58. For example, the second chain of the multi-domain binding molecule comprises or consists of the amino acid sequence set forth in SEQ ID NO: 58.

[0124] Alternatively, the second chain of the multi-domain binding molecule may have an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 60. The second chain of 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: 60. Preferably, the second chain of the multi-domain binding molecule comprises or consists solely of the amino acid sequence set forth in SEQ ID NO: 60.

[0125] The third chain of the multi-domain binding molecule may have an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 52. The third chain of 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: 52. Preferably, the third chain of the multi-domain binding molecule comprises or consists of the amino acid sequence shown in SEQ ID NO: 52.

[0126] Optionally, the above multi-domain binding molecule sequences may be further fused to one or more other polypeptide sequences.

[0127] The above sequence is GVYDGREHTV (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: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, or SEQ ID NO: 61 with the sequence of a different TCR of interest.

[0128] 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 (KD 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.

[0129] 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.

[0130] 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 are present.

[0131] 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.

[0132] "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)).

[0133] 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.

[0134] 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).

[0135] 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%.

[0136] 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.

[0137] 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.

[0138] 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.

[0139] 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.). 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 is one that exhibits an EC value in the nM-pM range, e.g., 500 nM or less, preferably 1 nM or less, or 500 pM or less. 50 It may have a value.

[0140] 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 be linked to 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 related to the activation of 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 described in Example 11. 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.

[0141] 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).

[0142] 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 alpha phase and a beta phase. To determine the in vivo half-life of a protein, the clearance rate in the beta phase is calculated and compared to the clearance rate of the wild-type or unmodified protein.

[0143] 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.

[0144] 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.

[0145] 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).

[0146] Suitable vectors can be selected 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., Molecular Cloning: A Laboratory Manual, 2nd ed., by Sambrook et al., 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 Short Protocols in Molecular Biology, 2nd ed., by Ausubel et al., John Wiley & Sons (1992).

[0147] 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.

[0148] 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 cells or 293T cells described in Graham, FL et al., J. Gen Virol. 36 (1977) 59-74), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., 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).

[0149] 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.

[0150] 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.

[0151] 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.

[0152] 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.

[0153] 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).

[0154] Exemplary promoters active in mammalian cells include the cytomegalovirus immediate early promoter (CMV-IE), the human elongation factor 1-α 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, a hybrid regulatory element comprising a CMV enhancer / β-actin promoter or an immunoglobulin promoter or an active fragment 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).

[0155] 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.

[0156] 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.

[0157] 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.

[0158] 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.

[0159] 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.

[0160] 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.

[0161] 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.

[0162] 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).

[0163] 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.

[0164] 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.

[0165] The binding molecules of the invention may have an extended in vitro half-life while maintaining a similar therapeutic window as the equivalent Fc-free molecule. As used herein, the term "therapeutic window" refers to the difference between on-target and off-target activity, e.g., the difference between on-target and off-target T cell activation.

[0166] 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.

[0167] 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.

[0168] 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.

[0169] 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.

[0170] 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.

[0171] The cancer to be treated may be a cancer associated with MAGEA4 expression. "Associated with MAGEA4 expression" means that the cancer contains cancer cells that express MAGEA4. In this regard, the cancer may be a MAGEA4-positive cancer. The cancer may be known to be associated with MAGEA4 expression, and therefore, MAGEA4 expression may not be assessed. Alternatively, MAGEA4 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 MAGEA4 expression can be detected by histological methods. Cancers associated with MAGEA4 expression include, but are not limited to, ovarian cancer, lung cancer, head and neck cancer, esophageal cancer, breast cancer, synovial sarcoma, gastric cancer, bladder cancer, and any tumor with squamous cell histology. The head and neck cancer may be head and neck squamous cell carcinoma (HNSCC). The lung cancer may be non-small cell lung cancer (NSCLC). The bladder cancer may be urothelial carcinoma. The esophageal cancer can be gastroesophageal junction (GEJ) adenocarcinoma. The ovarian cancer can be epithelial ovarian cancer, such as high-grade serous ovarian cancer.

[0172] 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.

[0173] 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.

[0174] 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 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 a tumor or an autoimmune disease or an infectious disease; 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.

[0175] 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.

[0176] 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.

[0177] 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 autoimmune disease, or an infectious disease) in a subject. The kit may further include (c) at least one additional therapeutically active compound or drug.

[0178] 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.

[0179] 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.

[0180] 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]

[0181] [Figure 1A] Schematic diagram of a T cell receptor (TCR):anti-CD3 fusion molecule according to the invention. (i) shows the components of the three individual chains, and (ii) shows the overall structure of the molecule. This format is defined herein as Mol020. [Figure 1B] Schematic diagram showing alternative TCR:anti-CD3 fusion molecule formats used in the Examples: (i) is a two-chain molecule fused to Fc, this format is defined herein as Mol014, and (ii) is a fusion molecule lacking the Fc domain, this format is defined herein as Mol001. [Figure 2] Figure 1 shows a comparison of T cell activation, defined as IFNγ release, in the presence and absence of antigen-positive cells for Mol020 and Mol014. In this example, the TCR portion of the molecule binds to a peptide from MAGEA4. T2 cells pulsed with MAGEA4 peptide or an irrelevant peptide were used as antigen-positive and antigen-negative cells, respectively. Mol020 generated a significantly stronger T cell response compared to Mol014, while maintaining a high degree of specificity. [Figure 3]Figure 1 shows a further comparison of T cell activation, defined as IFNγ release, in the presence and absence of antigen-positive cells for Mol020 and Mol014. In this example, the TCR portion of the molecule recognizes a peptide from PRAME. Human melanoma Mel624 cells were used as antigen-positive cells. Mel624 with a knockout of the MAGEA4 gene were used as antigen-negative cells. Mol020 was significantly more potent than Mol014 while maintaining a high degree of specificity. [Figure 4] Figure 1 shows a comparison of T cell activation, defined as IFNγ release, in the presence and absence of antigen-positive cells for MAGEA4-targeting Mol020 and Mol001 molecules. Human lung cancer NCI-H1755 cells were used as antigen-positive target cells. HLA-A2 B2M pre-transduced human melanoma Mel202 cells were used as antigen-negative cells. Mol020 demonstrated lower T cell activation against both antigen-positive and antigen-negative cells. Thus, the therapeutic window for Mol020 is maintained compared to the Fc-free fusion Mol001. [Figure 5] Pharmacokinetic (PK) evaluation of Mol020 and Mol001 in Tg32 SCID mice. Comparison of serum concentrations over time demonstrated a significantly increased in vivo half-life for Mol020 compared to Mol001. [Figure 6] Schematic diagram showing each glycosylation site mutant and indicating the location of the glycosylation site. Three glycosylation sites within the TCR variable domain are shown at positions N18 and N24 in the alpha chain and N84 in the beta chain. The corresponding relative yield is shown below each mutant, as well as the melting temperature (Tm) of the TCR portion of the molecule. The data show that removing all seven glycosylation sites in the TCR has a negative impact on yield and molecular stability. Retaining the single glycosylation site at position N18 in the alpha chain partially compensates for these effects. [Figure 7]1 shows the stability of the monoglycosylated (Mol020v14) and deglycosylated (Mol020v13) molecules over time in human serum. The dotted line indicates a 20% change from baseline. The data demonstrate that the monoglycosylated variant (v14) exhibits improved serum stability compared to the deglycosylated variant (v13). [Figure 8] Figure 1 shows pharmacokinetic (PK) evaluation of monoglycosylated (mol020v14) and deglycosylated (mol020v13) variants in Tg32 SCID mice. A fully glycosylated variant, which retains all seven glycosylation sites, was used as a control. [Figure 9] Figure 1 shows T cell activation, defined as IFNγ release, for the monoglycosylated (mol020v14) and deglycosylated (mol020v13) variants in the presence or absence of antigen-positive and antigen-negative primary cell lines. The data showed that both molecules drive potent T cell activation in the presence of antigen-positive cancer cells. [Figure 10] FIG. 1 is a schematic diagram of an exemplary TCR-PD-1 agonist binding molecule incorporating an Fc domain. [Figure 11] a) Graph showing inhibition of T cell signaling by TCR PD-1 agonist binding molecules in a Jurkat NFAT reporter assay; b) Graph comparing inhibition of T cell signaling by Fc-bearing and Fc-free TCR PD-1 agonists in a Jurkat NFAT reporter assay. [Figure 12] FIG. 1 depicts a graph showing in vivo concentrations of TCR PD-1 agonist binding molecules in SCID mice over a 3-week period following intravenous (IV) or subcutaneous (SC) administration. [Figure 13] FIG. 1 depicts a graph showing inhibition of IL2 release by primary CD4+ T cells in the presence of TCR PD-1 agonists. [Figure 14]FIG. 1 depicts graphs showing inhibition of β-cell killing (a) and IFNγ cytokine release (b) by two autoreactive T cell clones in the presence of TCR PD-1 agonist binding molecules. [Figure 15] FIG. 1 depicts a graph showing inhibition of stimulation in PD1-positive and PD-1-negative NK cells as indicated by % of cells positive for CD107a and IFNγ in the presence of TCR PD-1 agonist binding molecules (*p≦0.05, **p≦0.01, ns=non-significant). DETAILED DESCRIPTION OF THE INVENTION

[0182] Array Description SEQ ID NO: 1 HLA-A * 02 Restrictive peptide: GVYDGREHTV

[0183] SEQ ID NO: 5: Amino acid sequence of the TCR alpha chain variable domain of an exemplary TCR. The CDRs (CDR1, CDR2, and CDR3) are underlined and are designated SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively, and the framework regions (FR1, FR2, FR3, and FR4) are italicized and are designated SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively. This sequence includes the N24Q mutation (double underlined). TIFF2025526897000003.tif14170

[0184] SEQ ID NO: 11: 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: 8, SEQ ID NO: 9, and SEQ ID NO: 10, respectively, and the framework regions (FR1, FR2, FR3, and FR4) are italicized and are designated SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 23, respectively. This sequence includes the N84Q mutation (double underlined). TIFF2025526897000004.tif14170

[0185] SEQ ID NO: 24: 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: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively; the framework regions (FR1, FR2, FR3, and FR4) are italicized and are designated SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively. The constant region is shown in bold and is designated SEQ ID NO: 29. Within the constant region, the non-native cysteine residue introduced to create an interchain disulfide bond is double underlined (position 48 of the constant region). This sequence also includes the following substitutions (double underlined): N24Q, N146Q, N180Q, and N191Q. TIFF2025526897000005.tif26170

[0186] SEQ ID NO: 27: 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: 8, SEQ ID NO: 9, and SEQ ID NO: 10, respectively, and the framework regions (FR1, FR2, FR3, and FR4) are italicized and are designated SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 23, respectively. The constant region is shown in bold and is designated SEQ ID NO: 31. Within the constant region, the non-native cysteine residue introduced to create the interchain disulfide bond is double underlined (position 57 of the constant region). This sequence also includes the N84Q and N186Q substitutions (double underlined). TIFF2025526897000006.tif32170

[0187] SEQ ID NO: 62. An exemplary anti-CD3 scFv (immune cell engaging domain (ICE)) referred to herein as "U0." The light chain variable domain (VL) is in italics and is designated SEQ ID NO: 66. The light chain CDRs (CDR1, CDR2, and CDR3) are underlined and are designated SEQ ID NO: 68, SEQ ID NO: 69, and SEQ ID NO: 70. The heavy chain variable domain (VH) is shown in bold and is designated SEQ ID NO: 74. The heavy chain CDRs (CDR1, CDR2, and CDR3) are underlined and are designated SEQ ID NO: 75, SEQ ID NO: 72, and SEQ ID NO: 73. The glycine-serine linker connecting VL and VH is shown in plain text and is designated SEQ ID NO: 46. TIFF2025526897000007.tif30170

[0188] SEQ ID NO: 63: Another exemplary anti-CD3 scFv (immune cell engaging domain (ICE)) referred to herein as "U28." This sequence is the same as SEQ ID NO: 62 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: 66. The light chain CDRs (CDR1, CDR2, and CDR3) are underlined and are designated SEQ ID NO: 68, SEQ ID NO: 69, and SEQ ID NO: 70. The heavy chain variable domain (VH) is shown in bold and is designated SEQ ID NO: 67. The heavy chain CDRs (CDR1, CDR2, and CDR3) are underlined and are designated SEQ ID NO: 71, SEQ ID NO: 72, and SEQ ID NO: 73. The glycine-serine linker connecting VL and VH is shown in plain text and is designated SEQ ID NO: 46. TIFF2025526897000008.tif31170

[0189] SEQ ID NO: 51 Unmodified human IgG1 Fc region (CH2 and CH3 domains) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0190] SEQ ID NO:49: Another exemplary IgG1 Fc region sequence. This sequence has two double-underlined substitutions compared to the unmodified IgG1 Fc sequence above (SEQ ID NO:51). 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:50) containing the T366S, L368A, and Y407V substitutions (a hole-forming substitution). The numbering of the substitutions in this sequence follows the EU numbering scheme. TIFF2025526897000009.tif25170

[0191] SEQ ID NO: 50: Exemplary IgG1 Fc region sequence. This sequence has four substitutions (double underlined) compared to the unmodified IgG1 Fc sequence above (SEQ ID NO: 51). 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: 49) containing the T366W substitution (forming a knob). The numbering of the substitutions in this sequence follows the EU numbering scheme. TIFF2025526897000010.tif24170

[0192] SEQ ID NO: 76: Exemplary IgG1 Fc region sequence. This sequence has seven substitutions (double underlined) compared to the unmodified IgG1 Fc sequence above (SEQ ID NO: 51). These are the N297G substitution, which inhibits binding to FcγR; the T366W substitution (a substitution that forms a knob), the T366S substitution (a substitution that promotes dimerization with another Fc region (e.g., SEQ ID NO: 77), the L368A substitution, and the Y407V substitution (a substitution that forms a hole); and the substitutions M252Y, S254T, and T256E, which enhance binding to FcRn. The numbering of the substitutions in this sequence follows the EU numbering scheme. TIFF2025526897000011.tif24170

[0193] SEQ ID NO: 77: A further exemplary IgG1 Fc region sequence. This sequence has five double-underlined substitutions compared to the unmodified IgG1 Fc sequence above (SEQ ID NO: 51). 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: 76), and the M252Y, S254T, and T256E substitutions that enhance binding to FcRn.

[0194] The numbering of the substitutions in this sequence follows the EU numbering scheme. TIFF2025526897000012.tif24170

[0195] SEQ ID NO: 47 Exemplary IgG1 hinge sequence (comprising a C to S substitution at position 5 compared to the native human IgG1 sequence, numbered according to SEQ ID NO: 47, double underlined): TIFF2025526897000013.tif6170

[0196] SEQ ID NO: 33 Truncated IgG1 hinge sequence: DKTHTCPPCP

[0197] SEQ ID NO: 48 IgG4 hinge sequence: ESKYGPPCPSCP

[0198] SEQ ID NO: 52: Complete amino acid sequence of the third polypeptide chain of an exemplary multi-domain binding molecule. This polypeptide comprises a hIgG1 Fc chain with a shortened hinge (underlined). The Fc region sequence corresponds to the Fc sequence (in this case the FC2 region) in SEQ ID NO: 49 (italics), including the amino acid substitutions (double underlined) of N297G (effector attenuation) and T366W (knob-forming substitution). The numbering of substitutions within this sequence follows the EU numbering scheme. TIFF2025526897000014.tif26170

[0199] SEQ ID NO: 54: Complete amino acid sequence of the second polypeptide chain of an exemplary multi-domain binding molecule. This polypeptide comprises a TCR alpha chain (in this case VC2 shown in SEQ ID NO: 25 (bold letters)) containing amino acid substitutions N297G (effector attenuation, double underlined) and T366S, L368A, Y407V (hole-forming substitutions, double underlined) linked to an Fc region sequence (plain letters) (in this case the FCl region shown in SEQ ID NO: 50) via a shortened hinge (underlined) of IgG1 Fc. The TCR alpha chain sequence also includes the following substitutions (double underlined) relative to the TCR alpha chain of SEQ ID NO: 26: N18Q, N24Q, N146Q, N180Q, and N191Q. TIFF2025526897000015.tif45170

[0200] SEQ ID NO: 58: Alternative complete amino acid sequence of the second polypeptide chain of an exemplary multi-domain binding molecule. This polypeptide comprises a TCR alpha chain (in this case "VC2" shown in SEQ ID NO: 24 (bold letters)) containing amino acid substitutions N297G (effector attenuation, double underlined) and T366S, L368A, Y407V (hole-forming substitutions, double underlined) linked to an Fc region sequence (plain letters) (in this case the FCl region shown in SEQ ID NO: 50) via a shortened hinge (underlined) of IgG1 Fc. The TCR alpha chain sequence also includes the following substitutions (double underlined) relative to the TCR alpha chain of SEQ ID NO: 26: N24Q, N146Q, N180Q, and N191Q. In this polypeptide, the N-linked glycosylation site at N18 (relative to SEQ ID NO: 26) is retained. TIFF2025526897000016.tif44170

[0201] SEQ ID NO: 60: Further alternative complete amino acid sequence of the second polypeptide chain of an exemplary multi-domain binding molecule. This polypeptide comprises a TCR alpha chain (in this case "VC2" shown in SEQ ID NO: 26 (bold letters)) containing amino acid substitutions N297G (effector attenuating, double underlined) and T366S, L368A, Y407V (hole-forming substitutions, double underlined) linked via a shortened hinge (underlined) of IgG1 Fc to an Fc region sequence (plain letters) (in this case the F1 region shown in SEQ ID NO: 50). TIFF2025526897000017.tif45170

[0202] SEQ ID NO: 56: Complete amino acid sequence of the first polypeptide chain of an exemplary multi-domain binding molecule. This polypeptide comprises an immune cell engaging domain (ICE) (dotted underline) comprising the anti-CD3 scFv sequence ("U28") set forth in SEQ ID NO: 63. The VH (SEQ ID NO: 67, dotted underline, plain text) and VL (SEQ ID NO: 66, dotted underline, italics) of the scFv are linked by a glycine-serine linker (dotted underline, bold text) of SEQ ID NO: 46. The scFv is linked to a pMHC binding domain (not underlined) comprising the TCR β chain sequence (in this case "VC1") set forth in SEQ ID NO: 27 by a glycine-serine linker (underlined, bold text) of SEQ ID NO: 34. The TCR β variable domain is shown in italics and is designated SEQ ID NO: 11. The TCR β constant domain is shown in bold and is designated SEQ ID NO: 31. TIFF2025526897000018.tif55170

[0203] SEQ ID NO: 61: Complete amino acid sequence of the first polypeptide chain of an alternative exemplary multi-domain binding molecule. This polypeptide comprises an immune cell engaging domain (ICE) (dotted underline) comprising the anti-CD3 scFv sequence ("U28") set forth in SEQ ID NO: 63. The VH (SEQ ID NO: 67, dotted underline, plain text) and VL (SEQ ID NO: 66, dotted underline, italics) of the scFv are linked by a glycine-serine linker (dotted underline, bold text) of SEQ ID NO: 46. The scFv is linked to a pMHC binding domain (not underlined) comprising the TCR β chain sequence (in this case "VC1") set forth in SEQ ID NO: 28 by a glycine-serine linker (underlined, bold text) of SEQ ID NO: 34. The TCR β variable domain is shown in italics and is designated SEQ ID NO: 12. The TCR β constant domain is shown in bold and is designated SEQ ID NO: 32. TIFF2025526897000019.tif54170

[0204] Additional linker sequences: GGGGS (SEQ ID NO: 34), GGGSG (SEQ ID NO: 35), GGSGG (SEQ ID NO: 36), GSGGG (SEQ ID NO: 37), GSGGGP (SEQ ID NO: 38), GGEPS (SEQ ID NO: 39), GGEGGGP (SEQ ID NO: 40), GGEGGGSEGGGS (SEQ ID NO: 41), GGGSGGGG (SEQ ID NO: 42), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 43), EAAAK (SEQ ID NO: 44), EAAAAKEAAAKEAAAK (SEQ ID NO: 45), and GGGGSGGGGSGGGGSGGGGSGGGS (SEQ ID NO: 46).

[0205] TIFF2025526897000020.tif178170TIFF2025526897000021.tif240170TIFF2025526897000022.tif247170TIFF2025526897000023.tif245170 TIFF2025526897000024.tif204170TIFF2025526897000025.tif246170TIFF2025526897000026.tif246170TIFF2025526897000027.tif192170 [Example]

[0206] 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.

[0207] Example 1 - Multi-domain Fc fusion molecules with improved potency Multidomain TCR-anti-CD3 fusion protein molecules incorporating a half-life-extending Fc domain have previously been described in U.S. Patent No. 5,627,499 and shown to be functional. 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.

[0208] An initial panel of eight multi-domain Fc fusion molecules with various domain configurations incorporating either IgG1 or IgG4 Fc domains was constructed. In the first example, the TCR variable domain of the multi-domain molecule was linked to the HLA-A domain derived from MAGEA4. * The TCR variable domains were designed to specifically recognize and have high affinity for the 02-restricted peptide GVYDGREHTV. Such TCR variable domains are described in WO 2017 / 175006. Exemplary sequences of multi-domain Fc functional molecules according to the present invention are also shown in SEQ ID NOs: 52, 54, and 56, SEQ ID NOs: 52, 58, and 56, or SEQ ID NOs: 52, 60, and 61. The immune cell-engaging domain was an anti-CD3 scFv fragment. Such sequences can be found in (WO 2020 / 157210) and are also shown in SEQ ID NOs: 62 and 63. The ability of these various molecules to drive T cell activation in the presence of antigen-positive and antigen-negative cells was investigated using an ELISpot assay using IFNγ as a readout for T cell activation.

[0209] ELISpot assays were performed using a human IFN-γ ELISPOT kit (BD Biosciences) according to the manufacturer's instructions. Briefly, T2 cells pulsed with 100 nM MAGEA4 peptide were used as antigen-positive target cells, and T2 cells pulsed with an irrelevant peptide were used as antigen-negative target cells. PBMCs were used as effector cells. The effector-target ratio was 0.5:1.

[0210] A multi-domain molecule called Mol020, with the format shown in Figure 1A and with functional domains distributed across three polypeptide chains, was selected based on potency and specificity data.

[0211] Figure 2 shows a comparison of T cell activation in the presence and absence of antigen-positive cells for the two different formats tested (fused to IgG1). The multidomain molecule with the Mol020 format was significantly more potent than the equivalent molecule with an alternative two-chain format (Mol014, shown in Figure 1B(i)), while maintaining a high degree of specificity.

[0212] Subsequently, an expanded panel of 38 additional Fc fusion formats was generated and screened by ELISpot assay to see if more potent formats could be identified. None of the 38 formats tested showed better activity than Mol020.

[0213] To determine whether Mol020 would produce a high degree of efficacy in alternative TCRs, the MAGEA4-specific TCR variable domains of Mol020 and Mol014 were cloned into PRAME-derived HLA-A * The TCR variable domain was replaced with one that specifically recognizes the O2-restricted peptide SLLQHLIGL. Such high-affinity PRAME-specific TCRs are described in WO 2018 / 234319. IFNγ ELISpot assays were performed as described above, except that human melanoma Mel624 cells were used as antigen-positive target cells. Mel624 cells in which PRAME was knocked out were used as antigen-negative cells.

[0214] The data in Figure 3 demonstrate that Mol020 is again significantly more potent than the alternative two-chain format molecule (Mol014) while maintaining a high degree of specificity. Overall, these data demonstrate that Mol020 is superior to Mol014, regardless of TCR sequence.

[0215] Example 2 - Multi-domain Fc fusion molecules targeting MAGEA4 maintain a similar therapeutic window to Fc-free molecules The potency and specificity of a MAGEA4-targeting multi-domain Fc fusion molecule (Mol020) was compared to a comparable multi-domain molecule lacking an Fc domain (Mol001, shown in Figure 1B(ii)). Mol014 and Mol001 incorporate the same MAGEA4-specific TCR. Such TCR-anti-CD3 fusion molecules are further described in WO 2017 / 175006.

[0216] IFNγ ELISpot assays were performed according to the manufacturer's instructions. Briefly, human lung carcinoma NCI-H1755 cells were used as antigen-positive target cells, and human melanoma Mel202 cells were used as antigen-negative target cells. Mel202 cells were pre-transduced with HLA-A2 B2M, resulting in very high levels of HLA-A2 expression. This is known to result in artificially high levels of nonspecific reactivity, which facilitates comparison. PBMCs were used as effector cells. The effector-target ratio was 0.8:1 for Mel202 and 1:1 for NCI-H1755.

[0217] The data shown in Figure 4 indicate that Mol020 generates a weaker response than Mol001 against antigen-positive cells, while at higher concentrations there is also a corresponding reduction in nonspecific activity with Mol020 compared to Mol001. The difference between on-target and off-target T cell activation is the same for both molecules. Collectively, these data demonstrate that the therapeutic window is maintained for Mol020 compared to the Fc-free fusion, Mol001.

[0218] Example 3 - Multi-domain Fc fusion molecules targeting MAGEA4 exhibit extended in vivo half-life compared to Fc-free molecules The pharmacokinetic (PK) properties of Mol020 and Mol001 were evaluated in Tg32 SCID mice to determine their in vivo half-lives. Test articles were dosed at 1 mg / kg by IV bolus with 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 MAGEA4 peptide-HLA capture and sulfo-tagged anti-scFv antibody detection. Figure 5 shows serum concentrations over time. PK parameters were extracted by noncompartmental analysis. Mol001 showed rapid clearance, whereas Mol020 showed a slow t 1 / 2 showed a significant increase in HR, which was calculated to be 4.5 days.

[0219] Example 4 - Preparation of multi-domain Fc fusion molecules targeting MAGEA4 MAGEA4-derived HLA-A * A multi-domain Fc fusion molecule (MolO20) recognizing the O2-restricted peptide GVYDGREHTV was prepared using CHO cells and purified via a two-step process.

[0220] Briefly, material was produced by transient transfection of expiCHO cells according to the manufacturer's "Max-titre" protocol (Thermo Fisher). After incubation, the CHO culture was centrifuged at 10,000 rpm to remove cells and other debris, and the supernatant was filtered and loaded onto a HiTrap Protein L column using PBS as the running buffer. The protein was then eluted with 100 mM citric acid (pH 2.5) and concentrated in a 10,000 MWCO concentrator after raising the pH with 200 μl of 2 M Tris (pH 8.3) per ml of eluate. Further purification was then performed using a SUP 200 10 / 300 size-exclusion column using PBS as the running buffer. Peak fractions were collected and purity was determined by SDS-PAGE.

[0221] Example 5 - Effect of glycosylation on yield and thermal stability of multi-domain Fc fusion molecules targeting MAGEA4 To investigate the effect of glycosylation, sites within the TCR portion of the molecule were identified and mutated. Briefly, seven N-linked glycosylation sites were identified within the TCR variable domain and engineered for their systematic removal by replacing N with Q. The molecule was prepared as described in Example 4.

[0222] The concentration of purified material was measured by absorbance at 280 nm using a Nanodrop spectrophotometer, and extinction coefficient and molecular weight values were calculated from the sequence using Snap Gene software. The final mg of protein was then divided by the total expression volume in liters.

[0223] The UNCLE instrument (Unchained Labs) was used to assess the unfolding and thermal aggregation of purified proteins in PBS in parallel quartz capillaries ("Uni"). The machine uses static light scattering to monitor aggregation and centroid average of tryptophan fluorescence to monitor unfolding of protein samples. The temperature was increased at a rate of 0.5°C / min, and protein samples were run at both 50 μg / ml and 200 μg / ml to monitor denaturation and aggregation. The melting temperature of the TCR portion of the molecule was used as an indicator of thermal stability.

[0224] Analysis of glycosylation site mutants revealed that removing all seven N-linked glycosylation sites within the TCR portion of the molecule resulted in an approximately 75% reduction in yield. Further investigation demonstrated that removing four N-linked glycosylation sites within the TCR constant region did not appear to contribute to the reduction in yield.

[0225] Subsequently, double and single glycosylation site mutants in the TCR variable region were generated and tested. Figure 6 shows a schematic diagram illustrating each mutant and indicating the location of the glycosylation site. Three glycosylation sites within the TCR variable domain are shown at positions N18 and N24 in the alpha chain and N84 in the beta chain. The corresponding relative yield is shown below each mutant, along with the melting temperature (Tm) of the TCR portion of the molecule. The data demonstrate a stepwise decrease in yield with reduced glycosylation.

[0226] Example 6 - Binding affinity and kinetics of monoglycosylated and deglycosylated multi-domain Fc fusion molecules targeting MAGEA4 In the examples below, molecule Mol020v13 is deglycosylated, while molecule Mol020v14 contains a single glycosylation site at position N18 within the TCR alpha variable domain. The complete amino acid sequences of both molecules are shown in SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, and SEQ ID NO:52, SEQ ID NO:58, SEQ ID NO:56, respectively.

[0227] To verify that the TCR and anti-CD3 portions of the molecules bound to their respective targets, single-cycle kinetics was performed by surface plasmon resonance (SPR) on a T200 BIAcore followed by a single injection of CD3(γε). Binding to FcRn was also assessed.

[0228] CD3 and pHLA binding measurements were performed on an 8K Biacore instrument by first immobilizing streptavidin to the chip using EDC-NHS amine coupling, followed by binding of biotinylated CD3 or pHLA. Single-cycle kinetic experiments were performed with five injections at either 37°C (pHLA) or 25°C (CD3) in a running buffer of PBS (pH 7.4) containing 0.005% surfactant P20. Dissociation and association rates were fitted to a 1:1 binding kinetic analysis in Biaevaluation software.

[0229] FcRn binding measurements were performed using a Biacore T200 instrument. Fc-ImmTAC molecules were immobilized on a CM5 sensor chip by first amine-coupling streptavidin to the chip, then binding biotinylated pHLA to the treated chip, and then binding Fc-ImmTAC to the pHLA, effectively immobilizing these molecules by taking advantage of the long dissociation time of the TCR-pHLA interaction. Increasing concentrations of soluble FcRn (Immunitrak) were then injected across the sensor surface and allowed to dissociate. Responses were then plotted versus concentration and fitted to a "one-site total" equation in graphpad prism to determine the equilibrium K. D These experiments were carried out at 25°C in 20 mM MES (pH 6) containing 0.005% surfactant P20 and 137 mM NaCl.

[0230] Both Mol020v13 and Mol020v14 showed similar strong binding to pHLA and CD3, consistent with the Fc-free fusion Mol001. Additionally, Mol020v13 and Mol020v14 showed binding to FcRn.

[0231] TIFF2025526897000028.tif54170

[0232] Example 7 Serum stability of monoglycosylated and deglycosylated multi-domain Fc fusion molecules targeting MAGEA4 The stability of Mol020v14 and Mol020v13 over time was assessed in vitro in human serum at 37°C as a surrogate for in vivo stability. Bispecific binding activity was assayed at various time points over a total of 14 days by electrochemiluminescence immunoassay, captured with biotinylated MAGEA4 peptide-HLA and detected with a sulfo-tagged anti-scFv antibody. The percent change from baseline was calculated for each time point and plotted. The results are shown in Figure 7.

[0233] Example 8 - In vivo half-life of monoglycosylated and deglycosylated multi-domain Fc fusion molecules targeting MAGEA4 Further PK studies in Tg32 SCID mice were performed as described in Example 3 to compare the effect of glycosylation on in vivo half-life. In this example, the monoglycosylated mutant mol020v14 is compared to the deglycosylated mutant mol020v13. A fully glycosylated molecule (used in Example 3) that retains all seven TCR glycosylation sites was used as a control. The data shown in Figure 8 indicate that the monoglycosylated mutant (mol020v14) exhibited a further increase in in vivo half-life compared to the deglycosylated mutant (mol020v13). The molecule with all seven glycosylation sites intact is comparable to the deglycosylated molecule.

[0234] The PK analysis for Mol020v14 was repeated at three dose levels as shown in the table below. The data demonstrated consistent PK parameters across a wide dose range. Terminal phase t calculated by non-compartmental analysis 1 / 2 The mean time to maturity was 5.5 to 7.2 days.

[0235] TIFF2025526897000029.tif30170

[0236] Example 9 Multi-domain Fc fusion molecules that bind MAGEA4 exhibit potent and specific activity against antigen-positive cancer cell lines Further evaluation of the potency and specificity of v14 and v13 was performed using various cancer cell lines. T cell activation was assessed by IFNγ ELISpot assay, and cell killing was assessed using the xCELLigence assay.

[0237] ELISpot Assays were performed using a human IFNγ ELISPOT kit (BD Biosciences) according to the manufacturer's instructions. Briefly, target cells were cultured at 1 × 10 cells per ml in assay medium. 6 Cells were prepared at a density of 1000 and plated at 50,000 cells per well in a volume of 50 μl. PBMCs isolated from fresh donor blood were used as effector cells. Fusion molecules were titrated downward from 10 nM to obtain the final concentrations indicated. Samples were detected using AEC chromagen. Spot counting was performed using a CTL analyzer equipped with Immunospot software (Cellular Technology Limited). Ec50 values were calculated from the data.

[0238] Xcelligence Assays were performed using the xCELLigence platform with the appropriate 96-well plate for impedance readings (xCELLigence E-plate 96 PET, part number 300600900) and were performed according to the manufacturer's instructions. Target cells were plated at the required density 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-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. The final volume in each well was adjusted to 200 μl using assay medium. The percentage of cell lysis was determined using a normalized cell index (impedance measurement). In each case, assays were performed in triplicate, and measurements were taken every 2 hours over a 96-hour period. Calculation of Ec50 was derived from the curve of percent cell lysis at 72 hours.

[0239] The antigen positive (MAGEA4 positive; HLA-A * 02 positive) Cancer cell lineage, NCI-H1703(NSCLC) NCI-H1755(NSCLC) H314 (head and neck cancer) SCaBER (bladder cancer) UM-UC-3 (bladder cancer) It was.

[0240] Antigen-negative cell lines express MAGEA4 and HLA-A * The results were negative for all 02.

[0241] result The data shown in Figure 9 demonstrate that T cell activation was observed against all HLA-positive / Ag-positive cell lines from various indications, with Ec50 values in the pM range. Limited responses were observed in antigen-negative cells, even at the highest concentrations of the molecules tested.

[0242] The table below shows the average Ec50 values obtained from three different PBMC donors.

[0243] TIFF2025526897000030.tif31170

[0244] In the most sensitive cell lines (NCI-H1703 and NCI-H1755), cancer cell killing was observed at concentrations as low as 0.17 pM up to 370 pM.

[0245] Ec50 values were calculated from data obtained at 72 hours. Data from one T cell donor is shown in the table below. UM-UC-3 cells were not suitable for testing with the xCELLigence assay.

[0246] TIFF2025526897000031.tif36170

[0247] Overall, these data demonstrate that mol020v13 and mol020v14 generate equally potent and specific T cell responses against antigen-positive cancer cells, making them suitable for therapeutic use.

[0248] Example 10 - Multi-domain Fc fusion molecules that bind MAGEA4 show limited reactivity against a panel of normal cell lines Mol020v13 and mol020v14 were further tested for reactivity against a panel of cells derived from high-risk normal tissue types, including cardiac cells, muscle cells, excretory cells, gastrointestinal cells, lung cells, bone cells, and induced pluripotent stem cells (astrocytes, cardiomyocytes, hepatocytes). In each case, reactivity was determined using an ELISpot assay to detect the release of IFNγ and granzyme B at various test molecule concentrations up to 10 nM. The lowest concentration of test molecule at which IFNγ was detected was recorded.

[0249] For both molecules, a limited loss of specificity was observed at 1.1 nM, with a broad range of specificity loss observed from 3.3 nM onwards. The lowest concentrations producing reactivity were 0.55 nM and 1.1 nM for the monoglycosylated and aglycosylated forms, respectively.

[0250] Overall, the differences between the two molecules are minimal, and both molecules display acceptable profiles for therapeutic use.

[0251] Example 11 - HLA-A from preproinsulin (PPI) fused to a PD-1 agonist binding domain * Multidomain Fc fusion molecules containing TCR mutants that bind to O2-restricted peptides retain target specificity and are functional in vitro a) Production of multi-domain binding molecules Preproinsulin-derived HLA-A *TCRs that bound to the O2-restricted peptide ALWGPDPAAA were isolated by panning a TCR phage library, and the amino acid sequences of the corresponding TCR alpha and beta variable regions were determined. Construction and panning of native TCR phage libraries were previously described (WO 2015 / 136072, WO 2017 / 046201, WO 2017 / 046198). Soluble TCRs were generated by fusing the variable regions to truncated versions of the respective alpha and beta chain constant domains, incorporating non-native interchain disulfide bonds between constant domain residues, as previously described (WO 2003 / 020763). One of the identified soluble TCRs, TCR a2b3, was further mutated to remove potential glycosylation sites and further optimize the sequence for the manufacturing process. Five TCR sequence variants were tested. These are designated by the following molecular IDs:

[0252] PD-1 agonist antibody V HH The domain was fused via a short linker to the N-terminus of the beta chain of a soluble TCR to generate a TCR PD-1 agonist-binding molecule. To extend in vivo half-life, a functionally silent Fc domain was attached to the C-terminus of the TCR alpha chain via a truncated hinge region.

[0253] FIG. 10 shows a schematic of the resulting extended half-life TCR PD-1 agonist binding molecules.

[0254] b) Mammalian Expression Molecules according to item a) were expressed in CHO cells using Thermo's ExpiCHO™ transient expression protocol and subsequently purified using immobilized metal affinity chromatography and size exclusion chromatography.

[0255] c) Biophysical characterization TCR PD-1 agonist binding molecules with Fc domains were tested for binding to target and mimetic peptides. Experiments were performed using single-cycle kinetics as described above, except measurements were performed at 37°C.

[0256] TIFF2025526897000032.tif58170

[0257] The data demonstrate that Fc domain-containing TCR PD-1 agonist-binding molecules can be produced in high yields in mammalian cells while maintaining a suitable window of high-affinity target recognition and binding to mim1. This high level of specificity makes these molecules particularly suitable for therapeutic development as potential treatments for T1D.

[0258] d) In vitro functional - Jurkat NFAT cell reporter assay An NFAT reporter assay was developed to determine the ability of TCR PD-1 agonist binding molecules to inhibit signaling in activated T cells. Briefly, i) Melan A-derived HLA-A * Jurkat cells expressing a TCR specific for the O2-restricted peptide (ELAGIGILTV), ii) PD-1, and iii) a luciferase reporter driven by an NFAT response element were incubated with the PPI-positive beta cell line ECN90 pulsed with a Melan-A-derived peptide to induce TCR signaling and NFAT promoter-mediated luminescence. Control experiments were performed using a PPI-negative target cell line (Mel624, NCI-H1703) instead of ECN90.

[0259] Target cells were harvested and plated at 50,000 cells per well in Optiβ3 medium into the inner 60 wells of a white 96-well cell culture plate pre-coated with β-coat (Univercell Biosolutions). After 16–20 h of incubation at 37°C and 5% CO2, the medium was removed and assay buffer containing Melan-A peptide was added. No peptide was added to the Mel624 melanoma line, which naturally presents the melan-A peptide. After 2 h of pulsing at 37°C and 5% CO2, assay buffer alone or assay buffer containing titrations of TCR PD-1 agonist binding molecules was added to each well. The assay was initiated by immediately adding 50,000 Jurkat NFL Mel5 PD-1 effector cells and incubating for 16–20 h at 37°C and 5% CO2. Bioluminescent signals were detected and quantified using the Bio-Glo™ Luciferase Assay System (Promega) and a luminometer (CLARIOstar). NFAT activity was normalized to TCR-stimulated controls, and dose-response data were analyzed in Prism (GraphPad) using a four-parameter nonlinear least-squares fit to determine IC 50 value was determined.

[0260] The obtained IC 50 Values are shown in the table below for each TCR-PD1 agonist-binding molecule indicated. Values are based on the average from two independent experiments. Figure 11 shows data for one experiment from two molecules tested.

[0261] TIFF2025526897000033.tif37170

[0262] Using a similar NFAT-based reporter assay, we found that the Fc domain was IC 50 The impact on the value was evaluated.

[0263] In this case, HLA-A cells were pulsed with 20 μM of PPI peptide for 2 hours at 37°C and 5% CO2. * 02 human B lymphoblastoid cells (Raji) were used as target cells. Cells were harvested and plated at 50,000 cells per well in assay medium (R10 without antibiotics) into the inner 60 wells of a white 96-well cell culture plate. Subsequently, cells were treated with 2 μg / ml SEB (Staphylococcal enterotoxin B) for 1 hour at 37°C and 5% CO2. Assay buffer alone or assay buffer containing a titration of TCR PD-1 agonist binding molecules was added to each well. The assay was initiated by immediately adding 50,000 Jurkat NFL Mel5 PD-1 effector cells and incubating for 16–20 hours at 37°C and 5% CO2. Bioluminescent signals were detected as described above.

[0264] Data were obtained using the TCR-PD1 agonist binding molecule a18b16 with the Fc domain described above, and glycosylation variants with or without the Fc domain.

[0265] The data in Figure 11b show that the inclusion of the Fc domain has minimal effect on potency in vitro.

[0266] Data from reporter assays support that the TCR PD-1 agonist binding molecule can potently inhibit T cell activation and demonstrates therapeutic potential for the treatment of T1D.

[0267] Example 12 - TCR-PD1 agonist binding molecules provide extended half-life in vivo The pharmacokinetic properties of the Fc domain-bearing TCR PD-1 agonist binding molecule a18b16 (described in Example 11) were evaluated in SCID mice. The test article was dosed intravenously (IV) or subcutaneously (SC) at 1 mg / Kg, and blood samples were collected serially over 21 days. Four mice were sampled per time point per dosing route. The binding molecule was detected in serum using a bifunctional MSD (Meso Scale Diagnostics) assay. PK parameters were extracted by non-compartmental analysis.

[0268] The mean PK parameters are shown in the table below: Figure 12 shows the concentration of the reagent in serum over a 3-week period.

[0269] TIFF2025526897000034.tif35170

[0270] This study demonstrated that Fc-bearing TCR PD-1 agonists produced a terminal phase t 1 / 2 It has been shown to have a subcutaneous bioavailability of greater than 80%. These properties indicate its therapeutic potential to provide a convenient dosing schedule for the treatment of T1D.

[0271] Example 13 - TCR-PD-1 agonist binding molecules demonstrate robust efficacy in in vitro models a) Primary human T cell IL-2 assay The TCR PD-1 agonist binding molecules described in Example 11 were tested to determine their ability to bind primary human CD4 T cells by antigen-presenting cells (APCs). + The ability to inhibit T cell activation was determined. Free PD-1 agonist was used as a control, along with a non-targeting TCR PD-1 agonist control that does not bind to the PPI peptide.

[0272] HLA-A * 02 β2-microgobulin-transduced Raji cells were used as APCs (Raji-A2). Primary human CD4 +T cells were isolated from PBMCs using a pan T cell isolation kit (Miltenyi). T cells were preactivated by incubation with irradiated Raji A2 cells preloaded with 1 μg / ml SEB (Sigma). After preactivation, expanded T cells were primarily CD4 + T cells, typically 60%-70% PD-1 positive, Raji A2 cells, at 2 × 10 per ml in R10 6 Raji A2 cells were pulsed or not with 20 μM PPI peptide for 2 hours at 37°C and 5% CO2. Raji A2 cells were then loaded with 31.6 ng / ml SEB for 1 hour at 37°C and 5% CO2 and irradiated with 33 Gy. Raji A2 cells were plated at 100,000 cells per well, and test molecules were added. After 1 hour of preincubation, washed pre-activated T cells were added to Raji A2 cells at 100,000 cells per well and incubated for 48 hours at 37°C and 5% CO2. Supernatants were collected, and IL-2 levels were measured by ELISA (IL2 Ready-SET-Go! ELISA, Invitrogen). IL-2 release was normalized to the SEB-stimulated control, and dose-response data were analyzed in Prism (GraphPad) using a four-parameter nonlinear least-squares fit to determine IC 50 value was determined.

[0273] These results demonstrated that in the presence of PPI peptide-pulsed APCs, the TCR PD-1 agonist molecule, when present at picomolar concentrations, reduced IL-2 production from activated T cells by 40%–50% (Figure 13). Furthermore, the PD-1 agonist alone and the non-targeting TCR PD-1 agonist control did not reduce IL-2 levels, indicating that targeting of the PD-1 agonist to the immune synapse is required for functional activity.

[0274] These data suggest that targeted TCR PD-1 agonist molecules inhibit primary CD4 +These results demonstrate that the antibody is a potent inhibitor of T cells. Furthermore, the lack of activity seen with non-targeted molecules may avoid the risk of systemic activation in vivo.

[0275] b) Protection of pancreatic β cells co-cultured with autoreactive T cells The TCR PD-1 agonist binding molecules described in Example 11 were tested to demonstrate their ability to kill the pancreatic β-cell line EndoCβH2-A2 and inhibit autoreactive CD8 + The ability to inhibit cytokine release by T cells was determined.

[0276] EndoC-βH2 target cells labeled with mKate 2 (EndoC-βH2 Red) were generated by transducing EndoC-βH2 cells with an HLA-A2 β2-microglobulin lentiviral construct and NucLight red lentiviral reagent (Sartorius). Target cells were grown at 5 × 10 per well of a 96-well plate in Optib3 medium. 4 The cells were plated at 1000 x 1000 cells per well and incubated overnight at 37°C, 5% CO2. TCR PD-1 agonist molecules or control molecules were added at various concentrations and incubated for 2 hours. To initiate the assay, two β-cell-specific CD8 agonists with high or low affinity for the target cells were used. + One of the T cell clones was cultured at 5 × 10 per well. 4EndoC-βH2 red target cells were added at 1000 cells / mL. PD-L1-transduced EndoC-βH2 red target cells with or without anti-PD-L1 blocking antibodies were used as an additional control. Cell killing was determined by quantifying the number of EndoC-βH2 red cells over time using an IncuCyte S3 imaging system (Sartorius). The number of red nuclear-labeled cells at each time point was normalized to the initial number of subjects to account for variations in cell density within the visualized area. The number of events was averaged over four images. Cytokine release was measured using culture supernatants from the IncuCyte killing assay 24 hours after each time point using the V-PLEX Plus Proinflammatory Panel 1 (Human) kit according to the manufacturer's instructions (MSD, Meso Scale Diagnostics). For cytokine assays, unstimulated T cells alone were evaluated as an additional control. Cytokine release was normalized to stimulated controls and dose-response data were analyzed in Prism (GraphPad) using a four-parameter nonlinear least-squares method to determine IC 50 value was determined.

[0277] The data showed that when co-cultured in the presence of autoreactive T cells, increasing concentrations of TCR PD-1 agonist-binding molecules resulted in a dose-dependent increase in the relative number of β cells, demonstrating that these molecules can prevent β cell killing by autoreactive T cells. No effect was observed with the PPI TCR alone or a non-targeting control (Figure 14a).

[0278] Cell culture supernatants from both co-culture assays were assessed for cytokine production, demonstrating that TCR PD-1 agonists potently inhibited IFNγ production by autoreactive T cells (Fig. 14b).

[0279] These data demonstrate that TCR PD-1 agonist binding molecules inhibit killing and cytokine release by T cells that span the expected affinity range of the natural repertoire of autoreactive T cells, indicating the therapeutic potential of the molecules.

[0280] c) Inhibition of PD-1-positive NK cell stimulation The TCR PD-1 agonist binding molecules described in Example 11 were further investigated to determine their ability to inhibit stimulation of PD-1 positive NK cells. + To test whether NK cells could be specifically inhibited, NK cells were activated with the pancreatic β-cell line EndoC-βH2. Activation was monitored by expression of the cytotoxic marker CD107a and IFNγ production.

[0281] Primary human NK cells were isolated from PBMCs using an NK cell isolation kit (Miltenyi Biotec 130-092-657). NK cells were incubated for 6 days in R10 medium (RPMI-1640 supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, and 1 mM sodium pyruvate) containing dexamethasone (500 ng / mL, Merck, D2915), IL-12 (10 ng / mL, Miltenyi Biotec 130-096-704), IL-15 (25 ng / mL, Peprotech), and IL-18 (100 ng / mL, R&D Systems, 9124-IL-050). After 6 days, NK cells were washed in R10 and transfected with EndoC-βH2 HLA-A2 in R10 containing monensin, brefeldin A (GolgiPlug and GolgiStop BD), and anti-CD107a antibody with or without TCR-PD1 agonist binding molecules. +The cells were incubated at a 1 / 4 (effector / target) ratio for 4 hours (37°C, 5% CO2). After activation, NK cells were surface stained (anti-CD56, anti-CD3, anti-PD1, and dead cell markers) for 30 minutes, then fixed and permeabilized (eBioscience Foxp3 Transcription Factor Staining Buffer Set, Catalog No.: 00-5523-00) for IFNγ intracellular staining.

[0282] The data showed that in the presence of TCR PD-1 agonists, the expression levels of CD107 and IFNγ were reduced in PD-1-positive NK cells. No effect was observed on PD-1-negative NK cells. Therefore, TCR PD-1 agonists reduced the expression of PD-1. + Specifically reduces NK cell activation (Figure 15). Data shown are from two independent experiments.

[0283] These data demonstrate that TCR PD-1 agonist binding molecules inhibit the stimulation of PD-1+ NK cells, providing a potential additional therapeutic mechanism of action and differentiating them from other approaches.

[0284] d) Targeting beta cells in pancreatic tissue slices To assess the activity of the TCR-PD-1 agonist molecule in a physiologically relevant context, live pancreatic tissue sections from nondiabetic and diabetic tissue donors were treated with a fluorescently labeled TCR PD-1 agonist. Confocal images captured revealed that the molecule specifically targeted beta cells within the islets in both nondiabetic and diabetic donors. Furthermore, increased T cell mobility within the islets was observed, suggesting decreased beta cell-T cell interactions.

[0285] Overall, these data obtained across a variety of disease-relevant models demonstrate the therapeutic potential of TCR-PD-1 agonist molecules for the treatment of T1D.

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 immune effector domain comprising a linkage of an antibody light chain variable domain (TCE-VL) and an antibody heavy chain variable domain (TCE-VH), (iii) A half-life extension domain comprising a portion of the first IgG Fc region (FC1) and a portion of the second IgG1 Fc region (FC2), A multi-domain binding molecule, including The aforementioned multi-domain binding molecule is i) The T cell-engaging immune effector domain is linked to the N-terminus of VC1 in a first polypeptide chain, ii) A second polypeptide chain in which VC2 is linked to the N-terminus of FC1 via its C-terminus, iii) A third polypeptide chain containing FC2, including, and, The pMHC-binding domain and the T-cell-engaging immune effector domain are multi-domain binding molecules capable of binding to the pMHC complex and T cells, respectively.

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

3. The multiple-domain 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 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. (i) The pMHC-binding domain binds to a tumor-associated antigen peptide that forms a complex with MHC, optionally the tumor-associated antigen being MAGEA4, and optionally the pMHC-binding domain binds to the GVYDGREHTV (SEQ ID NO: 1) HLA-A*02 complex; and / or (ii) Two or more of the domains are linked via linkers and / or IgG hinge sequences, and optionally, the linkers(s) may have sequences selected from the group consisting of GGGGS (SEQ ID NO: 34), GGGSG (SEQ ID NO: 35), GGSGG (SEQ ID NO: 36), GSGGG (SEQ ID NO: 37), GSGGGGP (SEQ ID NO: 38), GGEPS (SEQ ID NO: 39), GGEGGGP (SEQ ID NO: 40), GGEGGGSSEGGGS (SEQ ID NO: 41), GGGGSGGGG (SEQ ID NO: 42), GGGGSGGGGGSGGGGGSGGGGGSGGGS (SEQ ID NO: 46), GGGGSGGGGGSGGGGGSGGGGGS (SEQ ID NO: 43), EAAAK (SEQ ID NO: 44), and EAAAKEAAAAKEAAAAK (SEQ ID NO: 45); and / or (iii) The half-life extension domain comprises one or more amino acid substitutions that promote dimerization of FC1 and FC2, optionally, (a) Either FC1 or FC2 comprises one or more amino acid substitutions selected from the group consisting of T366S, L368A, and Y407V (as numbered according to the EU numbering scheme), (b) The other of FC1 or FC2 comprises the amino acid substitution T366W (as numbered according to the EU numbering scheme); and / or (iv) FC1 and / or FC2 contain the amino acid substitution N297G (as numbered according to the EU numbering scheme); and / or (v) The multi-domain binding molecule according to claim 1 or 2, wherein FC1 and / or FC2 comprises the amino acid substitution M252Y / S254T / T256E (as numbered according to the EU numbering scheme).

6. VC1 and / or VC2 include one or more amino acid substitutions that remove a glycosylation site, optionally, (a) The substitution is a substitution from N to Q; and / or (b) (i) The TCRα chain contains one or more amino acid substitutions at positions selected from the group consisting of N18, N24, N146, N180, and N191 (numbered according to Sequence ID No. 25), and / or (ii) The TCRβ chain contains amino acid substitutions at positions N84 and N186 (numbered according to Sequence ID No. 27), A multi-domain binding molecule according to claim 1 or 2.

7. The aforementioned T cell-engaging immune effector domain is (a) (i) A TCE-VL region containing the CDRs of sequence numbers 68, 69, and 70 as CDR1, CDR2, and CDR3, respectively, (ii) A TCE-VH region containing the CDRs of sequence numbers 71, 72, and 73 as CDR1, CDR2, and CDR3, respectively, Including; and / or (b) The T cell-engaging immune effector domain comprises TCE-VL, which is at least 80% identical to the sequence of SEQ ID NO: 66, and TCE-VH, which is at least 80% identical to the sequence of SEQ ID NO: 67; and / or (c) (i) The TCRα variable domain includes the CDRs of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4 as CDR1, CDR2, and CDR3, respectively, (ii) The TCRβ variable domain includes the CDRs of SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10 as CDR1, CDR2, and CDR3, respectively; and / or (d) VC2 includes a TCRα variable region that is at least 80% identical to the sequence of SEQ ID NO: 6, and VC1 includes a TCRβ variable region that is at least 80% identical to the sequence of SEQ ID NO: 11; and / or (e) VC2 includes a TCRα constant region which is at least 80% identical to the sequence of SEQ ID NO: 29, and VC1 includes a TCRβ constant region which is at least 80% identical to the sequence of SEQ ID NO: 31; and / or (f) Either FC1 or FC2 has an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 49, and the other FC1 or FC2 has an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 50; and / or (g) (i) The first polypeptide chain of the molecule has a sequence that is at least 80% identical to the sequence of Sequence ID No. 56, (ii) The second polypeptide chain of the molecule has a sequence that is at least 80% identical to the sequence of Sequence ID No. 54, (iii) The third polypeptide chain of the molecule has a sequence that is at least 80% identical to the sequence of Sequence ID No.

52. A multi-domain binding molecule according to claim 1 or 2.

8. The aforementioned multi-domain binding molecule is (i) Starting from the N-terminus and moving toward the C-terminus, in the following order: a. Amino acid sequence of anti-CD3 scFv, b. Optionally followed by the linker sequence shown in sequence number 34, and c. Amino acid sequences of the variable and constant regions of TCRβ, A first polypeptide chain including, (ii) Starting from the N-terminus and moving toward the C-terminus, in the following order: a. Amino acid sequences of the variable and constant regions of TCRα, b. Optionally followed by the shortened hIgG1 hinge sequence shown in Sequence ID No. 33, and c. Fc region having the sequence shown in Sequence ID 50, A second polypeptide chain, including (iii) A third polypeptide chain comprising an Fc region having the sequence shown in Sequence ID No. 52, Including, here, The multi-domain binding molecule according to claim 1 or 2, wherein the TCRα chain and the TCRβ chain dimerize to form a peptide-major histocompatibility complex (pMHC) binding domain.

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

10. An expression vector comprising the nucleic acid described in claim 9.

11. A host cell comprising the nucleic acid described in claim 9 or the expression vector described in claim 10.

12. A method for producing a multi-domain binding molecule according to claim 1 or 2, comprising: maintaining a host cell according to claim 11 under conditions optimal for the expression of the nucleic acid according to claim 9 or the expression vector according to claim 10; and isolating a multi-domain antigen-binding polypeptide.

13. A pharmaceutical composition comprising a multi-domain binding molecule according to claim 1 or 2, a nucleic acid according to claim 9, an expression vector according to claim 10, or a host cell according to claim 11.

14. A pharmaceutical composition according to claim 13, to be used as a pharmaceutical.

15. The pharmaceutical composition according to claim 13, which is used in the treatment of cancer, an infectious disease or an autoimmune disease, wherein the cancer may express MAGEA4.

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