The present disclosure relates to proteins comprising T-cell receptor (TCR) constant domains with one or more stabilization mutations, nucleic acids encoding such proteins, and methods of making and using such proteins.

T cell receptors (TCRs) are the adaptive immune system’s tool for recognizing and eliminating “non-self’ intracellular antigens. These non-self antigens can emerge from viral infection or from genetic alteration. Genetic alterations are key to aberrant cellular function and can lead to diseases such as cancer. TCRs exist in a/b and g/d forms, which are structurally similar but express on different T cells. The a/b TCRs are expressed on both CD8+ effector and CD4+ helper T cells and recognize proteosomally degraded foreign antigens displayed on infected/cancerous cell surfaces when complexed with HLA/MHC (Davis, et ah, Nature, 1988. 334(6181): 395-402; Heemels, et ah, Annu Rev Biochem, 1995. 64: 463-91). When expressed on CD8+ T effector cells, a/b TCRs interact specifically with Type I MHC/peptide complexes and this interaction induces T cell activation and elimination of cells displaying recognizable non-self antigens.

Structurally, the extracellular portion of native a/b TCR consists of two polypeptides, a chain and b chain, each of which has a membrane-proximal constant domain (Ca or Cb domain), and a membrane-distal variable domain (Va or nb domain). Each of the constant and variable domains includes an intra-chain disulfide bond. The variable domains contain the highly variable loops analogous to the complementarity determining regions (CDRs) of antibodies. CDR3 of the TCR interacts with the peptide presented by MHC (major histocompatibility complex), and CDR1 and CDR2 interact with the peptide and the MHC. The diversity of TCR sequences is generated via somatic rearrangement of linked variable (V), diversity (D), joining (J), and constant (C) genes; and such rearrangement generates an incredible diversity that enables the TCR to recognize diverse peptide antigens displayed by MHCs.

Many approaches have been developed to harness the exquisite non-self-recognizing properties of the a/b TCRs for therapeutic use. Recombinant a/b TCRs have been transduced/transfected into bulk naive T cells as a means of redirecting these T cells to target tumor associated antigens (Riley, et al., Nat Rev Drug Discov, 2019. 18(3): 175-196; Parkhurst, et al., Clin Cancer Res, 2017. 23(10): 2491-2505). Alternately, the use of the soluble extracellular region of the TCR fused to an scFv that binds an activating T cell receptor, commonly CD3s, has been used to redirect endogenous T cells to target tumor cells (Liddy, et al., Nat Med, 2012. 18(6): 980-7). Unlike typical BiTE-like bispecific antibodies that rely on the direct recognition of overexpressed antigens on the cell surface (Baeuerle, et al., Cancer Res, 2009. 69(12): 4941-4), TCR or TCR-mimic bispecifics can recognize a much larger subset of intracellular and abnormal tumor or viral antigens and thus have broader potential applicability (Liddy, et al., Nat Med, 2012. 18(6): 980-7).

However, TCR assembly and expression is challenging (Wilson, et al., Curr Opin Struct Biol, 1997. 7(6): 839-48). For both research and therapeutic purposes, the common method of soluble a/b TCR production has been through the expression as inclusion bodies in Escherichia coli ( E . coli) followed by resolubilization, refolding/assembly/oxidation, and finally purification at relatively low yields (van Boxel, et al., J Immunol Methods, 2009. 350(1-2): 14-21). To simplify the assembly piece, some researchers have tried using only the variable domain regions of TCRs in a single chain format or scTv, like an antibody single chain Fv (scFv), for targeting specific HLA/peptide complexes (Stone, et al., Methods Enzymol, 2012. 503: 189-222). While scFvs have shown a propensity for instability, aggregation, and low solubility, scTCR variable domains have generally shown worse expression and stability issues. Great efforts have been made to stabilize scVa/nb proteins for therapeutic and diagnostic use (Stone, et al., Methods Enzymol, 2012. 503: 189-222). Unfortunately, the high diversity of Va/nb germlines (much higher than V _H /V _L germlines of antibodies), renders each set of stabilizing mutations within a scVa/nb to be unique to the individual Va/nb subunit and unlikely to find general use across multiple TCRs.

Given that most extracellular proteins are intrinsically glycosylated with complex disulfide pairings, mammalian expression is used to generate soluble TCRs. Industrial antibody production has predominately moved to mammalian expression systems (Shukla, et al., Bioeng Transl Med, 2017. 2(1): 58-69). However, a/b TCRs express poorly with less reliable assembly than antibodies when expressed in the commonly utilized Chinese hamster ovary (CHO) cells system. Many novel bispecific antibody formats, including those with relevance to soluble a/b TCR bispecifics, may require TCRs to express at antibody -like levels for proper molecular assembly, which is an obstacle for their production. Bispecific ImmTac moieties that recombinantly fuse soluble TCRs to antibody scFvs are typically expressed as insoluble inclusion bodies in bacteria, solubilized, refolded and assembled at low yield (Liddy, et al., Nat Med, 2012. 18(6): 980-7). Additionally, these moieties have intrinsically rapid serum clearance as they lack a recycling mechanism.

There exists a need for TCR stabilization engineering that is suitable for general use across different TCRs and have good expression and/or assembly level.

Provided herein are proteins comprising one or more stabilization mutations in the TCR constant domains (Ca/Ob domains) that increase the unfolding temperature of the TCR constant domains and improve the expression and/or assembly of TCRs. Provided herein are proteins comprising: a first polypeptide comprises a T cell receptor (TCR) alpha constant domain (Ca) comprising at least one of the following residues: phenylalanine at position 139, isoleucine at position 150, threonine at position 190 (residues numbered according to Rabat numbering); or a second polypeptide comprises a TCR beta constant domain (Ob) comprising at least one of the following residues: lysine at position 134, arginine at position 139, proline at position 155, aspartic acid or glutamic acid at position 170 (residues numbered according to Rabat numbering).

The numbering of the amino acid residues in the TCR Ca and Cb domains used herein follows the Rabat numbering system (Rabat, et al. Sequences of Immunological Interest Vol. 1 Fifth Edition 1991 US Department of Health and Human Services, Public Health Service, NIH), as illustrated in Fig. 2. The TCR Ca residues recited above based on Rabat numbering correspond to the following residues in SEQ ID NO: 5 or 6 (Ca): position 139 of Rabat numbering corresponds to position 22 of SEQ ID NO: 5 or 6; position 150 of Rabat numbering corresponds to position 33 of SEQ ID NO: 5 or 6; position 190 of Rabat numbering corresponds to position 73 of SEQ ID NO: 5 or 6. The TCR Ob residues recited above based on Kabat numbering correspond to the following residues in SEQ ID NO: 7 or 8 (Ob): position 134 of Kabat numbering corresponds to position 18 of SEQ ID NO: 7 or 8; position 139 of Kabat numbering corresponds to position 23 of SEQ ID NO: 7 or 8; position 155 of Kabat numbering corresponds to position 39 of SEQ ID NO: 7 or 8; position 170 of Kabat numbering corresponds to position 54 of SEQ ID NO: 7 or 8.

In one aspect, provided herein are proteins comprising: a first polypeptide comprising a TCR Ca comprising at least one of the following residues: phenylalanine at position 139, isoleucine at position 150, threonine at position 190 (residues numbered according to Kabat numbering); and/or a second polypeptide comprising a TCR Cb comprising at least one of the following residues: lysine at position 134, arginine at position 139, proline at position 155, aspartic acid or glutamic acid at position 170 (residues numbered according to Kabat numbering).

In another aspect, provided herein are proteins comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a TCR Ca comprising at least one (e.g., one, two, or three) of the following residues: phenylalanine at position 139, isoleucine at position 150, threonine at position 190 (residues numbered according to Kabat numbering); and/or the second polypeptide comprises a TCR Cb comprising at least one (e.g., one, two, three, or four) of the following residues: lysine at position 134, arginine at position 139, proline at position 155, aspartic acid or glutamic acid at position 170 (residues numbered according to Kabat numbering). In some embodiments, the protein comprises any one of the seven residues listed above. In some embodiments, the protein comprises any two of the seven residues listed above. In some embodiments, the protein comprises any three of the seven residues listed above. In some embodiments, the protein comprises any four of the seven residues listed above. In some embodiments, the protein comprises any five of the seven residues listed above. In some embodiments, the protein comprises any six of the seven residues listed above. In some embodiments, the protein comprises all seven residues listed above. In some embodiments, the proteins described herein are soluble proteins. In some embodiments, the proteins described herein have one or more superior properties, e.g., a higher unfolding temperature (Tm), increased stability, increased expression level when expressed under the same condition, or reduced glycosylation level, when compared to a protein comprising the same amino acid sequence except that: the Ca domain comprising serine at position 139, threonine at position 150, and alanine at position 190 (residues numbered according to Kabat numbering); and the Ob domain comprising glutamic acid at position 134, histidine at position 139, aspartic acid at position 155, and serine at position 170 (residues numbered according to Kabat numbering).

In some embodiments, the first polypeptide comprises a Ca domain comprising phenylalanine at position 139 (Kabat numbering). In some embodiments, the first polypeptide comprises a Ca domain comprising threonine at position 190 (Kabat numbering). In some embodiments, the first polypeptide comprises a Ca domain comprising isoleucine at position 150 (Kabat numbering). In some embodiments, the second polypeptide comprises a C domain comprising proline at position 155 (Kabat numbering). In some embodiments, the second polypeptide comprises a C domain comprising aspartic acid or glutamic acid at position 170 (Kabat numbering). In some embodiments, the second polypeptide comprises a C domain comprising lysine at position 134 (Kabat numbering). In some embodiments, the second polypeptide comprises a C domain comprising arginine at position 139 (Kabat numbering).

In some embodiments, provided herein are proteins comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a TCR Ca domain comprising the following residues: phenylalanine at position 139, isoleucine at position 150, threonine at position 190 (residues numbered according to Kabat numbering); and the second polypeptide comprises a TCR C domain comprising the following residues: lysine at position 134, arginine at position 139, proline at position 155, aspartic acid at position 170 (residues numbered according to Kabat numbering). In some embodiments, provided herein are proteins comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a TCR Ca domain comprising the following residues: phenylalanine at position 139 and threonine at position 190 (residues numbered according to Kabat numbering); and the second polypeptide comprises a TCR Ob domain comprising the following residues: lysine at position 134, arginine at position 139, proline at position 155, aspartic acid at position 170 (residues numbered according to Kabat numbering).

In some embodiments, the TCR Ca domain comprises SEQ ID NO: 5; and the TCR C domain comprises SEQ ID NO: 7. Accordingly, provided herein are proteins comprising a first polypeptide comprising a Ca domain comprising SEQ ID NO: 5, and a second polypeptide comprising a Ob domain comprising SEQ ID NO: 7. In some embodiments, the Ca domain consists of SEQ ID NO: 5; and the Ob domain consists of SEQ ID NO: 7. Accordingly, provided herein are proteins comprising a first polypeptide comprising a Ca domain consisting of SEQ ID NO: 5, and a second polypeptide comprising a Ob domain consisting of SEQ ID NO: 7.

In some embodiments, the first polypeptide is linked to the second polypeptide by one or more inter-chain disulfide bonds. Disulfide bonds can be formed by pairs of engineered cysteine residues in the TCR a and b chains, and such disulfide bonds link the TCR a and b chains together (see W003/020763, W02004/033685, W02004/074322, Li, et ah, Nat. Biotechnol. 2005, 23(3): 349-354; Boulter, et ah, Protein Eng. 2003, 16(9): 707-11). For example, disulfide bonds can be formed between the following pairs of engineered cysteines at the specified residues in the TCR a and b chains: Thr48Cys (a chain) and Ser57Cys (b chain), Thr45Cys (a chain) and Ser 77Cys (b chain), TyrlOCys (a chain) and Ser 17Cys (b chain), Thr45Cys (a chain) and Asp59Cys (b chain), and Serl5Cys (a chain) and Glul5Cys (b chain) (see W003/020763).

In some embodiments, the Ca domain further comprises a cysteine residue at position 166 (residue numbered according to Kabat numbering), and the Cb domain further comprises a cysteine residue at position 173 (residue numbered according to Kabat numbering), wherein the first polypeptide and the second polypeptide are linked by an inter-chain disulfide bond between the cysteine residue at position 166 of Ca and the cysteine residue at position 173 of Ob.

In some embodiments, the TCR Ca domain comprises SEQ ID NO: 6; and the TCR Ob domain comprises SEQ ID NO: 8. Accordingly, provided herein are proteins comprising a first polypeptide comprising a Ca domain comprising SEQ ID NO: 6, and a second polypeptide comprising a Ob domain comprising SEQ ID NO: 8. In some embodiments, the Ca domain consists of SEQ ID NO: 6; and the Ob domain consists of SEQ ID NO: 8. Accordingly, provided herein are proteins comprising a first polypeptide comprising a Ca domain consisting of SEQ ID NO: 6, and a second polypeptide comprising a Ob domain consisting of SEQ ID NO: 8.

In some embodiments, the first polypeptide further comprises a TCR a chain variable domain (Va); and the second polypeptide further comprises a TCR b chain variable domain (nb), wherein the Va and nb form an antigen binding domain that binds an antigen, e.g., a tumor antigen or a viral antigen. In some embodiments, the Va is fused to the N-terminus of Ca, forming a Va - Ca domain; and the nb is fused to the N-terminus of Cb, forming a nb - Cb domain.

The TCR variable regions (Va/nb) can bind any tumor or viral antigen, including but not limited to, a viral antigen, a neoantigen (the antigens expressed only in cancer cells but not in normal cells), a tumor-associated antigen (the processed fragments of proteins that are expressed at low levels in normal cells, but are overexpressed in cancer cells), or a cancer/testis (CT) antigen (derived from proteins usually only expressed by reproductive tissues, e.g. testes, fetal ovaries, and placenta, and have limited/no expression in all other adult tissues) (see Pritchard, et al., BioDrugs, 2018, 32:99-109). In some embodiments, the TCR variable region (Va/nb) binds an antigen selected from any one of the following: ERBB2, CD19, NY-ESO-1, MAGE (e.g., MAGE-A1, A2, A3, A4, A6, A10, A12), gplOO, MART - 1 /Mel an- A, gp75/TRP-l, TRP-2, Tyrosinase,

BAGE, CAMEL, SSX-2, b-Catenin, Caspase-8, CDK4, MUM-2 (TRAPPCl), MUM-3, MART-2, OS-9, pl4ARF (CDKN2A), GAS7, GAPDH, SIRT2, GPNMB, SNRP116, RBAF600, SNRPD1, PRDX5, CLPP, PPP1R3B, EF2 (see Pritchard, et al., BioDrugs, 2018, 32:99-109; and Wang, et al., Cell Research, 2017, 27:11-37).

In some embodiments, the proteins described herein further comprise a second antigen binding domain. Such second antigen binding domain can bind an antigen on the T cell surface, e.g., CD3, CD4, CD8. In some embodiments, the second antigen binding domain binds CD3.

In some embodiments, the second antigen binding domain is an antibody or antibody fragment, e.g., an scFv, Fab, Fab’, (Fab’)2, single domain antibody, or camelid VHH domain. In some embodiments, the second antigen binding domain is a Fab. In some embodiments, the Fab comprises a Fab heavy chain comprising a heavy chain variable domain (VH) and a human IgG CHI domain, and a Fab light chain comprising a light chain variable domain (VL) and a human light chain constant domain (CL), wherein the VH and VL domains form the second antigen binding domain that binds an antigen on the T cell surface, e.g., CD3, CD4, CD8.

In some embodiments, the proteins described herein comprise three polypeptides: a first polypeptide comprising Va - Ca - linker- VH - CHI; a second polypeptide comprising nb - Cb; and a third polypeptide comprising VL - CL; wherein the second and third polypeptides are linked to the first polypeptide by inter-chain disulfide bonds. In some embodiments, the proteins described herein have a TCR-CD3 Fab format.

In some embodiments, the proteins described herein comprise four polypeptides: a first polypeptide comprising Va - Ca - hinge - first Fc region; a second polypeptide comprising nb - Ob; a third polypeptide comprising VL - CL; and a fourth polypeptide comprising VH - CHI - hinge - second Fc region; wherein the second and fourth polypeptides are linked to the first polypeptide by inter-chain disulfide bonds; and the third polypeptide is linked to the fourth polypeptide by inter-chain disulfide bonds. In some embodiments, the proteins described herein have an IgG like format or TCR-CD3 Fab-Fc IgG format.

In some embodiments, the proteins described herein comprise four polypeptides: a first polypeptide comprising Va - Ca - linker - VH- CHI - hinge - first Fc region; a second polypeptide comprising nb - Cb; a third polypeptide comprising VL-CL; a fourth polypeptide comprising a second Fc region; and wherein the second, third, and fourth polypeptides are linked to the first polypeptide by inter-chain disulfide bonds. In some embodiments, the proteins described herein have a TCR-CD3 Fab-Fc tandem format.

In some embodiments, the proteins described herein comprise a VHH domain that binds human serum albumin (HSA). In some embodiments, the proteins described herein comprise three polypeptides: a first polypeptide comprising Va - Ca - linker- VH - CH1- VHH; a second polypeptide comprising nb - Cb; and a third polypeptide comprising VL - CL; wherein the second and third polypeptides are linked to the first polypeptide by inter chain disulfide bonds. In some embodiments, the proteins described herein have a TCR- CD3 Fab -VHH format.

In some embodiments, the proteins described herein comprise two TCR Va - Ca domains and two TCR nb - Cb domains. In some embodiments, the proteins described herein have a 2xTCR-CD3 Fab-Fc format.

In some embodiments, the proteins described herein comprise a linker comprising an amino acid sequence selected from any one of SEQ ID NOs: 41-46.

In some embodiments, the proteins described herein comprise an Fc region, e.g., a human IgG Fc region, e.g., a human IgGl, IgG2, IgG3, or IgG4 Fc region. In some embodiments, the Fc region is a modified human IgG Fc region with reduced effector function compared to the corresponding wild type human IgG Fc region. In some embodiments, the Fc region is a modified human IgGl Fc region. IgGl is well known to bind to the proteins of the Fc-gamma receptor family (FcyR) as well as Clq. Interaction with these receptors can induce antibody-dependent cell cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Therefore, certain amino acid substitutions are introduced into human IgGl Fc region to ablate immune effector function. In some embodiments, the Fc region is a modified human IgGl Fc region comprising one or more of the following mutations: N297A, N297Q, D265A, L234A, L235A, C226S, C229S, P238S, E233P, L234V, P238A, A327Q, A327G, P329A, K322A, L234F, L235E, P331S, T394D, A330L, M252Y, S254T, T256E (residues numbered according to the EU Index Numbering). In some embodiments, the Fc region is a modified human IgGl Fc region comprising the following mutations: L234A, L235A and N297Q (residues numbered according to the EU Index Numbering). In some embodiments, the Fc region is a modified human IgGl Fc region comprising SEQ ID NO: 48. In some embodiments, the proteins described herein further comprise a human IgGl hinge region (e.g., SEQ ID NO: 47), at the N-terminus of the modified human IgGl Fc region.

In some embodiments, the Fc region is a modified human IgG4 Fc region comprising one or more of the following mutations: E233P, F234V, F234A, L235A, G237A, E318A, S228P, L236E, S241P, L248E, T394D, M252Y, S254T, T256E, N297A, N297Q (residues numbered according to the EU Index Numbering). In some embodiments, the Fc region is a modified human IgG4 Fc region comprising the following mutations: F234A and L235A (residues numbered according to the EU Index Numbering). In some embodiments, the Fc region is a modified human IgG4 Fc region comprising SEQ ID NO: 50. In some embodiments, the proteins described herein further comprise a modified human IgG4 hinge region comprising the S228P mutation (according to the EU Index Numbering, e.g., SEQ ID NO: 49), at the N-terminus of the modified human IgG4 Fc region. Such modified human IgG4 hinge region reduces the IgG4 Fab-arm exchange in vivo (see Labrijn, et al., Nat Biotechnol 2009, 27(8):767).

In some embodiments, the proteins described herein comprise a hinge region comprising SEQ ID NO: 47 or 49. In some embodiments, the proteins described herein comprise an Fc region comprising SEQ ID NO: 48 or 50. In some embodiments, the proteins described herein comprise a hinge region comprising SEQ ID NO: 47 and a first and second Fc region comprising SEQ ID NO: 48. In some embodiments, the proteins described herein comprise a hinge region comprises SEQ ID NO: 49 and a first and second Fc region comprising SEQ ID NO: 50.

In some embodiments, the first and second Fc regions comprise a set of heterodimerization mutations, e.g., a set of CH2 and/or CH3 heterodimerization mutations.

In some embodiments, the first and second Fc regions comprise a set of CH3 heterodimerization mutations, e.g., knobs-in-holes (Ridgway, et al., Protein Eng. 1996, 9:617-621), electrostatic mutations, and other CH3 dimerization mutations described in Verdino, et al., Current Opinion in Chemical Engineering 2018, 19:107-123;

WO2016118742; US Patent Nos. 9605084, 9701759, 10106624; US Patent Application Publication No. 20180362668.

In some embodiments, one of the first or second Fc region comprises a CH3 domain comprising an alanine at residue 407; and the other of the first or second Fc region comprises a CH3 domain comprising a valine or methionine at residue 366 and a valine at residue 409 (residues numbered according to the EU Index Numbering). In some embodiments, one of the first or second Fc region comprises a CH3 domain comprising an alanine at residue 407, a methionine at residue 399, and an aspartic acid at residue 360; and the other of said first or second Fc region comprises a CH3 domain comprising a valine at residue 366, a valine at residue 409, and an arginine at residues 345 and 347 (residues numbered according to the EU Index Numbering).

In some embodiments, the proteins described herein are linked to a detectable label.

In some embodiments, such detectable label can be a fluorescent label, a radioactive label, a chemiluminescent label, a bioluminescent label, a paramagnetic label, an MRI contrast agent, an organic dye, or a quantum dot.

In some embodiments, the proteins described herein are linked to a therapeutic agent, e.g., a cytotoxic agent, an anti-inflammatory agent, or an immunostimulatory agent. In some embodiments, the proteins described herein are soluble proteins. In some embodiments, the proteins described herein are transmembrane proteins.

In some embodiments, the first polypeptide of the protein further comprises the transmembrane and intracellular domains of the TCR a chain; and the second polypeptide further comprises the transmembrane and intracellular domains of the TCR b chain.

Also provided herein are T cells comprising a TCR that comprises a Ca domain comprising at least one (e.g., one, two, or three) of the following residues: phenylalanine at position 139, isoleucine at position 150, threonine at position 190 (residues numbered according to Rabat numbering); and/or a Ob domain comprising at least one (e.g., one, two, three, or four) of the following residues: lysine at position 134, arginine at position 139, proline at position 155, aspartic acid or glutamic acid at position 170 (residues numbered according to Rabat numbering). Such TCR can also include an antigen binding domain, e.g., an antibody fragment or TCR Va / nb domain.

In another aspect, provided herein are nucleic acids encoding a polypeptide of the proteins described herein. Such nucleic acid can encode a polypeptide comprising a TCR Ca domain comprising at least one (e.g., one, two, or three) of the following residues: phenylalanine at position 139, isoleucine at position 150, threonine at position 190 (residues numbered according to Rabat numbering). Such a nucleic acid can encode a polypeptide comprising a TCR Cb domain comprising at least one (e.g., one, two, three, or four) of the following residues: lysine at position 134, arginine at position 139, proline at position 155, aspartic acid or glutamic acid at position 170 (residues numbered according to Rabat numbering). In some embodiments, provided herein are nucleic acids encoding a polypeptide comprising SEQ ID NO: 5 or 7. In some embodiments, provided herein are nucleic acid encoding a polypeptide comprising SEQ ID NO: 6 or 8.

In another aspect, provided herein are vectors comprising nucleic acids encoding a polypeptide of the proteins described herein. Such vectors can further include an expression control sequence operably linked to the nucleic acid encoding a polypeptide of the proteins described herein. Expression vectors capable of direct expression of genes to which they are operably linked are well known in the art. Expression vectors can encode a signal peptide that facilitates secretion of the polypeptides from a host cell. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide. The expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Expression vectors can contain selection markers, e.g., tetracycline, neomycin, and dihydrofolate reductase, to permit detection of those cells transformed with the desired DNA sequences. The vectors containing the polynucleotide sequences of interest can be transferred into the host cell by well-known methods (e.g., stable or transient transfection, transformation, transduction or infection), which vary depending on the type of cellular host. In some embodiments, provided herein are vectors comprising a nucleic acid encoding a polypeptide comprising SEQ ID NO: 5 and a nucleic acid encoding a polypeptide comprising SEQ ID NO: 6. In some embodiments, provided herein are vectors comprising a nucleic acid encoding a polypeptide comprising SEQ ID NO: 7 and a nucleic acid encoding a polypeptide comprising SEQ ID NO: 8.

Also provided herein are host cells, e.g., mammalian cells, comprising a nucleic acid or vector described herein; such host cells can express the proteins described herein. Mammalian host cells known to be capable of expressing functional proteins include CHO cells, HEK293 cells, COS cells, and NSO cells. The present disclosure further provides a process for producing a protein described herein by cultivating the host cell described above under conditions such that the protein is expressed, and recovering the expressed protein.

In another aspect, provided herein are pharmaceutical compositions comprising a protein, nucleic acid, vector, or cell described herein. Such pharmaceutical compositions can also comprise one or more pharmaceutically acceptable carriers, diluents, or excipients.

In another aspect, provided herein are methods of treating cancer or infection in a subject in need thereof by administering to the subject a therapeutically effective amount of a protein, nucleic acid, vector, cell, or pharmaceutical composition described herein.

Also provided are proteins, nucleic acids, vectors, cells, and pharmaceutical compositions described herein for use in a therapy. Furthermore, the present disclosure also provides proteins, nucleic acids, vectors, cells, or pharmaceutical compositions described herein for use in the treatment of cancer or infection. Additionally, the present disclosure provides the use of a protein, nucleic acid, vector, cell, or pharmaceutical composition described herein in the manufacture of a medicament for the treatment of cancer or infection.

As used herein, the term “a,” “an,” “the” and similar terms used in the context of the present invention (especially in the context of the claims) are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context.

The term “antibody,” as used herein, refers to monoclonal immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter connected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region comprises three domains, CHI, CH2, and CH3. Each light chain is comprised of a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDR regions in VH are termed HCDR1, HCDR2, and HCDR3. The CDR regions in VL are termed LCDR1, LCDR2, and LCDR3. The CDRs contain most of the residues which form specific interactions with the antigen. Assigning the residues to the various CDRs may be done by algorithms, such as Rabat, Chothia, or North. The Rabat CDR definition (Rabat et al, “Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, Md. (1991)) is based upon antibody sequence variability. The Chothia CDR definition (Chothia et al, “Canonical structures for the hypervariable regions of immunoglobulins”, Journal of Molecular Biology, 196, 901-917 (1987); Al- Lazikani et al, “Standard conformations for the canonical structures of immunoglobulins”, Journal of Molecular Biology, 273, 927-948 (1997)) is based on three-dimensional structures of antibodies and topologies of the CDR loops. The North CDR definition (North et al, “A New Clustering of Antibody CDR Loop Conformations”, Journal of Molecular Biology, 406, 228-256 (2011)) is based on affinity propagation clustering with a large number of crystal structures.

The term “antigen -binding domain” or “antibody fragment” refers to a portion of an antibody that retains the ability to specifically interact with (e.g., by binding, steric hinderance, stabilizing/destabilizing, spatial distribution) an epitope of an antigen. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, scFv antibody fragments, disulfide-linked Fvs (sdFv), a Fd fragment consisting of the VH and CHI domains, linear antibodies, single domain antibodies such as sdAb (either VL or VH), camelid VHH domains, multi-specific antibodies formed from antibody fragments such as a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, and an isolated CDR or other epitope binding fragments of an antibody.

The term “bind” as used herein means the ability of a protein or molecule to form a type of chemical bond or attractive interaction with another protein or molecule, which results in the close proximity of the two proteins or molecules as determined by common methods known in the art.

The term “Fc region” as used herein refers to a polypeptide comprising the CH2 and CH3 domains of a constant region of an immunoglobulin, e.g., IgGl, IgG2, IgG3, or IgG4. Optionally, the Fc region may include a portion of the hinge region or the entire hinge region of an immunoglobulin, e.g., IgGl, IgG2, IgG3, or IgG4. In some embodiments, the Fc region is a human IgGFc region, e.g., a human IgGl Fc region, IgG2 Fc region, IgG3 Fc region or IgG4 Fc region. In some embodiments, the Fc region is a modified IgG Fc region with reduced effector function compared to the corresponding wild type IgG Fc region. The numbering of the residues in the Fc region is based on the EU index as in Kabat. Kabat et al, Sequences of Proteins of Immunological Interest , 5th edition, Bethesda, MD: U.S. Dept of Health and Human Services, Public Health Service, National Institutes of Health (1991). The boundaries of the Fc region of an immunoglobulin heavy chain might vary, and the human IgG heavy chain Fc region is usually defined as the stretch from the amino acid residue at position 231 (according to the EU index) to the carboxyl-terminus of the immunoglobulin. The term “polypeptide”, as used herein, refers to a polymer of amino acid residues. The term applies to polymers comprising naturally occurring amino acids and polymers comprising one or more non-naturally occurring amino acids.

The term “therapeutically effective amount,” as used herein, refers to an amount of a protein or nucleic acid or vector or cell or composition of the invention that will elicit the biological or medical response of a subject, for example, reduction or inhibition of an enzyme or a protein activity, or ameliorate symptoms, alleviate conditions, slow or delay disease progression, or prevent a disease, etc. In one non-limiting embodiment, the term “a therapeutically effective amount” refers to the amount of a protein or nucleic acid or vector or cell or composition of the invention that, when administered to a subject, is effective to at least partially alleviate, inhibit, prevent and/or ameliorate a condition, or a disorder or a disease.

As used herein, “treatment” or “treating” refers to all processes wherein there may be a slowing, controlling, delaying or stopping of the progression of the disorders disclosed herein, or ameliorating disorder symptoms, but does not necessarily indicate a total elimination of all disorder symptoms. Treatment includes administration of a protein or nucleic acid or vector or cell or composition of the present invention for treatment of a disease or condition in a patient, particularly in a human.

Brief Description of the Drawings:

Figs. 1A-1D show the characterization of TCR fragments and stabilizing mutations. Fig. 1A shows DSF denaturation curves with a recombinant TCR (1G4 122, A), Va/nb fragment from the same TCR (¨) with an a42/b110 engineered disulfide (homologous to the VH44/VL100 disulfide in dsFvs), and a CaAI$ fragment (·) with both the standard stalk disulfide and a stabilizing a166/b173 disulfide. Fig. IB is a bar graph showing the midpoints of thermal unfolding (T _m s) of the ‘wild-type’ (WT) CaAI$ subunit and the OcL b subunit with indicated individual stabilizing mutations. Fig. 1C shows DSF curves of wild-type OcL b (·) and CaAI$ with 4 (¨) or 7 ( _■ ) stabilizing mutations. Fig. ID shows the SDS- PAGE characterization of wild-type Ca/Ob (left lane) and Ca/Ob with 4 (middle lane) or 7 (left lane) stabilizing mutations.

Fig. 2 shows the Kabat numbering system of the TCR Ob and Ca domains based on two different TCRs. The upper Ob and Ca sequences were derived from the first human T- lymphocyte receptor Human HPB-MLT sequences in Kabat, et al., (Sequences of Immunological Interest Vol. 1 Fifth Edition 1991 US Department of Health and Human Services, Public Health Service, NIH, pages 1004 and 1005) (human HPB-MLT ϋb, SEQ ID NO: 51; human HPB-MLT Ca, SEQ ID NO: 52); and the bottom 1G4 122 NYES01 Eb and Ca sequences (1G4_122_NYES01 Cb, SEQ ID NO: 4; 1G4_122_NYES01 Ca, SEQ ID NO: 3) were derived from the 2F53 pdb crystal structure (Dunn, et al., Protein Sci 2006. 15: 710-21). Boxed squares indicate differences between the constant domains of 2F53 and the HPB-MLT native sequences. 0b_81730 and Ca_T166S in the 2F53 sequence are the cysteines that form the stabilizing disulfide bond (Boulter, et al., Protein Eng. 2003. 16: 707- 11). The shaded residues underneath the alignment are the stabilizing mutations described here that dramatically stabilize the Ca/Ob subunit and the different full-length TCRs that harbor the stabilized Ca/Cb subunit.

Fig. 3A shows the structural characterization of mutations involving polar amino acids. Each row features a single mutation. The first (left) column features a zoomed in schematic ribbon diagram of the Ca/Eb backbone structure along with a stick depiction of the original residue that is mutated superimposed with the structural diagram of the model harboring the stabilizing mutation. The second (middle) column shows a superposition of the stabilizing mutation within the Ca/Eb model diagram superimposed upon the crystal structure diagram of the same mutation. The third column (right) column highlights a feature of that mutation that makes it stabilizing.

Fig. 3B shows the structural characterization of mutations involving hydrophobic amino acids. Each row features a single mutation. The first (left) column features a zoomed in schematic ribbon diagram of the Ca/Eb backbone structure along with a stick depiction of the original residue that is mutated superimposed with the structural diagram of the model harboring the stabilizing mutation. The second (middle) column shows a superposition of the stabilizing mutation within the Ca/CP model diagram superimposed upon the crystal structure diagram of the same mutation. The third column (right) column highlights a feature of that mutation that makes it stabilizing. The third column of the first row shows the Ramachandran plot for proline overlaid on the Ramachandran plot for aspartic acid with CP 155 represented by the dark box. The plot shows that CP D155 naturally adopts a backbone phi/psi dihedral angle preferred by proline. The third column of the following two rows exhibit how well the mutations pack with surrounding residues, with Ca T150I slightly darker to make it more visible.

Fig. 3C shows comparison of the crystal structure of the 7-mutant TCR and the output of Rosetta when it is used to predict the conformations of the amino acid side chains given the backbone coordinates of the 7-mutant Ca/CP structure.

Figs. 4A-4G show characterization of four diverse, recombinant a/b TCRs with and without a stabilized Ca/CP subunit. Fig. 4A shows DSC curves of the 1G4_122 TCR with zero (WT, dotted line) or seven (solid line) stabilizing Ca/CP mutations in the absence of the a166/b173 stabilizing disulfide. Fig. 4B shows DSC curves of four, diverse TCRs with zero (WT, dotted line) or seven (solid line) stabilizing mutations all in the presence of the a166/b173 disulfide. Fig. 4C shows the non-reduced SDS-PAGE of four, diverse TCRs with zero (WT, dotted line) or seven (solid line) stabilizing mutations all in the presence of the a166/b173 disulfide. Fig. 4D shows the analytical SEC of four, diverse TCRs with zero (WT, dotted line) or seven (solid line) stabilizing mutations all in the presence of the a166/b173 disulfide. Fig. 4E shows the only significant areas of backbone protection from deuterium exchange were in Ca. The heat map under each peptide region indicates the level of protection observed at 10s, 30s, 2min, lOmin, lhr, and 4hr. Lack of a heat map indicates no significant differences in deuterium exchange observed in the region. Fig. 4F shows representative deuterium uptake plots of peptides from the wild-type CE10 TCR (black squares) and stabilized CE10 TCR (grey circles). Fig. 4G shows extracted ion chromatograms of the peptide containing the Va_N67 N-linked glycosylation site. The peptide at 18.6 min was not glycosylsated while the peak 19.9 min shows up after enzymatic deglycosylati on/conversion to Asp. The peptide from the wild-type TCR is a dotted line and the peptide from the stabilized TCR is a solid line.

Figs. 5A-5E show the characterization of both stabilizing and destabilizing Va/nb mutants within the 1G4_122 TCR with or without the stabilized Oa/Ob subunit. Fig. 5A shows cartoon depiction of the 1G4 122 (pdb 2F53) with various Va/nb mutations shown in lavender. DSF curves of the 1G4 122 TCR in the presence of various Va/nb mutant combinations and in the absence (Fig. 5B) and presence (Fig. 5C) of a stabilized Oa/Ob subunit. Normalized expression levels (Fig. 5D) and lowest Tm (Fig. 5E) of the 1G4_122 TCR with different variable domain mutations in the absence (·) and presence (¨) of a stabilized Oa/Ob subunit.

Figs. 6A-6E show the characterization of IgG-like and Tandem-Fab like TCR/CD3 proteins. Fig. 6A is a schematic diagram of the TCR/CD3 IgG-like and Tandem-Fab like bispecific molecules. Fig. 6B shows non-reduced and reduced SDS-PAGE of TCR/CD3 IgG-like and Tandem-Fab like bispecific proteins. Fig. 6C shows analytical SEC characterization of the TCR/CD3 IgG-like BsAbs using the wild-type NY-ESO-1 TCR and a stabilized NY-ESO-1 TCR as well as a tandem Fab BsAb using only the stabilized NY-ESO-1. Fig. 6D shows flow cytometry cell binding titrations of the BsAb molecules to Saos-2 (top) and 624.38 (middle) HLA-A2+ tumor cells pulsed with the NY-ESO-1 peptide and to Jurkat (bottom) CD3+ cells. Fig. 6E shows T cell redirected killing of Saos-2 tumor cells pulsed with NY-ESO-1 peptide.

Figs. 7A-7B shows additional TCR/CD3 bifunctionals and mouse pharmacokinetics of the IgG-like TCR/CD3 BsAbs. Fig. 7A shows the reduced and non-reduced SDS-PAGE of the IgG-llike TCR/CD3 BsAbs with different affinity or with Ca deglycosylated by mutation of the canonical N-linked glycosylation sites. Fig. 7B shows the serum concentrations of the 1G4_122/SP34 IgG4ike BsAb with or without the N-linked glycosylation in Ca following a single 5 mg/kg intravenous injection in Balb-c mice.

EXAMPLES

Recombinant TCRs can be used to redirect naive T cells to eliminate virally infected or cancerous cells; however, they are plagued by low stability and uneven expression. Molecular modeling is used to identify mutations in the TCR constant domains (Ca/C subunits) to improve Ca/Ob stability, increase Ca/Ob expression, and increase the unfolding temperature of Oa/Ob. When 7 of these mutations are collectively added to the Ca/Ob subunit, an increase in the midpoint of thermal unfolding by 20 °C is observed. Adding the stabilized Ca/Ob subunit improves the expression and assembly of four separate a/b TCRs by 3- to 10-fold. Additionally, the mutations rescued the expression of TCRs with destabilizing mutations in the variable domains. Interestingly, the improved stability and folding of the TCRs led to reduced glycosylation. The CaA^ variant enabled antibody-like expression, allowing development of a new class of bispecific molecules that combine an anti-CD3 antibody with the stabilized TCR. These TCR/CD3 bispecific proteins can redirect T cells to kill tumor cells expressing the HLA/peptide antigens.

General stabilization of the Ca/Cb subunit may improve the overall stability and folding of a/b TCRs. Recent studies have shown that strong thermodynamic cooperativity exists between the subunits of a/b TCRs. Ca requires pairing with Cb in the ER for folding similar to what has been observed for antibody C _H 1/CK subunits (Feige, et ah, Mol Cell, 2009. 34(5): 569-79; Toughiri, et ah, MAbs, 2016. 8(7): 1276-1285). Additionally, many Va/nb subunits are intrinsically unfolded in isolation and require the CaA^ subunit for proper folding (Feige, M.J., et ak, J Biol Chem, 2015. 290(44): p. 26821-31). In support of the hypothesis, adding a disulfide between the CaA^ domains has been shown to have a positive impact on many a/b TCRs (Boulter, J.M., et ak, Protein Eng, 2003. 16(9): p. 707- 11). Therefore, a more robust Ca/Eb subunit is engineered for general TCR stabilization with the goal of generating TCRs at antibody -like levels that assemble properly. A stabilized TCR should be a more amenable building block for recombinant fusion to antibodies in different geometries including those that include an antibody -Fc moiety to enhance their pharmacokinetic properties.

Protein simulations were performed with the molecular modeling software Rosetta to identify mutations that stabilize the Ca and Ob domains (Leaver-Fay, et ak, Methods Enzymol, 2011. 487: 545-74). An alternative strategy for finding mutations that will stabilize a protein is to assemble a multiple sequence alignment (MSA) for the protein family and search for highly conserved amino acids that are not conserved in the protein of interest (Magliery, et ak, Curr Opin Struct Biol, 2015. 33: 161-8). Here, mutations were tested based on Rosetta calculations as well as use conservation analysis to filter the results from the simulations.

Results

Stabilizing the Ca/CP TCR subunit

First, the thermodynamic properties of the a/b TCR are examined by generating a soluble form of the a/b TCR, 1G4_122, and its Va/nb and Ca/CP subunits; 1G4_122 binds to the NY-ESO-1 antigen (Li, Y., et ak, Nat Biotechnol, 2005. 23(3): 349-54). Using a mammalian expression system, both the Va/nb and Ca/CP subunits were generated in the presence or absence of flexible (Gly4Ser)4 linkers (SEQ ID NO: 43) that link Va to Ύb or Ca to Cb. The subunit expression and assembly were tested with or without stabilizing interdomain disulfides. Most of the Va/nb and Ca/CP constructs either failed to express or failed to assemble, including the single chain variants. The best Va/nb subunit expression was obtained by adding a na44/nb110 disulfide homologous to the VH44/VLI _OO disulfide used to stabilize antibody variable domain fragments or Fvs (Brinkmann, et ak, Proc Natl Acad Sci USA, 1993. 90(16): 7538-42), while the best Ca/CP expression was obtained using the known stabilizing disulfide (Cal66/Cpi73) on top of the native Ca/CP disulfide at the C -terminus of the domains (Ca213/CP247) (Boulter, et ak, Protein Eng, 2003. 16(9): 707- 11). Differential scanning calorimetry (DSC) experiments showed the (Cal66/Cpi73) disulfide increased the midpoint of thermal unfolding (T _m ) of the full length TCR (both subunits) by 8 °C. When comparing the Va/nb and Ca/Ob subunit unfolding to the unfolding of the intact extracellular TCR domain, the individual subunits were clearly both destabilized in the absence of one another (Fig. 1A), agreeing with the thermodynamic cooperativity observed previously (Feige, et al., J Biol Chem, 2015. 290(44): 26821-31).

Molecular modeling simulations were also used to identify mutations that stabilize the constant domains. For each simulation, the TCR was fixed in space and only residues in close proximity to the mutation were allowed to make small movements (“relax”) to accommodate the mutation. These local relaxations were repeated using the native sequence without any mutations. The change in energy was determined by comparing the Rosetta score of the mutation with the Rosetta score of the native sequence. The mutations were split into two lists: a list of mutations that are observed in a multiple sequence alignment (MSA) of TCRs and a list of mutations that are absent from the MSA. The mutations with the best predicted energies from both lists were picked for experimental screening.

Using the isolated Oa/Ob fragment containing the Oa166/Ob173 disulfide bond (Fig. 1A), the library of single, computationally selected mutants was generated and expressed in two separate blocks. Increased expression level was used blindly to select mutants for scale- up, purification, and characterization by differential scanning fluorimetry (DSF, Table 1). Increased expression correlated well with protein stability. Roughly half of the single mutants chosen for further evaluation based on expression demonstrated a significant increase in stability over the wild-type (native) Ca/Ob protein (Table 1). The T _m s for seven of the identified stabilizing mutants are shown in Fig. IB and range from +1 to +7 °C over wild-type Oa/Ob. Five of these mutations were substitutions that are observed in a multiple sequence alignment of TCRs, but the two mutations that produced the biggest gains in T _m , D155P (+7.1 °C) and S139F (+4.4 °C), are not observed in the multiple sequence alignment.

Next, the stabilizing mutations were combined into a variant harboring four of the mutations and a variant with all seven mutations. Modeling results suggested each of the mutations was likely compatible with each another (i.e., unlikely to interfere with each other). Including all seven stabilizing mutations led to a 7-fold increase in expression and a 20 °C increase in the T _m over the Ca/Ob subunit harboring only the Oa166/Ob173 disulfide (denoted ‘wild-type’) (Table 2, Fig. 1C). Assembly of the wild-type and mutant Ca/C subunits was probed on a non-reducing SDS-PAGE gel. The Ca/C variant with seven mutations assembled more efficiently as evidenced by the lack of disulfide laddering and was smaller/more compact, likely due to less spurious glycosylation (described in detail below) via maintainance of a more discretely folded and compact structure (Fig. ID). Even for the stabilized variant, multiple bands are clear on the SDS-PAGE gel (Fig. ID) that likely reflect partial glycan occupancy of the three N-linked glycosylation sites in Ca and single N-linked glycosylation site in Ob.

A primary sequence depiction of the exact locations of each mutation along with their proper numbering according to Kabat notation (Kabat, et al. Sequences of Immunological Interest Vol. 1 Fifth Edition 1991 US Department of Health and Human Services, Public Health Service, NIH) is shown in Fig. 2. Table 1. Expression of Ca/Eb subunits with various mutations derived by modeling. ^a all CaCP fragments contain the aT166C/pS173C disulfide.

Lastly, the impact of the mutations on potential immunogenicity of the TCR proteins was assessed. The EpiVax server was used to investigate whether any increase MHC -peptide complexes were likely for each of the mutations (De Groot, et al., Clin Immunol, 2009. 131(2): 189-201). Overall, a slight increase in the EpiMatrix score was observed for both Ca and Cp. Where increases were observed, these tended to be slight increases in the baseline scores that did not reach the level predicted to elicit significant MHC binding as described by Epibars. Where Epibars were observed in the native sequences, there were some subtle increases in the scores, but few that led to the Epibars moving into a higher category of risk. An outlier of concern includes the Ca_T150I variant, whose EpiMatrix score goes up substantially for one 9-mer and there is a strong, existing EpiMatrix cluster in a second peptide for the wild-type Ca protein that also exists for the mutant. TCRs recognizing the wild-type peptide may be eliminated during immune development and tolerance. This mutation provides the smallest contribution to the stability. The CP_E 134K H 139R mutants are found together on multiple single peptides and there is a slight increase in the EpiMatrix scores for these two residues. The Epibars that are introduced for this double mutant are primarily in the low risk category with no increases to the high risk category. Interestingly, the CP_D 155P and CP_S 170 D variants, which are the two most impactful mutants from a T _m perspective, lower the overall EpiMatrix scores versus wild-type Ca/Cp. Table 2. Expression and thermal stability of wild-type (WT) TCRs and TCRs with the 7 stabilizing mutations (Des). ^a Unless stated otherwise, all TCRs contain the aT166C/ S173C disulfide. Structural characterization of the seven stabilizing Ca/CP mutations.

Next, attempts to crystallize both the wild-type and stabilized forms of the Ca/CP subunit were performed. The proteins were deglycosylated enzymatically before crystallization. Consistent with the SDS-PAGE gel results with the proteins (Fig. ID), only the stabilized version yielded protein crystals. A 1.76 A crystal structure of the stabilized Ca/CP subunit was obtained. The side chains for all seven mutants are resolved in the structure and provide an indication of how the mutations are stabilizing the protein (Fig. 3A and Fig. 3B)

In the crystal structure and the Rosetta model, phenylalanine 139 (Ca S139F) fills a partially buried cleft in the structure and forms tight packing interactions with the side chain of Ca 129Q. Notably, the orientation of the interaction leaves the polar groups on the glutamine open for forming hydrogen bonds with water. Despite being solvent exposed, CP D155P has the largest impact on protein stability of the seven mutations. The Rosetta energy calculations favor the proline because the residue has backbone torsion angles (ø = -73.4°, y = 52.3°) compatible with the closed ring of the proline. Fig. 3B shows overlapping Ramachandran plots for both aspartic acid and proline and highlights that CP 155 is in the allowed region for both amino acids. One benefit of mutating to proline, however, is that proline is more restrictive than aspartic acid in Ramachandran space and therefore loses less entropy when the protein folds. Isoleucine 150 (Ca T150I) packs tightly with surrounding residues and removal of the threonine does not leave any buried polar groups without a hydrogen bond partner.

Ca A190T is at the beginning of a turn in a solvent-exposed loop. In addition to increasing solubility on the surface, Rosetta predicted that this threonine would form a stabilizing hydrogen-bond to Ca 129Q. Instead, the crystal structure shows the threonine adopting a different rotamer to form a hydrogen bond with the backbone on the other end of the loop’s turn (backbone nitrogen of Ca 193N). Rosetta predicted that CP S170D would form a bidentate hydrogen bond across the interface with the sidechain of Ca 171R. The crystal structure shows CP S170D hydrogen bonding with the backbone nitrogen atom of Ca 171R (which was not previously participating in a hydrogen bond) and allowing the arginine’s sidechain to become more solvent-exposed. CP H139R creates an interface-spanning hydrogen bond with Ca 124D. Rosetta modeled the arginine and aspartic acid to form a bidentate hydrogen bond, however the crystal structure shows that they form just one hydrogen bond so that the arginine can better pack with neighboring residues. The new lysine at residue 134 on the P chain (CP E134K) has high b-factors and is likely interacting with several negatively charged amino acids in the vicinity.

Investigation was carried out to see why Rosetta did not predict the observed sidechain conformations. One possibility is that the sidechain and backbone sampling protocol in Rosetta was unable to sample these conformations. Alternatively, Rosetta may have sampled them but predicted them to be higher in energy. To test these hypotheses, the backbone coordinates from the crystal structure of the stabilized mutant was used as the starting point for fixed-backbone sidechain prediction simulations. The backbone conformation in the new crystal structure is similar to the crystal structure of the wild-type protein, but there are small deviations that may affect sidechain positioning and ability to form hydrogen bonds. This is what has been observed, given the crystal structure backbone Rosetta correctly predicted most of the side chain conformations of the mutants (Fig. 3C). This result suggests that Rosetta did not correctly predict the sidechain conformations of these mutations in the original model because it did not sample or favor the small backbone perturbations observed in the crystal structure.

Impact of the Ca/CP Mutations on Full Length TCRs.

To evaluate the impact of the Ca/CP mutations on the expression and stability of unrelated, full-length, soluble TCRs, the 1G4 122 anti-NY-ESO-1 TCRs were generated with and without the novel Ca/CP mutations. Without the disulfide, the 1G4 122 TCR expressed extremely poorly in mammalian cells, but with the Ca/CP mutations, the expression increased over 10-fold and the T _m measured by DSC was 8 °C higher (Table 2, Fig. 4A). The stabilizing Ca/CP mutations were also evaluated in the presence of the CaTT66C/CpS173C disulfide in four unrelated (and sequence diverse) a/p TCRs including additional TCRs targeting MAGE A3 (pdb 5BRZ; NCBI: a=ABU74337.1/b=AOZ48691.1, HIV (pdb 3VXT; NCBI: a=ABB89050.1/b=AOU74607.1), and WT-1 antigens (Raman, et al., Sci Rep, 2016. 6: 18851; Shimizu, et al., Sci Rep, 2013. 3: 3097; Richman, S.A., et al., Mol Immunol, 2009. 46(5): 902-16). Even in the presence of the CaT166CA^S173C disulfide, each of the TCRs experienced a roughly 3 -fold increase in titer and an increase in thermal stability by DSC in the presence of the stabilized CaA^ subunit (Table 2, Fig.4B). Interestingly, all four TCRs were more compact as assessed by SDS-PAGE analysis and analytical size exclusion chromatography (SEC, Figs. 4C-4D).

To investigate further the more compact nature of the TCRs in the presence of the Ca/Cb mutations, both hydrogen deuterium exchange (HDX) analyses and glycan occupancy studies were performed using peptide mass mapping with the WT-1 TCR (CE10) with and without the stabilizing CaA^ mutations. HDX patterns of the CE10 TCR with and without the CaA^ mutations was nearly identical except for significant regions of the Ca chain (Fig. 4E). HDX protection in the presence of the Ca domain upon stabilization was not localized to the sites of mutation, but more globally to various regions of the Ca fold, including some protection at the C-terminus. The wild-type and stabilized CE10 TCR was also evaluated for the presence/occupancy of N-linked glycans before and after enzymatic N-linked and O- linked deglycosylation. A single N-linked glycosylation motif exists in the Va domain, three in the Ca domain, and one in the Cb domain. CaA^ stabilization led to a significant reduction in N-linked glycosylation within the CE10 TCR including within Va, suggesting the stabilized CaAi^ mutations also stabilize the Va/nb domains (Table 3, Fig 4F). O- linked glycosylation was probable at the C-terminus of Ca since the C-terminal peptide could not be resolved in the TCR lacking the stabilizing mutations. This issue was eliminated in the presence of the CaA^ mutations with a well-resolved C-terminal peptide. The Ca C- terminal region is variably present in different TCR crystal structures and absent in the NY- ESO-1 structure being used. Interestingly, the Cb H139R mutation forms charge-charge interactions as well as a potential p-stacking interaction that may stabilize/lock-down the conformation of the Ca C-terminus, which also shows enhanced protection from HDX. Thus, the universal decrease in hydrodynamic radius observed for all the stabilized TCRs by SDS- PAGE and SEC was confirmed as a reduction in glycosylation.

Next the ability of the Ca/C mutations to enable TCRs to withstand variable domain mutation was assessed. Using Rosetta, a limited number of mutations in both the Va and nb domains were selected. Ultimately, one (bN66E) was stabilizing, multiple mutations were neutral, and one was significantly destabilizing (b849A) within the wild-type 1G4_122 anti- NY-ESO-1 TCR (Fig. 5A). The Va and nb mutations were combined and this small library of TCR variants were expressed in the absence and presence of the CaA^ stabilizing mutations. A wide range of T _m s were observed by DSF for the 1G4 122 TCRs in the absence of the CaAi^ mutations, while the stability of the 1G4_122 variants was virtually constant when the CaA^ mutations were present (Figs. 5B-5C). As observed with the TCRs described above, the entire library of 1G4 122 variants achieved a roughly 3 -fold increase in titer with the CaA^ mutations present (Fig. 5D). Interestingly, the T _m s of the 1G4_122 variants ranged from 49 - 65 °C, whereas the CaAi^ stabilized library all had T _m s tightly clustered around 62 °C (Fig. 5E) and were impervious to variable domain instability. These results suggest the Ca/Cb mutations may rescue the expression and stability of TCRs with low intrinsic stability.

Table 3. Glycan occupancy of the CE10 TCR in the absence and presence of the stabilizing CaA^ mutations. ^a Peptide could not be resolved. Possible O-linked glycosylation at CaT204 occludes peptide resolution.

Utility of the Ca/CP mutations in producing well-assembled, soluble TCR/CD3 bispecifics for redirecting T cells to target tumor cells.

A major benefit to stabilizing a/b TCRs would be the novel forms of bispecifics that could be enabled. The first generation TCR bispecifics combine the specificity of soluble a/b TCRs with an anti-CD3 scFv in the ImmTac format (Liddy, et al., Nat Med, 2012. 18(6): 980-7). This format enables exquisite redirected lysis potency; however, low yield bacterial expression and refolding as well as rapid serum clearance of this format make the in vivo dosing challenging. With the significant increase in expression and proper folding of a/b TCRs in mammalian cells afforded by the Ca/CP mutations, new bispecific formats should be accessible that include moieties with enhanced pharmacokinetics (PK) as well as avidity to the tumor cells.

IgG-like and Tandem Fab-like bispecific antibodies (BsAbs) were generated (Fig. 6A). The BsAbs utilized the 1G4 122 a/b TCR that binds the HLA-A2/NY-ESO-1 peptide complex and was engineered to bind with high affinity (1 nM) (Li, Y., et al., Nat Biotechnol, 2005. 23(3): 349-54). The IgG-like format requires the expression of two heavy chains that require a set of antibody Fc heterodimerization mutations to achieve proper heavy chain assembly. The previously published 7.8.60 heterodimer mutations were chosen (Leaver-Fay, et al., Structure, 2016. 24(4): 641-651). The TCR/CD3 IgG-like BsAb was expressed with or without the Ca/CP mutations. As observed with the soluble TCRs, the TCR/CD3 IgG-like BsAb containing the stabilizing Ca/CP mutations expressed over 2-fold higher than the construct lacking the Ca/CP mutations. The IgG-like BsAb protein lacking the stabilizing mutations was also found to be a mixture of TCR/CD3 BsAb and anti-CD3 half-antibody. The anti-CD3 half-antibody was observable as a low-molecular weight (LMW) peak by analytical SEC after IgG-Fc affinity purification (Figs. 6B-6C). The IgG-like BsAb and Tandem Fab-like BsAbs with the stabilizing Ca/CP mutations were secreted as >90% monodisperse (i.e., pure) proteins coming directly from the cell culture based on their profile after a single affinity chromatography step (Figs. 6B-6C).

Next, the pharmacokinetic (PK) properties of the TCR/CD3 IgG-like BsAbs were evaluated in mice (the Tandem Fab-like BsAbs were not tested). The residual glycosylation might result in systemic (particularly liver) uptake of the TCR/CD3 moiety by binding asialoglycan receptors (Sethuraman, et ah, Curr Opin Biotechnol, 2006. 17(4): 341-6; Lepenies, et ah, Adv Drug Deliv Rev, 2013. 65(9): 1271-81). Previously, a similar construct containing a non-stabilized Ca/Ob demonstrated a substantial a-phase clearance, which might be due to asialoglycan receptor uptake (Wu, et ah, MAbs, 2015. 7(2): p. 364-376). Since the Ca/Ob mutations described here considerably reduce the level of glycosylation on soluble TCRs, the mutations may result in improved PK properties. As a control, a second IgG BsAb was generated that had the N-linked glycosylation sites within the Ca domain mutated as described previously (Wu, et ah, MAbs, 2015. 7(2): p. 364-76). Interestingly, when the N-linked glycosylation sites were eliminated within non-stabilized Ca/Ob subunits, the expression was decreased by more than an order of magnitude (Wu, et ah, MAbs, 2015. 7(2): p. 364-76). Knock-out of the N-linked glycosylation sites in the stabilized Ca/Ob construct had no impact on expression (Fig. 7A). Both the stabilized and the stabilized/aglycosyl TCR/CD3 IgG-like BsAbs were evaluated for their PK by intravenous injection into B ALB/c mice (Fig. 7B). The IgG-like BsAbs demonstrated near identical PK in Balb-c mice with a b-half-life of roughly 3-8 days depending on the timepoints included in the fits. The Fc-moeity clearly leads to a long, antibody -like serum half-life and the presence of residual TCR glycosylation is not impacting the clearance to any great extent. This PK is much better than would be expected for ImmTacs. At the one week timepoint, a non-linear drop in molecule concentration for both TCR/CD3 IgG-like BsAb was observed. The drop in concentration was more dramatic at 10 days and by 14 days BsAb levels were largely below the level of detection (Fig. 7B). The sera were tested for the presence of mouse anti-drug antibodies (ADA) and a low level was observed at 7 days that increased substantially at 10 days and 14 days, perhaps explaining the accelerated clearance beginning at 7 days. The reason behind the early onset ADA is not clear. The TCR/CD3 BsAb was next evaluated for their function. Flow cytometry was performed with Jurkat cells to assess CD3 binding and 624.38 and Saos-2 tumor cells to assess HLA-A2/peptide binding. Both the IgG-like and Tandem Fab-like BsAbs were capable of binding both the CD3 and HLA-A2/peptide cellular antigens (Fig. 6D). Both BsAbs showed a 10- to 30-fold decrease in binding potency to Jurkat cells compared to the bivalent anti-CD3 antibody due to a loss of avidity. The Tandem Fab-like BsAb appeared to bind slightly better to two separate HLA-A2+ tumor cells compared to the IgG-like BsAb. Given the weaker binding to tumor cells observed for the IgG-like BsAb, a couple higher potency TCR (Li, et ak, Nat. Biotechnol, 2005, 23(3): 349-54) versions of the IgGBsAb were generated to offset the potency loss (Fig. 7A). The 1G4 107 TCR variant did demonstrate improved potency binding to tumor cells over the 1G4 122 variant by flow cytometry (Fig. 6D).

Next, the BsAbs were titrated onto peptide-labeled tumor cells in the presence of unstimulated primary T cells. Weak T cell redirected of tumor cells was observed on the Saos-2 cell line (Fig. 6E) for the IgG BsAb. Increasing the potency towards the HLA/peptide complex did not improve the weak activity (Fig. 6E). The Tandem Fab BsAb potently redirected T cells to kill Saos-2 cells.

Based on the data presented here, the novel Ca/C mutations will likely improve the expression and stability of most recombinant TCRs including those with low stability variable domains. Compared to traditional bacterial methods of production in inclusion bodies followed by solubilization, refolding and purification (Wilson, Curr Opin Struct Biol, 1997. 7(6): 839-48), the mammalian expression/secretion of fully assembled/oxidized TCRs described here should dramatically increase their ease of production. The stabilizing mutations led to antibody-like expression levels with or without a stabilizing disulfide bond (Boulter, et ak, Protein Eng., 2003. 16(9): p. 707-11) - an achievement not provided by the disulfide bond in isolation. This increased expression enables the robust production of various TCR/antibody bifunctional molecules. An interesting possible application would be to include stabilizing mutations within full-length TCRs transgenically induced within primary T cells. Difficulty with chain associations and expression have seen minor improvements via the introduction of the disulfide bond, the order of expression of the a/b chains, or introduction of hydrophobics in the transmembrane region (Jin, et al., JCI Insight, 2018. 3(8); Scholten, et al., Cell Oncol, 2010. 32(1-2): 43-56). It would be interesting to see if the Ca/Ob mutations described here generally improve cell-surface TCR expression for cell-based therapy approaches.

The stabilized TCRs also displayed reduced overall glycosylation (both variable and constant domains) compared to their non-stabilized counterparts. Glycosylation can naturally stabilizes and solubilizes many different proteins including antibody Fc and, in specific cases, antibody Fab moieties (Zhou, J Pharm Sci, 2019. 108(4):1366-1377).

Glycoengineering has even been used as an approach to stabilize proteins, reduce their propensity to aggregate/misfold, or improve their solubility (Zhou, J Pharm Sci, 2019.

108(4): 1366-1377). The loss of glycosylation of Ca was shown to dramatically reduce the expression of IgG-like proteins containing a Oa/Ob subunit (Wu, et al., MAbs, 2015. 7(2): 364-76). Thus, it is surprising to find that the stabilizing mutations significantly reduced the N-linked glycosylation by roughly 50% per site throughout the entire TCR. Putative O-linked glycosylation at the C-terminus of Ca was also absent in the stabilized form. It is hypothesized that stabilizing interactions reduce the conformational fluctuation of these sites within the folding/folded protein, reducing access of the enzymes to decorate the canonical N-linked motifs and O-linked site(s). This includes an N-linked site within Va, providing further evidence that the CaA^ mutations generally stabilize the entire TCR. Further, full- elimination of the N-linked glycans on Ca via mutation had no impact on protein expression within the stabilized constructs.

Clear geometrical constraints exist for the TCR/CD3 BsAb’s ability to redirect T cells to lyse tumor cells. Such constraints have been demonstrated previously for bispecific antibody redirection (Bluemel, et ak, Cancer Immunol Immunother, 2010. 59(8): 1197-209; Wu, et al., MAbs, 2015. 7(2): 364-76; Li, et al., Cancer Cell, 2017. 31(3): 383-395). No similar data has been shown for TCR-based therapeutics. The flexibility and spacing of the soluble TCR and anti-CD3 Fab subunits appears to be important to achieve high redirected lysis potency considering the substantial difference in overall activity and potency between the IgG-like and tandem Fab-like BsAbs containing identical soluble TCR and anti-CD3 arms. Many discussions regarding the size of the antigen and/or bispecific construct and their ability to fit within the immune synapse have been raised (Davis, et al., Nat. Immunol, 2006, 7(8): 803-9). However, differences were observed in binding to the tumor cells between the IgG-like and Tandem Fab-like BsAb formats in the absence of the T cells, which complicates the size/immune synapse interpretation. The individual binding characteristics to each cell type may play a bigger role in the case of these BsAbs. Overall, the significant advantages afforded by the stabilized Ca/Eb mutations could be valuable not only for soluble TCR- based therapeutics, but also for improving recombinant a/b TCR expression in cellular systems including those that use TCRs as part of cell based therapies.

Materials and Methods

Molecular Biology

NY-ESO-1 1G4_122 soluble TCR, and the individual NY-ESO-1 Va/nb and Ea/Eb subunits were originally generated using gBlocks (Integrated DNA Technologies, IDT) and subcloned into mammalian expression vectors (Lonza) using recombinase cloning (In- Fusion, Clontech). Sequences for 1G4_122,107, and 113 were derived using the 2F53 crystal structure (Dunn, et al., Protein Sci, 2006. 15(4): 710-21) and the CDRs described elsewhere (Li, et al., Nat Biotechnol, 2005. 23(3): 349-54). A murine kappa light chain leader sequence was used to drive secretion of each protein and 8xHistags were added recombinantly to the N-termini of the a-chains. DNA sequences were derived using IDT optimized codon algorithms.

Single mutant libraries were created using a site-directed mutagenesis protocol. Briefly, the protocol employs the supercoiled double-stranded DNA vector and two synthetic oligonucleotide primers (generated at IDT) containing the desired mutation(s). The oligonucleotide primers, each complementary to opposite strands of the vector, are extended during thermal cycling by the DNA polymerase (HotStar HiFidelity Kit, Qiagen) to generate an entirely new mutated plasmid. Following temperature cycling, the product is treated with Dpn I enzyme to eliminate the methylated, non-mutated plasmid that was prepared using E. coli (New England BioLabs). Each newly generated mutant plasmids is then transformed into Top 10 E. coli competent cells (Life Technologies). Colonies were picked, cultured, miniprepped (Qiagen), and sequence verified. Mutant combinations were generally synthesized as gBlocks.

TCR/CD3 constructs were created using overlapping PCR of each fragment and recombinase cloning. The chimeric SP-34 anti-CD3 heavy and light chain sequences were published previously (Wu, et ak, MAbs, 2015. 7(2): 364-76).

Protein Expression and Purification. a/b constructs were co-transfected for transient expression in either 24 well plates (1 mL scale) or individual flasks (100-200 mL scale) as described previously (Rajendra, et ak, Biotechnol Prog, 2017. 33(2): 469-477). Supernatants were harvested for purification as previously described (Lewis, et ak, Nat Biotechnol, 2014. 32(2): 191-8; Froning, et ak, Protein Sci, 2017. 26(10): 2021-2038). Small scale expressions of TCR and TCR fragments were quantified using Ni ²⁺ -NTA tips (Pall Forte Bio) reading on the Octet RED384 System. TCR/TCR fragment His-tag proteins were purified using a Ni ²⁺ immobilized resin (cOmplete™ His-Tag, Millipore Sigma) and an AKTA Pure system (GE Healthcare). The column was equilibrated with at least 5 column volumes of binding buffer (20mM Tris-HCl pH 8.0, 0.5M NaCl) at a flow rate of 1 mL/min for lmL columns and 5mL/min for 5mL columns, respectively. After passing the CHO expression supernatant over the column to capture the histagged TCRs or TCR fragments, the TCR proteins were eluted with ten column volumes of elution buffer (20mM Tris-HCl pH8.0, 0.5M NaCl, 250mM Imidazole). Imidazole was later removed by passing the proteins through a preparative size exclusion column (SRT-10C SEC300) or via dialysis against a phosphate buffered saline solution (PBS) at pH 7.2-7.4 using VIVASPIN 6 concentrators with a 10,000 MW cutoff. IgG-Fc containing proteins were purified using mAbSure resin (GE Healthcare) and preparative size exclusion. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed using 4-12% Bis Tris gels according to the manufacturer (Life Technologies). Reductions and alkylations were performed with 1 mM dithiothreitol (DTT) and 1 mM N- ethylmaleimide (NEM).

Differential Scanning Fluorimetry and Calorimetry (DSF and DSC).

Protein unfolding was monitored on the Lightcycler 480II RT PCR machine (Roche) using SYPRO Orange dye (Invitrogen, S6651). Excitation and emission filters were set at 465 nm and 580 nm, respectively. The temperature was ramped from 25 °C to 95 °C at a rate of 1 °C/s. Stock solutions of protein were stored in running buffer (1 X PBS pH 7.4).

Final assay conditions were 0.2 mg/mL protein and a 500-fold Sypro dilution in running buffer. The experiment was performed by diluting the proteins to 0.4 mg/ml and diluting Sypro 250-fold, then combining the diluted protein and Sypro in equal parts in a 96- well plate (final volume: 30 pL per well). The samples were divided into 6 pL triplicates on a 384-multiwell assay plate (Roche, 04-729-749-001).

The midpoint of thermal unfolding (T _m ) determination for each protein was performed on the LightCycler Thermal Shift Analysis Software (Roche) using the first derivative method. The analysis software smoothed the raw fluorescence data and the T _m was collected by determining the temperature where the upward slope of fluorescence vs. temperature was the steepest (i.e., temperature where first derivative of melt curve was maximal).

DSC data was collected and analyzed as described previously (Dong, et ah, J Biol Chem, 2011. 286(6): 4703-17).

Rosetta Energy Predictions.

The TCR crystal structure with PDB code 2F53 was used with chains A-C removed, leaving only the alpha and beta chains. The remaining structure was relaxed using the FastRelax protocol in Rosetta with coordinate constraints (energy penalties that grow as a backbone atom deviates from its starting position) on every residue’s CA atom (Conway, et. al., Protein Sci, 2014. 23(1): 47-55). All possible mutations (except for mutations to cysteine) were also simulated using the FastRelax protocol. FastRelax consists of two sub -protocols: (1) fixed-backbone rotamer substitutions (i.e. side chain conformation sampling) and (2) all-atom minimization of backbone and side chain torsion angles. The first sub-protocol generates a rotamer library for each residue position and stochastically samples rotamers at user-allowed positions. The lowest-energy combination of rotamers is assigned to the protein after this sampling is repeated many times (hundreds of thousands). The second sub-protocol uses gradient-based minimization of torsion angles to relax the structure into its local minimum in the Rosetta energy landscape. The energy function REF2015 was used for this study (Park, J Chem Theory Comput, 2016, 12(12): 6201-6212; O’Meara, et al., J Chem Theory Comput, 2015. 11(2): 609-22). Certain considerations were made to prevent noise in the Rosetta energy predictions. For FastRelax’s first sub-protocol, only residues within lOA of the mutation were used to sample alternate side chain conformations. Coordinate constraints were added to all residue positions in the second sub-protocol to prevent the backbone from varying much from the starting structure. After FastRelax completed, one final run of all-atom minimization without coordinate constraints was performed; allowing the protein to relax into its local, unconstrainted energy minimum. Ten independent trajectories were run for every possible mutation and used the average score of the three lowest-scoring trajectories as the representative score for that mutation. The version identifier (git shal) of our Rosetta version was 360d0b3a2bc3d9e08489bb9c292d85681bbc0cbd, released on June 4th, 2017.

Multiple Sequence Alignment.

A list of 39 CA and 53 CB sequences were assessed by hand-curating a multiple sequence alignment (MSA) of TCR sequences from various organisms. Two lists of TCR- similar sequences were assembled by passing the sequences of the CA and CB chains of the 2F53 structure into BLAST using NCBI’s database of non-redundant protein sequences (169,859,411 sequences in database). Both lists were purged by hand of any sequences that did not appear to be a TCR. The list of mutations deemed stabilizing by Rosetta were partitioned into two lists: (1) mutations present in the MSA and (2) mutations absent from the MSA. 30 mutations were selected from list 1 and 25 mutations were selected from list 2 for experimental testing. Selection decisions were made primarily by Rosetta score but some candidates were removed for the sake of increasing diversity. By splitting the mutations into two lists, mutations that were deemed lower-tier by Rosetta can be promoted if they have been shown to be compatible with any T-Cell Receptor structure.

Modeling Ca/CP mutant combinations.

After the best performing point mutations were determined experimentally, Rosetta simulations were run to verify that each mutation was compatible with every other mutation. Each mutation was separately applied to the pre-relaxed 2F53 structure and ran fixed- backbone sidechain repacking. The same simulations were performed with every pair of mutations and ensured that the Rosetta energy of the pair was not dissimilar to the sum of the energies of the mutations individually.

Protein crystallization.

Purified protein was crystallized at 8 °C using the sitting-drop vapor-diffusion method. Crystals were obtained by mixing one part protein solution at 15 mg/mL (lOmM Tris-HCl pH 7.5, and 150 mM sodium chloride) with one part reservoir solution containing 23% PEG 4K, 300 mM magnesium sulfate, and 10% glycerol. Drops were immediately seeded with crushed crystals grown from 15% PEG 3350, and 100 mM magnesium formate. Single, plate-shaped crystals were observed within one week. These were harvested into a drop of reservoir solution containing 15% glycerol, and flash frozen in liquid nitrogen.

X-ray data collection and structure determination.

Synchrotron X-ray diffraction data were collected on a single crystal at the Advanced Photon Source on beamline 31-ID-D (LRL-CAT). The resulting data were integrated using autoPROC (Vonrhein, et al., Acta Crystallogr D Biol Crystallogr, 2011. 67(Pt 4): 293-302), and merged and scaled with SCALA (Evans, Acta Crystallogr D Biol Crystallogr, 2011. 67(Pt 4): 282-92). The structure was solved by Molecular Replacement with Phaser (McCoy, et al., J Appl Crystallogr, 2007. 40(Pt 4): 658-674), using the TCR constant domains of a previously solved TCR-pMHC complex (Protein Databank accession code 2F53) as the search model (Dunn, et al., Protein Sci, 2006. 15(4): 710-21). Several rounds of model building and refinement were conducted with COOT (Emsley, et al., Acta Crystallogr D Biol Crystallogr, 2010. 66(Pt 4): 486-501) and REFMAC5 (Murshudov, et al., Acta Crystallogr D Biol Crystallogr, 2011. 67(Pt 4): p. 355-67), as implemented in the CCP4 software package (Winn, et al., Acta Crystallogr D Biol Crystallogr, 2011. 67(Pt 4): 235-42). The final model was validated with MolProbity (Williams, et al., Protein Sci, 2018. 27(1): 293-315). Additional data collection and refinement details can be found in Table 4 and Table 5.

Table 4 * Values in parentheses are for the highest resolution shell.

Table 5

Peptide Mapping.

The wild-type and stabilized CE10 TCRs were denatured, reduced, cysteine- alkylated, and digested for the analyses. Briefly, 100 pg of TCR variant was denatured in 6 M guanidine prior to reduction with 10 mM dithiothreitol at 37 °C for 30 minutes. Next, 15 mM iodoacetamide was added and the reaction was allowed to proceed for 45 minutes in the dark. Each sample was then buffer-exchanged into 10 mM TRIS, pH 7.5 using spin filters. 5 pg of trypsin was added to the reduced/alkylated sample and incubated for 4 hours at 37 °C. The trypsinized samples were divided into two equal parts. The first sample was deglycosylated by adding 1 pL PNGaseF (ProZyme) and 5 uL lOx digestion buffer and incubating overnight at room temperature, while the second sample was left untreated.

To determine the glycan occupancy, an aliquot of PNGase F treated and untreated tryptic digest was injected onto an Agilent 1290 UPLC coupled to an Agilent 5210 QTOF Mass Spectrometer for peptide mapping and mass alignment. Briefly, 5 pg TCR digest was injected on to a Waters 150mm x 2.1mm 1.7 pm Cl 8 CSH UPLC column and gradient separated from 2% B ( 0.1% formic acid in acetonitrile) to 35% B over 30 minutes. The eluting peptides were injected into the mass spectrometer for mass analysis with a scan range from 200 to 2000 m/z, source temperature of 350 °C, capillary voltage of 4 kV, fragmentor at 250 V, capillary exit at 75 V, source gas at 40 PSI, and cone gas at 12 PSI. The peptide mass spectra was aligned to the protein sequence by Mass Hunter/Bioconfirm 7.0 software with a mass accuracy of 5 ppm resulting in over 90% sequence coverage. To measure glycan occupancy, deamidation of Asn was added as a variable modification and each predicted glycan was checked manually by extracted ion chromatogram (EIC) with 5 ppm mass accuracy. The ratio of Asn to Asp was calculated using the EIC and reported as percent glycan occupancy: Asn being unoccupied and Asp being occupied since the enzymatic deglycosylation converts Asn to Asp.

Hydrogen/Deuterium Exchange coupled with mass spectrometry (HDX-MS).

HDX-MS experiments were performed on a Waters nanoACQUITY system with HDX technology (Wales, et al., Anal Chem, 2008. 80(17): 6815-20), including a LEAP HDX robotic liquid handling system. The deuterium exchange experiment was initiated by adding 55 pL of D2O buffer containing O.lx PBS to 5 mΐ of each protein (concentration was 25 mM) at 15 °C for various amounts of time (0s, 10s, 1 min, lOmin, 60min, and 240 min). The reaction was quenched using equal volume of was 0.32 M TCEP, 0.1 M phosphate pH 2.5 for two minutes at 1 °C. 50 pL of the quenched reaction was injected on to an on-line pepsin column (Waters BEH Enzymate) at 14°C, using 0.2% formic acid in water as the mobile phase at a flow rate of 100 pL/ min for 4 min. The resulting peptic peptides were then separated on a C18 column (Waters, Acquity UPLC BEH C18, 1.7 pm, 1.0 mm x 50 mm) fit with a Vanguard trap column using a 3 to 85% acetonitrile (containing 0.2% formic acid) gradient over 10 min at a flow rate of 50 pL/min. The separated peptides were directed into a Waters Xevo G2 time-of-flight (qTOF) mass spectrometer. The mass spectrometer was set to collect data in the MS ^E , ESI ⁺ mode; in a mass acquisition range of mlz 255.00-1950.00; with a scan time of 0.5 s. The Xevo G2 was calibrated with Glu-fibrinopeptide prior to use. All acquired data was mass corrected using a 2 pg/ml solution of LeuEnk in 50% ACN, 50%

H2O and 0.1% FA at a flowrate of 5 pl/min every 30 s {m/z of 556.2771). The peptides were identified by Waters Protein Lynx Global Server 3.02. The processing parameters were set to low energy threshold at 100.0 counts, an elevated energy threshold at 50.0 counts and an intensity threshold at 1500.0 counts. The resulting peptide list was imported to Waters DynamX 3.0 software, with threshold of 5 ppm mass, 20% fragments ions per peptide based on peptide length. The relative deuterium incorporation for each peptide was determined by processing the MS data for deuterated samples along with the non-deuterated control in DynamX 3.0 (Waters Corporation).

Pharmacokinetic measurements.

Pharmacokinetics measurements of the TCR/CD3 IgG-like BsAbs were performed as described previously (Lewis, et ah, Nat Biotechnol, 2014. 32(2): 191-8). To evaluate whether anti-drug antibodies were being generated in the mice against the BsAbs, ELISAs were carried out by coating a High Bind U-well 96-well plate (Greiner Microlon) with 2 pg/mL BsAb in 50 mM sodium carbonate, pH 9.3 overnight at 4 °C. The plates were washed with PBS+0.1% Tween20 (PBST), blocked for 1 hour with Blocker™ Casein in PBS (Thermo Scientific) at room temperature (RT), and washed with PBST. Mouse plasma dilutions starting at 1:50 with 1:3 serial dilutions in Blocker™ Casein in PBS were added to the plate for 1 hour at room temperature. The plates were washed with PBST and adding a 1:1000 goat anti -mouse-kappa- AP polyclonal (Southern Biotech Cat#1050- 04):Blocker ™ Casein primary antibody was added and incubated at 1 hour. The detection reagent was washed off followed by the addition of PNPP Substrate (Thermo Scientific). Colorimetric readout was performed by reading the absorbance at 450 nm on a SpectraMax 190 plate reader.

T cell redirected lysis assays.

Saos-2 cells were from ATCC and the 624.38 cells were from the NCI/NIH DTP,DCTD Tumor Repository and both were cultured in Complete Media (RPMI 1640, Coming). Primary naive T cells were from ALLCELLS. For the assay, the tumor cells were removed from their culture flask using Accutase and spun down for 10 min at 1200 RPM. The tumor cells were re-suspended in Complete Media and seeded at 5000 cells/well in 96- well black, clear bottom plates (Perkin Elmer) and incubated for 16-24 hours in a 5% CO2 incubator. The cells were then pulsed with 6 pg/mL SLLMWITQC (NY-ESO-1) peptide (synthesized by CPC Scientific), 50 pL total volume, for 3 hours. TCR/CD3 bifunctionals were then titrated onto the cells using 10X dilutions starting at 100 nM and ending at 0.001 nM in triplicate (50 pL/sample) for 30 minutes. During this period, primary T cells were thawed and washed twice in Complete Media and gentamicin. T cells (100 pL) were then added at 50K cells/well (200 mL total volume). Cells were incubated for 48 hours at 5% CO2 and 37 °C. At 48 hours, the plates were washed gently (twice) with serum free RPMI 1640. Next 100 pL RPMI 1640 and 100 pL Cell Titer Glo reagent (Promega) were added, mixed for two minutes on a shaker (slowly) and incubated for 10 minutes in the dark. Lastly, the luminescence was read on an EnVision 2130 multilabel reader.

Flow cytometry.

Cell culture was performed as described above. The day before running flow cytometry, T25 flasks were seeded with Jurkat, Saos-2 or 624.38 cells in Growth buffer (RPMI/10% fetal bovine serum (FBS)/gentamicin - Gibco). For the tumor cells, the next morning, cells were washed once with Growth buffer. Peptide was added to the tumor cells (or not as a negative control) at 6 pg/mL for 3 hours at 37 °C, 5% CO2. Next, excess peptide was aspirated off and the cells were washed 3X with PBS buffer. All subsequent steps were peformed on ice. The tumor cells were lifted from the T25 flasks using Accutase (Innovative Technologies). The Jurkat cells grow in suspension. Cells were transferred to centrifuge tubes and pelleted by centrifugation at 1200 RPM for 7 minutes. Henceforth, ‘Wash buffer’ was PBS/2% FBS/0.05% NaN _3. ‘Blocking buffer’ was Wash buffer supplemented with 10% Normal Goat Serum/10% FBS/Human BD Fc Block (BD). The cells were resuspended in Block buffer for 15 minutes, pelleted, Washed 3X, and resuspended in Block buffer before adding 50 pL of the cells (0.2 x 10 ⁶ ) to 96-well plates (Coming 3799). The TCR/CD3 bifunctionals and control mAbs were added to the wells at 30, 3, 0.3, and 0.03 pg/mL and incubated 45 minutes. The cells were pelleted, washed, and the supernatants were aspirated again before adding 100 pL diluted R-Phycoerythrin-conjugated AffmiPure Goat Anti- Human IgG-Fc (Jackson) or Anti-Human Lambda LC (Jackson) in Block buffer for 45 minutes. The cells were pelleted and washed again. Finally, the cells were resuspended in Block buffer with 1 : 1000 PI (Molecular Probes) and covered with foil. The cells were then sorted on a Fortessa H647780000006 flow cytometer acquiring with Diva software (BD) and data was analyzed using FloJo version 10.0.08.

Data availability

The coordinates for the TCR constant domain structure, and the corresponding structure factors, have been deposited in the Protein Data Bank (http://www.rcsb.org) under accession code 6U07.

Sequence Listing

SEQ ID NO.l WT TCRa constant domain (Ca)

PYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMD FKSNS AVAWSNKSDFACANAFNNSIIPEDTFFPS PESSC

SEQ ID NO. 2 WT TCRp constant domain (Cp)

EDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVS TDPQP LKEQPALNDSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVS AE AWGRADC

SEQ ID NO. 3 WT TCRa constant domain (Ca) with disulfide mutation

PYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMD FKSNS AVAWSNKSDFACANAFNNSIIPEDTFFPS PESSC

SEQ ID NO. 4 WT TCRP constant domain (CP) with disulfide mutation

EDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVC TDPQP LKEQPALNDSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVS AE AWGRADC

SEQ ID NO. 5 Ca for TCR with 3 stabilizing mutations minus disulfide Alpha constant domain minus disulfide_S139F_T150I_A190T

PYIQNPDPAVYQLRDSKSSDKFVCLFTDFDSQINVSQSKDSDVYITDKTVLDMRSMD FKSNS AVAWSNKSDFTCANAFNNSIIPEDTFFPS PESSC

SEQ ID NO. 6 Ca for TCR with 3 stabilizing mutations with disulfide mutation Alpha constant domain with disulfide_S139F_T150I_A190T

PYIQNPDPAVYQLRDSKSSDKFVCLFTDFDSQINVSQSKDSDVYITDKCVLDMRSMD FKSNS AVAWSNKSDFTCANAFNNSIIPEDTFFPS PESSC

SEQ ID NO. 7 CP for TCR with 4 stabilizing mutations minus disulfide

Beta constant El 34K H139R D155P S170D EDLKNVFPPEVAVFEPSKAEISRTQKATLVCLATGFYPPHVELSWWVNGKEVHDGVSTDP QP LKEQPALNDSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVS AE AWGRADC

SEQ ID NO. 8 Ob for TCR with 4 stabilizing mutations with disulfide mutation Beta constant E134K H139R D155P S170D

EDLKNVFPPEVAVFEPSKAEISRTQKATLVCLATGFYPPHVELSWWVNGKEVHDGVC TDPQP LKEQPALNDSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVS AE AWGRADC

SEQ ID NO.9 NY-ESO-1 Coe WT without disulfide

QEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLISPWQREQT SGRLN ASLDKSSGRSTLYIAASQPGDSATYLCAVRPLLDGTYIPTFGRGTSLIVHPYIQNPDPAV YQ LRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDF TC ANAFNNSIIPEDTFFPSPESSC

SEQ ID NO.10 NY-ESO-1 Coe WT with disulfide

QEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLT SLLLISPWQREQTSGRLN ASLDKSSGRSTLYIAASQPGDSATYLCAVRPLLDGTYIPTFGRGTSLIVHPYIQNPDPAV YQ LRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDF TC ANAFNNSIIPEDTFFPSPESSC

SEQ ID NO.ll NY-ESO-1 Coe with disulfide_S139F_T150I_A190T

QEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLT SLLLISPWQREQTSGRLN ASLDKSSGRSTLYIAASQPGDSATYLCAVRPLLDGTYIPTFGRGTSLIVHPYIQNPDPAV YQ LRDSKSSDKFVCLFTDFDSQINVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDF TC ANAFNNSIIPEDTFFPSPESSC

SEQ ID N0.12 NY-ESO-1 Coe without disulfide_S139F_T150I_A190T

QEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLT SLLLISPWQREQTSGRLN ASLDKSSGRSTLYIAASQPGDSATYLCAVRPLLDGTYIPTFGRGTSLIVHPYIQNPDPAV YQ LRDSKSSDKFVCLFTDFDSQINVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDF TC ANAFNNSIIPEDTFFPSPESSC

SEQ ID N0.13 NY-ESO-1 Ob WT without disulfide GVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVAIQTTDQGEVPN GY NVSRSTIEDFPLRLLSAAPSQTSVYFCASSYVGDTGELFFGEGSRLTVLEDLKNVFPPEV AV FEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVH SGVSTDPQPLKEQPALNDSRYA LSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADC SEQ ID N0.14 NY-ESO-1 Ob WT with disulfide

GVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVAIQTTDQGE VPNGY NVSRSTIEDFPLRLLSAAPSQTSVYFCASSYVGDTGELFFGEGSRLTVLEDLKNVFPPEV AV FEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVH SGVCTDPQPLKEQPALNDSRYA LSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADC SEQ ID N0.15 NY-ESO-1 Ob with disulfide_ E134K H139R D155P S170D

GVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVAIQTTDQGE VPNGY NVSRSTIEDFPLRLLSAAPSQTSVYFCASSYVGDTGELFFGEGSRLTVLEDLKNVFPPEV AV FEPSKAEISRTQKATLVCLATGFYPPHVELSWWVNGKEVHD GVCTDPQPLKEQPALNDSRYA LSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADC SEQ ID N0.16 NY-ESO-1 Ob without disulfide_ E134K H139R D155P S170D

GVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVAIQTTDQGE VPNGY NVSRSTIEDFPLRLLSAAPSQTSVYFCASSYVGDTGELFFGEGSRLTVLEDLKNVFPPEV AV FEPSKAEISRTQKATLVCLATGFYPPHVELSWWVNGKEVHD GVSTDPQPLKEQPALNDSRYA LSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADC SEQ ID N0.17 CE10 Coe WT with disulfide

QQVKQNSPSLSVQEGRISILNCDYTNSMFDYFLWYKKYPAEGPTFLIS ISSIKDKNEDGRFT VFLNKSAKHLSLHIVPSQPGDSAVYFCAASGDFNKFYFGSGTKLNVKPYIQNPDPAVYQL RD SKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACA NA FNNSIIPEDTFFPSPESSC SEQ ID N0.18 CE10 Coe with disulfide_S139F_T150I_A190T

QQVKQNSPSLSVQEGRISILNCDYTNSMFDYFLWYKKYPAEGPTFLIS ISSIKDKNEDGRFT VFLNKSAKHLSLHIVPSQPGDSAVYFCAASGDFNKFYFGSGTKLNVKPYIQNPDPAVYQL RD SKSSDKFVCLFTDFDSQINVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFTCA NA FNNSIIPEDTFFPSPESSC SEQ ID N0.19 CE10 Ob WT with disulfide EAGVAQSPRYKIIEKRQSVAFWCNP ISGHATLYWYQQILGQGPKLLIQFQNNGW DDSQLPK DRFSAERLKGVDSTLKIQPAKLEDSAVYLCASSLGTGLGEQYFGPGTRLTVTEDLKNVFP PE VAVFEPSEAEISHTQKATLVCLATGFYPDHVE LSWWVNGKEVHSGVCTDPQPLKEQPALNDS RYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADC

SEQ ID NO.20 CE10 Ob with disulfide_ E134K H139R D155P S170D

EAGVAQSPRYKIIEKRQSVAFWCNP ISGHATLYWYQQILGQGPKLLIQFQNNGW DDSQLPK DRFSAERLKGVDSTLKIQPAKLEDSAVYLCASSLGTGLGEQYFGPGTRLTVTEDLKNVFP PE VAVFEPSKAEISRTQKATLVCLATGFYPPHVE LSWWVNGKEVHDGVCTDPQPLKEQPALNDS RYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADC

SEQ ID NO.21 anti NEF134-10 HIV TCR Ca WT with disulfide

QQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMS IYSNGDKEDGRF TAQLNKASQYVSLLIRDSQPSDSATYLWGTYNQGGKLIFGQGTELSVKPYIQNPDPAVYQ LR DSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFAC AN AFNNSIIPEDTFFPSPESSC

SEQ ID NO.22 anti NEF134-10 HIV TCR Ca with disulfide_S139F_T150I_A190T

QQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKS PELIMSIYSNGDKEDGRF TAQLNKASQYVSLLIRDSQPSDSATYLWGTYNQGGKLIFGQGTELSVKPYIQNPDPAVYQ LR DSKSSDKFVCLFTDFDSQINVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFTC AN AFNNSIIPEDTFFPSPESSC

SEQ ID NO.23 anti NEF134-10 HIV TCR C WT with disulfide

QVTQNPRYLITVTGKKLTVTCSQNMNHEYMSWYRQDPGLGLRQI YYSMNVEVTDKGDVPEGY KVSRKEKRNFPLILESPSPNQTSLYFCASSGASHEQYFGPGTRLTVTEDLKNVFPPEVAV FE PSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVH SGVCTDPQPLKEQPALNDSRYALS SRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADC

SEQ ID NO.24 anti NEF134-10 HIV TCR C with disulfide_ E134K_H139R_D155P_S170D

QVTQNPRYLITVTGKKLTVTCSQNMNHEYMSWYRQDPGLGLRQI YYSMNVEVTDKGDVPEGY KVSRKEKRNFPLILESPSPNQTSLYFCASSGASHEQYFGPGTRLTVTEDLKNVFPPEVAV FE PSKAEISRTQKATLVCLATGFYPPHVELSWWVNGKEVHD GVCTDPQPLKEQPALNDSRYALS SRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADC

SEQ ID NO.25 MAGE-A3 Ca WT with disulfide KQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLYVRPYQREQTSG RL NASLDKSSGRSTLYIAASQPGDSATYLCAVRPGGAGPFFW FGKGTKLSVIPYIQNPDPAVY QLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSD FA CANAFNNSIIPEDTFFPSPESSC

SEQ ID NO.26 MAGE-A3 Ca with disulfide_S139F_T150I_A190T

KQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLYVRPYQREQ TSGRL NASLDKSSGRSTLYIAASQPGDSATYLCAVRPGGAGPFFW FGKGTKLSVIPYIQNPDPAVY QLRDSKSSDKFVCLFTDFDSQINVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSD FT CANAFNNSIIPEDTFFPSPESSC

SEQ ID NO.27 MAGE-A3 Ob WT with disulfide

GVTQTPRYLIKTRGQQVTLSCSPISGHRSVSWYQQTPGQGLQFLFEYFSETQRNKGN FPGRF SGRQFSNSRSEMNVSTLELGDSALYLCASSFNMATGQYFGPGTRLTVTEDLKNVFPPEVA VF EPSEAEISHTQKATLVCLATGFYPDHVEL SWWVNGKEVHSGVCTDPQPLKEQPALNDSRYAL SSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADC

SEQ ID NO.28 MAGE-A3 Ob with disulfide_ E134K H139R D155P S170D

GVTQTPRYLIKTRGQQVTLSCSPISGHRSVSWYQQTPGQGLQFLFEYFSETQRNKGN FPGRF SGRQFSNSRSEMNVSTLELGDSALYLCASSFNMATGQYFGPGTRLTVTEDLKNVFPPEVA VF EPSKAEISRTQKATLVCLATGFYPPHVEL SWWVNGKEVHDGVCTDPQPLKEQPALNDSRYAL SSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADC

SEQ ID NO.29 IgGl AA_N297Q NY-ESO-1 WT Ca with disulfide Fc 7.8.60A

QEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLISPWQREQT SGRLN ASLDKSSGRSTLYIAASQPGDSATYLCAVRPLLDGTYIPTFGRGTSLIVHPYIQNPDPAV YQ LRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDF AC ANAFNNSIIPEDTFFPSPESSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPE VT CW VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRW SVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTDNQVSLTCLVKGFYPSDIAVEWE SN GQPENNYKTTPPVLMSDGSFFLASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PG K

SEQ ID NO.30 IgGlAA_N297Q NY-ESO-1 Ca _S139F_T150I_A190T with disulfide and CH3 7.8.60 A heterodimer mutations

QEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLISPWQREQT SGRLN ASLDKSSGRSTLYIAASQPGDSATYLCAVRPLLDGTYIPTFGRGTSLIVHPYIQNPDPAV YQ LRDSKSSDKFVCLFTDFDSQINVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDF TC ANAFNNSIIPEDTFFPSPEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTP EV TCVW DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRW SVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTDNQVSLTCLVKGFYPSDIAVEW ES NGQPENNYKTTPPVLMSDGSFFLASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SP GK

SEQ ID N0.31 IgGl AA_N297Q SP34 mVH with 7.8.60B heterodimer mutations

EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNN YATYY

ADSVKDRFTISRDDSQSLLYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGT LVTVS

AASTKGPSVFPLAPSSKSTSGGTAALGCLVADYFPEPVTVSWNSGALTSGVHTFPAV LQSSG

LYSLASW TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSV

FLFPPKPKDTLMISRTPEVTCVW DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRV

VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRRPRVYTLPPSREEMT KNQVS

LVCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSVLTVDKSRWQQGN VFSCS

VMHEALHNHYTQKSLSLSPGK

SEQ ID NO.32 Ch SP34 mVL Lambda

QAW TQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGVPA RFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNLWVFGGGTKLTVLGQPKAAPSVTLFP PS SEELQANKATLVCLISDFYPGAVTVAWKAD SSPVKAGVETTTPSKQSNNKYAASSYLSLTPE QWKSHRSYSCQVTHEGSTVEKTVAPTEC

SEQ ID NO.33 NY-ESO-1 Ca _S139F_T150I_A190T with disulfide_(G4S)4 linker_Ch SP34 mVH tandem FAb

HHHHHHHHGSQEVTQIPAALSVPEGENLVLNCSFTDSAI YNLQWFRQDPGKGLTSLLLISPW QREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPLLDGTYIPTFGRGTSLIVH PY IQNPDPAVYQLRDSKSSDKFVCLFTDFDSQINVSQSKDSDVYITDKCVLDMRSMDFKSNS AV AWSNKSDFTCANAFNNSIIPEDTFFPSPESSCGGGGSGGGGSGGGGSGGGGSEVQLVESG GG LVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRF TI SRDDSQSLLYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSAASTKGPS VF PLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSW TV PSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG

SEQ ID NO.34 IgGlAA_N297Q NY-ESO-1 Ca _S139F_T150I_A190T Deglycosylated with disulfide 7.8.60 A QEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLISPWQREQTSGR LN ASLDKSSGRSTLYIAASQPGDSATYLCAVRPLLDGTYIPTFGRGTSLIVHPYIQNPDPAV YQ LRDSKSSDKFVCLFTDFDSQIQVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKADF TC ANAFQNSIIPEDTFFPSPEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTP EV TCVW DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRW SVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTDNQVSLTCLVKGFYPSDIAVEW ES NGQPENNYKTTPPVLMSDGSFFLASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SP GK

SEQ ID NO.35 IgGlAA_N297Q NY-ESO-1 #107 Ca _S139F_T150I_A190T with disulfide 7.8.60A

QEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLT SLLLISPWQREQTSGRLN ASLDKSSGRSTLYIAASQPGDSATYLCAVRPFTGGGYIPTFGRGTSLIVHPYIQNPDPAV YQ LRDSKSSDKFVCLFTDFDSQINVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDF TC ANAFNNSIIPEDTFFPSPEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTP EV TCW VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRW SVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTDNQVSLTCLVKGFYPSDIAVEW ES NGQPENNYKTTPPVLMSDGSFFLASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SP GK

SEQ ID NO. 36 NY-ESO-1#107 Ob with disulfide_ E134K_H139R_D155P_S170D

GVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVAIQTTDQGE VPNGY NVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGNTGELFFGEGSRLTVLEDLKNVFPPEV AV FEPSKAEISRTQKATLVCLATGFYPPHVEL SWWVNGKEVHDGVCTDPQPLKEQPALNDSRYA LSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADC

SEQ ID NO.37 IgGlAA_N297Q NY-ESO-1 #113 Ca _S139F_T150I_A190T with disulfide 7.8.60A

QEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLT SLLLITPWQREQTSGRLN ASLDKSSGRSTLYIAASQPGDSATYLCAVRPLLDGTYIPTFGRGTSLIVHPYIQNPDPAV YQ LRDSKSSDKFVCLFTDFDSQINVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDF TC ANAFNNSIIPEDTFFPSPEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTP EV TC\AADVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRW SVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTDNQVSLTCLVKGFYPSDIAVEW ES NGQPENNYKTTPPVLMSDGSFFLASKLTVDKSRWQQGNVFSCSA/MHEALHNHYTQKSLS LSP GK

SEQ ID NO.38 NY-ESO-l#113 Ob with disulfide_E134K_H139R_D155P_S170D GVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVAIQTTDRGEVPN GY NVSRSTIEDFPLRLLSAAPSQTSVYFCASSYLGNTGELFFGEGSRLTVLEDLKNVFPPEV AV FEPSKAEISRTQKATLVCLATGFYPPHVEL SWWVNGKEVHDGVCTDPQPLKEQPALNDSRYA LSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADC

SEQ ID NO.39 IgGlAA_N297Q NY-ESO-1 Ca _S139F_T150I_A190T with di sul fi de_3 XG4 S_ SP34mVH 7.8.60B

QEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLISPWQREQT SGRLN ASLDKSSGRSTLYIAASQPGDSATYLCAVRPLLDGTYIPTFGRGTSLIVHPYIQNPDPAV YQ LRDSKSSDKFVCLFTDFDSQINVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDF TC ANAFNNSIIPEDTFFPSPESSCGGGGSGGGGSGGGGSGSEVQLVESGGGLVQPKGSLKLS CA ASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSQSLLYL QM NNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSG GT AALGCLVADYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLASW TVPSSSLGTQTYICN VNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC W VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRW SVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPRRPRVYTLPPSREEMTKNQVSLVCLVKGFYPSDIAVEWESNG QP ENNYKTTPPVLDSDGSFFLYSVLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO.40 NY-ESO-1 Ca _S139F_T150I_A190T with disulfide_5XG4S_Ch SP34 mVH tandem FAb

HHHHHHHHGSQEVTQIPAALSVPEGENLVLNCSFTDSAI YNLQWFRQDPGKGLTSLLLISPW QREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPLLDGTYIPTFGRGTSLIVH PY IQNPDPAVYQLRDSKSSDKFVCLFTDFDSQINVSQSKDSDVYITDKCVLDMRSMDFKSNS AV AWSNKSDFTCANAFNNSIIPEDTFFPSPESSCGGGGSGGGGSGGGGSGGGGSGGGSEVQL VE SGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSV KD RFTISRDDSQSLLYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSAAST KG PSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SS W TVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG

SEQ ID N0.41 (G4S linker

GGGGS GGGGS GGGGS

SEQ ID NO.42 (G4Q linker

GGGGQGGGGQGGGGQ

SEQ ID NO.43 (G4S) ₄ linker GGGGS GGGGS GGGGS GGGGS

SEQ ID NO.44 (G4S) ₅ linker

GGGGS GGGGS GGGGS GGGGS GGGGS

SEQ ID NO.45 (G4Q) ₄ linker

GGGGQGGGGQGGGGQGGGGQ

SEQ ID NO.46 (G4Q) ₅ linker

GGGGQGGGGQGGGGQGGGGQGGGGQ

SEQ ID NO. 47 IgGl hinge region

EPKSCDKTHTCPPCP

SEQ ID NO. 48 IgGl Fc region with L234A_L235A_N297Q

APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVW DVSHEDPEVKFNWYVDGVEVHNAKTKPR

EEQYQSTYRW SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS

RDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSR

WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO. 49 IgG4 (StoP) hinge region

ESKYGPPCPPCP

SEQ ID NO. 50 IgG4AA Fc region

APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVW DVSQEDPEVQFNWYVDGVEVHNAKTKPR

EEQFNSTYRW SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPS

QEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLT VDKSR

WQEGNVFSCSVMHEALHNHYTQKSLSLSLG

SEQ ID NO: 51 Human HPB-MLT Ob

EDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVS TDPQP LKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVS AE AWGRADC

SEQ ID NO: 52 Human HPB-MLT Coe NIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSN SA VAWSNKSDFACANAFNNSIIPEDTFFPSPESSC