T cell receptor fusion protein with specificity for MAGE A4

Abstract

The present disclosure provides T cell receptor (TCR) fusion proteins comprising a TCR that binds to the GVYDGREHTV (SEQ ID NO: 34) HLA-A * 02 complex covalently linked to a T cell conjugation domain and an antibody Fc domain that bind to a protein expressed on the cell surface of a T cell, as well as polynucleotides, vectors, kits, host cells, pharmaceutical compositions, methods and uses related to the fusion proteins.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Application Serial No. 63 / 399,095, filed on August 18, 2022, which is incorporated herein by reference in its entirety.

[0003] References to electronic sequence listings

[0004] The contents of the electronic sequence listing (146392054140seqlist.xml; size: 35,722 bytes; creation date: August 9, 2023) are incorporated herein by reference in their entirety. Technical Field

[0005] The present disclosure relates to T cell receptor (TCR) fusion proteins comprising a TCR that binds the GVYDGREHTV (SEQ ID NO: 34) HLA-A*02 complex covalently linked to an antigen binding domain that binds a protein expressed on the cell surface of T cells and an antibody Fc domain, as well as related polynucleotides, vectors, kits, host cells, pharmaceutical compositions, methods, and uses. Background Art

[0006] T cell receptor (TCR) is naturally expressed by CD4+ and CD8+ T cells. TCR is designed to identify short peptide antigens (Davis et al., (1998), Annu Rev Immunol 16:523-544.) compounded with major histocompatibility complex (MHC) molecules (in humans, MHC molecules are also referred to as human leukocyte antigens, or HLA) displayed on the surface of antigen presenting cells. CD8+ T cells are also referred to as cytotoxic T cells, specifically recognize peptides that bind to MHC class I, and are generally responsible for searching for and mediating the destruction of diseased cells. CD8+ T cells can destroy cancerous cells and virally infected cells; however, as a result of thymic selection, the affinity of TCR expressed by cancer-specific T cells in natural reservoirs (repertoire) is generally very low, which means that cancerous cells frequently escape detection and destruction. A novel immunotherapy method designed to promote T cell recognition of cancer provides a highly promising strategy for developing effective anticancer treatments.

[0007] MAGE A4 belongs to the MAGE family of germline-encoded cancer antigens (De Plaen et al., (1994), Immunogenetics 40(5):360-369) and has a UniProt accession number of P43358. This class of antigens has been found to be frequently expressed in a variety of cancers, while its expression in normal tissues is restricted to adult testis and other immune-privileged sites, including the placenta. The cancer-specific nature of these genes makes them ideal targets for anticancer therapy. The exact function of MAGE A4 is unknown, but it is believed to play a role in embryonic development. High levels of MAGE A4 expression have been reported in several tumor types, including melanoma, esophageal cancer, head and neck cancer, lung cancer, breast cancer, and bladder cancer (Bergeron, (2009), Int J Cancer 125(6):1365-1371; Cabezon et al., (2013), Mol Cell Proteomics 12(2):381-394; Cuffel et al., (2011), Int J Cancer 128(11):2625-2634; Forghanifard et al., (2011), Cancer Biol Ther 12(3):191-197; Karimi et al., (2012), Clin Lung Cancer 13(3):214-219; Svobodova et al., (2011), Eur J Cancer 47(3):460-469). The 10-mer peptide GVYDGREHTV (SEQ ID NO: 34) corresponds to amino acids 230-239 of the full-length MAGE A4 protein. This peptide binds to HLA-A*02, and the peptide-HLA complex has been shown to stimulate cytotoxic T cells, leading to lysis of MAGE A4-positive, HLA-A*02-positive tumor cells (Duffour et al., (1999), Eur J Immunol 29(10): 3329-3337 and WO2000020445). Therefore, the GVYDGREHTV (SEQ ID NO: 34) HLA-A*02 complex provides a useful target antigen for immunotherapeutic intervention.

[0008] Soluble TCRs and TCR fusion proteins that bind to the GVYDGREHTV (SEQ ID NO: 34) HLA-A*02 complex are described, for example, in US PG Publication No. US20190092834 and International Publication No. WO2017175006. However, there remains a need for TCR fusion proteins that bind to the MAGE A4 peptide:HLA complex with favorable properties, such as stability, binding affinity, cell killing potency, and / or in vivo pharmacokinetics.

[0009] All references cited herein, including patent applications and publications, are hereby incorporated by reference in their entirety. Summary of the Invention

[0010] In certain aspects, provided herein is a T cell receptor (TCR) fusion protein comprising a TCR that binds a GVYDGREHTV (SEQ ID NO: 34) HLA-A*02 complex, wherein the TCR is a soluble TCR covalently linked to: (1) a T cell engaging domain that binds a protein expressed on the cell surface of a T cell, and (2) an antibody Fc domain; wherein the TCR comprises: (a) a TCR alpha chain comprising an alpha chain variable region, wherein the alpha chain variable region comprises (i) a CDR1 comprising the amino acid sequence of VSPFSN (SEQ ID NO: 1), (ii) a CDR2 comprising the amino acid sequence of LTFSENT (SEQ ID NO: 2), and (iii) a CDR3 comprising the amino acid sequence of VVNSAQGLYIPTF (SEQ ID NO: 3); and (b) a TCR beta chain comprising a beta chain variable region, wherein the beta chain variable region comprises (i) a CDR1 comprising the amino acid sequence of LDHEN (SEQ ID NO: 4), (ii) a CDR2 comprising the amino acid sequence of SRFATG (SEQ ID NO: 5). NO:5), and (iii) a CDR2 comprising the amino acid sequence of ASSSDQNSGDPYEQYF (SEQ ID NO:6); wherein the TCR is glycosylated at a single N-linked glycosylation site, wherein the N-linked glycosylation site is at residue N18 in the α chain variable region according to the numbering of SEQ ID NO:7.

[0011] In some embodiments according to any of the embodiments described herein, the TCR comprises an amino acid replacement at each potential N-glycosylation site except residue N18. In some embodiments, the TCR comprises an amino acid replacement at the following positions: (a) residue N24 of the alpha chain variable region, numbered according to SEQ ID NO: 32; (b) residues N33, N67, and N78 of the alpha chain constant region, numbered according to SEQ ID NO: 10; (c) residue N84 of the beta chain variable region, numbered according to SEQ ID NO: 33; and (d) residue N70 of the beta chain constant region, numbered according to SEQ ID NO: 15. In some embodiments, the amino acid replacement is N→Q. In some embodiments, the TCR comprises the following amino acid replacements: (a) N24Q in the α chain variable region, numbered according to SEQ ID NO:32; (b) N33Q, N67Q, and N78Q in the α chain constant region, numbered according to SEQ ID NO:10; (c) N84Q in the β chain variable region, numbered according to SEQ ID NO:33; and (d) N70Q in the β chain constant region, numbered according to SEQ ID NO:15.

[0012] In some embodiments according to any of the embodiments described herein, the TCR comprises one or more engineered cysteine residues in the α chain constant region and / or the β chain constant region to form a non-native disulfide bond between the α chain and the β chain. In some embodiments, according to SEQ ID NO: 15, the TCR comprises a cysteine residue at position 57 of the β chain constant region.

[0013] In some embodiments according to any of the embodiments described herein, the alpha chain variable region comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 7. In some embodiments, the beta chain variable region comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 13. In some embodiments, the alpha chain variable region comprises the amino acid sequence of SEQ ID NO: 7, and the beta chain variable region comprises the amino acid sequence of SEQ ID NO: 13. In some embodiments, the TCR alpha chain further comprises an alpha chain constant region, and wherein the alpha chain constant region comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the TCR beta chain further comprises a beta chain constant region, and wherein the beta chain constant region comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the TCR alpha chain further comprises an alpha chain constant region comprising the amino acid sequence of SEQ ID NO: 9, and the TCR beta chain further comprises a beta chain constant region comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the α chain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 11. In some embodiments, the β chain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 16. In some embodiments, the α chain comprises the amino acid sequence of SEQ ID NO: 11, and the β chain comprises the amino acid sequence of SEQ ID NO: 16.

[0014] In some embodiments according to any of the embodiments described herein, the antibody Fc domain is a human Fc domain. In some embodiments, the antibody Fc domain is a human IgG1, human IgG2, or human IgG4 Fc domain. In some embodiments, the antibody Fc domain comprises one or more mutations that weaken the effector function of the Fc domain. In some embodiments, according to the EU index numbering, the antibody Fc domain is a human IgG1 Fc domain comprising a mutation at residue N297. In some embodiments, according to the EU index numbering, the antibody Fc domain is a human IgG1 Fc domain comprising an N297G substitution. In some embodiments, according to the EU index numbering, the antibody Fc domain is a human IgG1 Fc domain comprising one or more mutations at residues E233, L234, L235, and / or G236. In some embodiments, according to the EU index numbering, the antibody Fc domain is a human IgG1 Fc domain comprising substitutions N297G, E233P, L234V, L235A, and a deletion at G236. In some embodiments, the antibody Fc domain is a human IgG1 Fc domain comprising one or more mutations at residues L234, L235, and P329, according to the EU index numbering. In some embodiments, the antibody Fc domain is a human IgG1 Fc domain comprising substitutions L234A, L235A, and P329G, according to the EU index numbering. In some embodiments, the antibody Fc domain is fused to the TCR via a hinge sequence. In some embodiments, the hinge sequence comprises the amino acid sequence of DKTHTCPP (SEQ ID NO: 31) or DKTHTCPPC (SEQ ID NO: 36). In some embodiments, the TCR fusion protein further comprises a second antibody Fc domain, wherein the second antibody Fc domain is associated with the first antibody Fc domain by: (1) one or more covalent linkages; and / or (2) one or more amino acid substitutions on one or both of the antibody Fc domains that promote heterodimerization.

[0015] In some embodiments, the first antibody Fc domain and the second antibody Fc domain both comprise an antibody CH2 domain and a CH3 domain. In some embodiments, the first antibody Fc domain is fused to the TCR via a first hinge sequence, and the second hinge sequence is connected to the N-terminus of the second antibody Fc domain. In some embodiments, the first hinge sequence and the second hinge sequence are connected via one or more interchain disulfide bonds between the first hinge sequence and the second hinge sequence. In some embodiments, the first hinge sequence and the second hinge sequence both comprise the amino acid sequence of DKTHTCPP (SEQ ID NO: 31) or DKTHTCPPC (SEQ ID NO: 36). In some embodiments, one of the first antibody Fc domain and the second antibody Fc domain comprises one or more knob-forming mutations, and the other of the first antibody Fc domain and the second antibody Fc domain comprises one or more corresponding hole-forming mutations to promote heterodimerization of these antibody Fc domains. In some embodiments, one of the first and second antibody Fc domains comprises a T366W substitution, and the other of the first and second antibody Fc domains comprises a T366S, L368A, and Y407V substitution, as numbered by the EU index. In some embodiments, one of the first and second antibody Fc domains comprises the amino acid sequence of SEQ ID NO: 27, and the other of the first and second antibody Fc domains comprises the amino acid sequence of SEQ ID NO: 26. In some embodiments, the first antibody Fc domain covalently linked to the TCR comprises the amino acid sequence of SEQ ID NO: 27, and the second antibody Fc domain comprises the amino acid sequence of SEQ ID NO: 26.

[0016] In some embodiments according to any embodiment described herein, the T cell engagement domain binds to human CD3 expressed on the cell surface of T cells. In some embodiments, the T cell engagement domain comprises an antibody antigen binding domain. In some embodiments, the T cell engagement domain (e.g., an antibody antigen binding domain) is a single chain variable fragment (scFv). In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO:17. In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO:35. In some embodiments, the T cell engagement domain is covalently linked to the TCR via a joint. In some embodiments, the joint comprises an amino acid sequence selected from SEQ ID NO:18-25. In some embodiments, the C-terminus of the T cell engagement domain is covalently linked to the N-terminus of the TCR β chain variable domains. In some embodiments, the C-terminus of the T cell engagement domain is covalently linked to the N-terminus of the TCR β chain variable domains via a joint. In some embodiments, the joint comprises an amino acid sequence selected from SEQ ID NO:18-25. In some embodiments, the N-terminus of the antibody Fc domain is covalently linked to the C-terminus of the TCR α chain constant domain. In some embodiments, the N-terminus of the antibody Fc domain is covalently linked to the C-terminus of the TCR α chain constant domain via a hinge sequence. In some embodiments, the TCR fusion comprises three polypeptides, including: (a) a first polypeptide comprising the TCR α chain variable region, the α chain constant region, the first hinge sequence, and the first antibody Fc domain from the N-terminus to the C-terminus; (b) a second polypeptide comprising a single-chain variable fragment (scFv) that binds to human CD3 expressed on the cell surface of T cells, a linker, the β chain variable region, and the β chain constant region from the N-terminus to the C-terminus; and (c) a third polypeptide comprising a second hinge sequence and a second antibody Fc domain from the N-terminus to the C-terminus. In some embodiments, the first polypeptide and the second polypeptide are linked by one or more disulfide bonds between the α chain constant region and the β chain constant region. In some embodiments, the first polypeptide and the third polypeptide are linked by: (1) one or more interchain disulfide bonds between the first hinge sequence and the second hinge sequence; and / or (2) one or more corresponding knob-forming mutations and hole-forming mutations on the antibody Fc domains. In some embodiments, the first polypeptide comprises the amino acid sequence of SEQ ID NO: 29, wherein the second polypeptide comprises the amino acid sequence of SEQ ID NO: 30, and wherein the third polypeptide comprises the amino acid sequence of SEQ ID NO: 28.

[0017] In other aspects, provided herein is a T cell receptor (TCR) fusion protein comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 29, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 30, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 28.

[0018] In other aspects, polynucleotides are provided herein, which encode the TCR fusion protein according to any one of the above embodiments. A polynucleotide kit is further provided herein, which comprises a first polynucleotide encoding the first polypeptide according to any one of the above embodiments, a second polynucleotide encoding the second polypeptide according to any one of the above embodiments, and a third polynucleotide encoding the third polypeptide according to any one of the above embodiments. In other aspects, a vector is provided herein, which comprises the polynucleotide according to any one of the above embodiments. A vector kit is further provided herein, which comprises a first vector encoding the first polypeptide according to any one of the above embodiments, a second vector encoding the second polypeptide according to any one of the above embodiments, and a third vector encoding the third polypeptide according to any one of the above embodiments. In some embodiments, the vector (these) is an expression vector.

[0019] In other aspects, provided herein is a host cell comprising a test kit of (one or more) polynucleotides, polynucleotides, (one or more) vectors or vectors according to any one of the above embodiments. In some embodiments, the host cell is a mammalian cell. In some embodiments, the mammalian cell is a Chinese hamster ovary (CHO) cell.

[0020] In other aspects, provided herein are methods for producing a TCR fusion protein, comprising culturing a host cell according to any one of the embodiments above under conditions suitable for producing the TCR fusion protein. In some embodiments, the method further comprises recovering the TCR fusion protein from the host cell. Further provided herein are TCR fusion proteins produced by the method according to any one of the embodiments above.

[0021] In other aspects, provided herein is a pharmaceutical composition comprising the TCR fusion protein according to any one of the above embodiments and a pharmaceutically acceptable carrier.

[0022] In other aspects, provided herein are methods for treating cancer, comprising administering an effective amount of a TCR fusion protein according to any one of the above embodiments or a pharmaceutical composition according to any one of the above embodiments to an individual. Further provided herein are TCR fusion proteins according to any one of the above embodiments for medical use, preferably for human subjects. Further provided herein are TCR fusion proteins according to any one of the above embodiments for use in treating cancer, preferably for human subjects. Further provided herein are uses of the TCR fusion proteins according to any one of the above embodiments in the manufacture of a medicament for treating cancer.

[0023] In some embodiments according to any of the embodiments described herein, the individual is human. In some embodiments, the individual has a cancer that expresses MAGE-A4. In some embodiments, the individual is of the HLA-A*02 subtype. In some embodiments, the TCR fusion protein or composition is administered intravenously or by intratumoral injection. In some embodiments, the method further comprises administering a second anticancer agent to the individual.

[0024] It should be understood that one, some or all of the properties of the various embodiments described herein can be combined to form other embodiments of the present invention. These and other aspects of the present invention will be apparent to those skilled in the art. These and other embodiments of the present invention are further described by the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Figures A and B show schematic diagrams of T cell receptor (TCR): anti-CD3 fusion molecules according to some embodiments. Figure 1 In the example shown in Figure A, effector function is provided by an anti-CD3 single-chain variable fragment (scFv) fused to the N-terminus of the TCRβ chain, targeting is provided by a soluble monoclonal high-affinity TCR, and in vivo half-life extension is provided at least in part by an antibody Fc domain (in this case, a human IgG1 Fc domain with the N297G mutation) fused to the C-terminus of the TCRα chain. Figure 1 Figure B shows more detailed features, including the knobs-into-holes (KIH) mutations that provide Fc heterodimerization (in this example, T366W on one chain and T366S / L368A / Y407V on the other chain), the disulfide bond in the hinge region, the engineered disulfide bond between the TCR α and β chains, the TCR constant region (Ca and Cβ on the α and β chains, respectively), and the TCR variable region (Va and Vβ on the α and β chains, respectively).

[0026] Figure 2Panels A and B illustrate N-glycosylation of TCR chains. Figure 2 A shows a schematic diagram of all seven N-glycosylation sites on TCR (left) and the occupancy of each N-glycosylation site (right). Figure 2 B shows variant TCRs with different sets of substitution mutations at different glycosylation sites (e.g., N→Q), resulting in unglycosylated TCRs (left) or monoglycosylated TCRs (right). The arrow indicates the single remaining N-glycosylation site.

[0027] Figure 2 Panels C and D illustrate the effect of deglycosylation on the yield of TCR:anti-CD3 fusion molecules. Figure 2 C shows that removal of N-glycosylation sites from the TCR variable region (using N→Q substitution) resulted in a significant decrease in yield, whereas removal of N-glycosylation sites from the TCR constant region had no effect on yield. Figure 2 D shows that retaining N-glycosylation at residue N18 in the α chain variable region is the most critical site for enhancing production.

[0028] Figure 3 The AEs demonstrated the in vivo pharmacokinetic properties of the TCR:anti-CD3 fusion molecule. Figure 3 Panel A shows the in vivo pharmacokinetic properties of various formats of TCR:anti-CD3 fusion molecules with or without an Fc domain (as indicated). Figure 3 Panel B shows the time course of serum concentrations of N297G control, unglycosylated, or monoglycosylated TCR:anti-CD3 fusion molecules with the indicated Fc formats in a SCID mouse model. Figure 3 C and D show the SCID mouse model. Figure 3 B shows the time course of serum concentrations of monoglycosylated or unglycosylated TCR:anti-CD3 fusion molecules (respectively). Figure 3 The E shows Figure 3 B shows the half-life and clearance of unglycosylated or monoglycosylated TCR:anti-CD3 fusion molecules at the indicated dose levels.

[0029] Figure 4 The AD showed the potency and selectivity of unglycosylated or monoglycosylated TCR:anti-CD3 fusion molecules. Figure 4 Panel A shows the name and type of each cell line, the average copy number and mRNA expression of MAGE-A4 for each cell line, HLA-A2 expression for each cell line, and the observed EC50 for cell killing or IFNγ release when treated with a monoglycosylated (“mono”) or unglycosylated (“aglyc”) TCR:anti-CD3 fusion molecule. ** indicates data are averaged from three different PBMC donors. Figure 4BD shows the effect of NCI-H1755 ( Figure 4 B), SCaBER( Figure 4 C) or NCI-H441( Figure 4 D) Changes in % cytolysis of cell lines over time. Figure 4 The values shown in BD refer to the lowest concentration of TCR:anti-CD3 fusion molecules that elicited a killing response.

[0030] Figure 5 AC shows the loss of potency upon Fc fusion, which was partially compensated by using variant anti-CD3 scFv. Figure 5 Figures A and B show the expression of TCR: anti-CD3 fusion molecules without Fc, TCR: anti-CD3 fusion molecules with Fc, and TCR: anti-CD3 fusion molecules with Fc and variant anti-CD3 scFv against NCI-H1755 ( Figure 5 A) or A375( Figure 5 B) Cell killing efficacy of the cell lines. Figure 5 Panel C shows the potency of cell killing against MAGE-A4+NCI-H1755 cells and MAGE-A4-MEL202A2B2M cells, illustrating the window between on-target and off-target activity.

[0031] Figure 6 Panels A and B show the results of an in vitro safety assay examining TCR:anti-CD3 fusion molecules. Figure 6 Panel A shows the results of testing a monoglycosylated TCR:anti-CD3 fusion molecule against a panel of normal cell lines, showing no detectable reactivity against normal cells. Figure 6 Panel B shows the results of testing TCR:anti-CD3 fusion molecules with variant anti-CD3 scFv and Fc domains versus the same molecules without the Fc domain or variant scFv, demonstrating that the therapeutic window was maintained for both on-target and off-target cells.

[0032] I. General Technology

[0033] The techniques and procedures described or referenced herein are generally well known to those skilled in the art and are routinely used using conventional methods, for example, the widely used methods described in Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd ed. (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (FM Ausubel et al., eds., (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor, eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (RI Freshney, ed. (1987)); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Manual, ed. (2003); Notebook (JE Cellis, ed., 1998) Academic Press; Animal Cell Culture (RI Freshney, ed., 1987); Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JB Griffiths and DG Newell, eds., 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MPCalos, ed., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., ed., 1994); Current Protocols in Immunology (J. E. Coligan et al., ed., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty, ed., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds., Harwood Academic Press, 1999); Publishers, 1995); and Cancer: Principles and Practice of Oncology (VT DeVita et al., eds., JB Lippincott Company, 1993).

[0034] II. Definitions

[0035] The description of the TCR sequences defined herein refers to the IMGT nomenclature, which is well known and available to those working in the field of TCRs. For example, see: LeFranc and LeFranc, (2001). "T cell Receptor Factsbook", Academic Press; Lefranc, (2011), Cold Spring Harb Protoc 201 1(6):595-603; Lefranc, (2001), Curr Protoc Immunol Appendix 1:Appendix 10O; and Lefranc, (2003), Leukemia 17(1):260-266. Briefly, TCR consists of two disulfide-linked chains. Each chain (α chain and β chain) is generally considered to have two domains, namely a variable domain and a constant domain. A short connecting region connects the variable domain and the constant domain and is generally considered to be part of the α variable region. In addition, the β chain generally contains a short multivariable region next to the connecting region, which is also generally considered to be part of the β variable region.

[0036] The variable domain of each chain is located at the N-terminus and contains three complementary determining regions (CDRs) embedded in the framework sequence. These CDRs contain the recognition sites for peptide-MHC binding. There are several genes encoding the α chain variable (Va) region, and several genes encoding the β chain variable (Vβ) region, which are distinguished by their framework, CDR1 and CDR2 sequences, and partially defined CDR3 sequences. The Va gene and the Vβ gene are referred to in the IMGT nomenclature by the prefixes TRAV and TRBV, respectively (Folch and Lefranc, (2000), Exp Clin Immunogenet 17(1):42-54; Scavner and Lefranc, (2000), Exp Clin Immunogenet 17(2):83-96; LeFranc and LeFranc, (2001), "T cell Receptor Factsbook", Academic Press). Likewise, there are several joining or J genes for the α and β chains, termed TRAJ or TRBJ, respectively, and a diversity or D gene for the β chain, termed TRBD (Folch and Lefranc, (2000), Exp Clin Immunogenet 17(2):107-114; Scavner and Lefranc, (2000), Exp Clin Immunogenet 17(2):97-106; LeFranc and LeFranc, (2001), "T cell Receptor Factsbook", Academic Press). The enormous diversity of T cell receptor chains is due to combinatorial rearrangements and joining diversity between various V, J, and D genes (including allelic variants) (Arstila et al., (1999), Science 286(5441):958-961; Robins et al., (2009), Blood 114(19):4099-4107). The constant or C regions of the TCR alpha and beta chains are referred to as TRAC and TRBC, respectively (Lefranc, (2001), Curr Protoc Immunol Appendix 1: Appendix 10).

[0037] "Engineered TCR" and "mutant TCR" are used synonymously herein to refer to a TCR with one or more introduced mutations relative to a native MAGE A4 TCR, particularly in its α and / or β chain variable domains. The mutation(s) generally improve the TCR's binding affinity for the GVYDGREHTV (SEQ ID NO: 34) HLA-A*02 complex, but may also or alternatively confer other advantages, such as improved stability and improved specificity in isolated form. Mutations at one or more positions may additionally or alternatively affect the interaction of adjacent positions with the cognate pMHC complex, for example by achieving a more favorable angle of interaction. To improve TCR binding to the GVYDGREHTV (SEQ ID NO: 34) HLA-A*02 complex, mutations are preferably made in one or more of the CDR regions.

[0038] Phenotypic silent variants of any TCR disclosed herein are within the scope of the present disclosure. As used herein, it is understood that the term "phenotypically silent variant" refers to a TCR that incorporates one or more further amino acid changes (including substitutions, insertions, and deletions) in addition to those amino acid changes set forth above, and that has a phenotype similar to that of the corresponding TCR without the (one or more) changes. For the purposes of this application, TCR phenotypes include antigen binding affinity (K D When measured under the same conditions (e.g., at 25°C and / or on the same SPR chip), the K of the phenotypically silent variants against the GVYDGREHTV (SEQ ID NO: 34) HLA-A*02 complex was D and / or binding half-life can be measured in the absence of the corresponding TCR(s) with the change(s) K D and / or binding half-life within 50%, or more preferably within 20%. As known to those skilled in the art, TCRs can be generated that incorporate changes in the variable domains without altering the affinity of the interaction with the GVYDGREHTV (SEQ ID NO: 34) HLA-A*02 complex compared to those described in detail above. In particular, such silent mutations can be incorporated into portions of the sequence known not to be directly involved in antigen binding (e.g., CDRs, or portions of CDRs that do not contact peptide antigens). Such subtle variants are encompassed within the scope of the present disclosure.

[0039] Phenotypically silent variants may contain one or more conservative substitutions and / or one or more tolerant substitutions. When measured under the same conditions (e.g., at 25°C and / or on the same SPR chip), the tolerant substitutions and conservative substitutions may result in a K of 100 for the GVYDGREHTV (SEQ ID NO: 34) HLA-A*02 complex.D and / or changes in binding half-life compared to the measured K of the corresponding TCR without said (one or more) conservative substitutions and / or tolerated substitutions D and / or within 50% of the binding half-life, or more preferably within 20%, even more preferably within 10%, provided that K D A change in will not result in an affinity less than (ie weaker than) 200 μM. Tolerated substitutions are those that do not fall within the definition of conservative as provided below, but are still phenotypically silent.

[0040] The TCRs of the present disclosure may comprise one or more conservative substitutions that have similar amino acid sequences and / or retain the same function (i.e., phenotypic silence as defined above). It is understood by those skilled in the art that various amino acids have similar properties and are therefore "conservative." One or more such amino acids of a protein, polypeptide, or peptide can often be replaced by one or more other such amino acids without eliminating the desired activity of the protein, polypeptide, or peptide.

[0041] Thus, the amino acids glycine, alanine, valine, leucine, and isoleucine can often be substituted for one another (amino acids with aliphatic side chains). Of these possible substitutions, glycine and alanine are preferred for substitution (because they have relatively short side chains), and valine, leucine, and isoleucine are preferred for substitution (because they have larger, hydrophobic aliphatic side chains). Other amino acids that can often be substituted for one another include: phenylalanine, tyrosine, and tryptophan (amino acids with aromatic side chains); lysine, arginine, and histidine (amino acids with basic side chains); aspartic acid and glutamic acid (amino acids with acidic side chains); asparagine and glutamine (amino acids with amide side chains); and cysteine and methionine (amino acids with sulfur-containing side chains). It should be understood that amino acid substitutions within the scope of the present disclosure can be performed using naturally occurring or non-naturally occurring amino acids. For example, it is contemplated herein that the methyl group on alanine can be replaced by an ethyl group, and / or that minor changes can occur in the peptide backbone. Regardless of whether natural or synthetic amino acids are used, it is preferred that only L-amino acids are present.

[0042] As known in the art, "identity" is as determined by comparing sequences, the relationship between two or more polypeptide sequences or two or more polynucleotide sequences. In the art, identity also refers to as determined by the matching between such sequence strings (string), the degree of sequence correlation between polypeptide or polynucleotide sequences, as the case may be. Although there are several methods to measure the identity between two polypeptides or two polynucleotide sequences, the method generally used to determine identity is incorporated into a computer program. Determine the preferred computer program for the identity between two sequences including, but not limited to, GCG program package (Devereux et al., Nucleic Acids Research, 12, 387 (1984)), BLASTP, BLASTN and FASTA (Altschul et al., J.Molec.Biol. 215, 403 (1990)).

[0043] Can use program (such as CLUSTAL program) to compare amino acid sequences.This program compares amino acid sequences and finds the best alignment by appropriately inserting spaces in any sequence.Can calculate the amino acid identity or similarity (identity plus the conservation of amino acid type) of the best alignment.The program similar to BLASTx will compare the longest continuous fragment (stretch) of similar sequences and assign a value to this matching (fit).Therefore, can obtain the comparison result that has several similarity regions, and each region has different scores.These two types of identity analysis are considered in the present disclosure.

[0044] The percent identity of two amino acid sequences or two nucleic acid sequences is determined by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced in the first sequence for optimal alignment with the sequences) and comparing the amino acid residues or nucleotides at corresponding positions. An "optimal alignment" is the alignment of the two sequences that yields the highest percent identity. The percent identity is determined by the number of identical amino acid residues or nucleotides in the compared sequences (i.e., % identity = number of identical positions / total number of positions x 100).

[0045] The determination of percent identity between two sequences can be accomplished using mathematical algorithms known to those skilled in the art. An example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, as modified in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. The N BLAST and XBLAST programs of Altschul et al. (1990) J. Mol. Biol. 215:403-410 have incorporated such algorithms. BLAST nucleotide searches can be performed with the NBLAST program with score = 100 and word length = 12 to obtain nucleotide sequences homologous to nucleic acid molecules. BLAST protein searches can be performed with the XBLAST program with score = 50 and word length = 3 to obtain amino acid sequences homologous to protein molecules, which are used for the purposes of the present disclosure. In order to obtain a gapped alignment for comparison purposes, gapped BLAST as described in Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402 can be utilized. Alternatively, PSI-Blast can be used to perform an iterative search (ibid.) for detecting long-range relationships between molecules. When utilizing BLAST, gapped BLAST, and PSI-Blast programs, the default parameters of each program (e.g., XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov. Another example of a mathematical algorithm for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). The ALIGN program (version 2.0), which is part of the CGC sequence alignment software package, has incorporated this algorithm. Other algorithms known in the art for sequence analysis include ADVANCE and ADAM as described in Torellis and Robotti (1994) Comput. Appl. Biosci., 10: 3-5; and FASTA as described in Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85: 2444-8. Within FASTA, ktup is a control option that sets the sensitivity and speed of the search.

[0046] Mutations (including conservative and tolerated substitutions, insertions, and deletions) can be introduced into the provided sequences using any suitable method, including, but not limited to, those based on the polymerase chain reaction (PCR), restriction endonuclease-based cloning, or ligation-independent cloning (LIC) procedures. These methods are described in detail in many standard molecular biology texts.

[0047] The TCR of the present disclosure can be an αβ heterodimer. The TCR of the present disclosure can be a single-chain format. The single-chain format includes, but is not limited to, Vα-L-Vβ, Vβ-L-Vα, Vα-Cα-L-Vβ, Vα-L-Vβ-Cβ or Vα-Cα-L-Vβ-Cβ type αβTCR polypeptides, wherein Vα and Vβ are TCRα variable regions and β variable regions, respectively, Cα and Cβ are TCRα constant regions and β constant regions, respectively, and L is a linker sequence (Weidanz et al., (1998) J Immunol Methods. December 1; 221(1-2): 59-76; Epel et al., (2002), Cancer Immunol Immunother. November; 51(10): 565-73; WO 2004 / 033685; WO9918129). One or both of these constant regions may be full-length, or they may be truncated as described above, and / or contain mutations. Due to natural polymorphism, the α chain extracellular constant region may have an asparagine (N) or lysine (K) residue at position 4. In certain embodiments, the single-chain TCR of the present invention may have an introduced disulfide bond between each constant domain, as described in WO 2004 / 033685. Single-chain TCRs are described in WO 2004 / 033685; WO 98 / 39482; WO 01 / 62908; Weidanz et al. (1998) J Immunol Methods 221(1-2):59-76; Hoo et al. (1992) Proc Natl Acad Sci US A 89(10):4759-4763; Schodin (1996) Mol Immunol 33(9):819-829.

[0048] The term "antibody" includes monoclonal antibodies (including full-length antibodies having an immunoglobulin Fc domain), antibody compositions with multiple epitope specificities, multispecific antibodies (e.g., bispecific antibodies, diabodies, and single-chain molecules), and antibody fragments (e.g., Fab, F(ab')2, and Fv). The terms "immunoglobulin" (Ig) and "antibody" are used interchangeably herein.

[0049] The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies are composed of 5 basic tetramer units and an additional polypeptide chain (called J chain) and contain 10 antigen binding sites, while IgA antibodies are composed of 2-5 basic 4-chain units that can be combined with J chains and polymerized to form a multivalent assembly (assemblage). In the case of IgG, the 4-chain unit is generally about 150,000 daltons. Each L chain is connected to the H chain by a covalent disulfide bond, and the two H chains are connected to each other by one or more disulfide bonds, specifically depending on the H chain isotype. Each H chain and L chain also has intrachain disulfide bridges distributed at regular intervals. Each H chain has a variable domain (V H ), followed by three constant domains each for the α and γ chains (C H ), and four C for the μ and ε isoforms H Each L chain has a variable domain (V L ), followed by a constant domain at its other end. L With the V H Comparison, and the C L The first constant domain of the heavy chain (C H 1) Alignment. Specific amino acid residues are believed to form an interface between the light and heavy chain variable domains. H and V L The pairing together forms a single antigen binding site. For the structure and properties of different classes of antibodies, see, for example, Basic and Clinical Immunology, 8th edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds.), Appleton & Lange, Norwalk, CT, 1994, page 71 and chapter 6. Based on the amino acid sequence of the constant domain, the L chains from any vertebrate species can be assigned to one of two distinct types (called kappa and lambda). Depending on the constant domain of the heavy chain (C H ) amino acid sequences, immunoglobulins can be assigned to different classes, or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, which have heavy chains designated α, δ, ε, γ, and μ, respectively. The γ and α classes are further divided into subclasses based on relatively minor differences in CH sequence and function. For example, humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgA2.

[0050] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable domain of the heavy chain and the variable domain of the light chain can be referred to as "VH" and "VL", respectively. These domains are generally the most variable parts of the antibody (relative to other antibodies of the same class) and contain the antigen-binding site.

[0051] The term "variable" refers to the fact that certain segments of the variable domain differ widely in sequence between antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for its specific antigen. However, variability is not evenly distributed across the entire span of the variable domain. Instead, it is concentrated in three segments called hypervariable regions (HVRs) in both the light chain variable domain and the heavy chain variable domain. The most highly conserved part of the variable domain is called the framework region (FR). The variable domains of native heavy and light chains each contain four FR regions, which primarily adopt a β-sheet conformation and are connected by three HVRs; this forms a loop connecting the β-sheet structure (and in some cases forming part of it). The HVRs in each chain are held together in close proximity by the FR region and, together with the HVRs from the other chain, participate in the formation of the antigen-binding site of the antibody (see Kabat et al., Sequences of Immunological Interest, 5th edition, National Institute of Health, Bethesda, MD (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

[0052] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies (i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation)). Monoclonal antibodies are highly specific, being directed against only a single antigenic site. Unlike polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against only a single epitope on the antigen. In addition to specificity, an advantage of monoclonal antibodies is that they are synthesized by hybridoma cultures and are not contaminated by other immunoglobulins. The modifier "monoclonal" indicates the characteristic of the antibody being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used according to the present disclosure can be made by a variety of techniques, including, for example, the hybridoma method (Kohler and Milstein., Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14(3):253-260 (1995), Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., In: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981)), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), phage display technology (see, e.g., Clackson et al., Nature, 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004)) and techniques for producing human antibodies or human-like antibodies in animals that have partial or complete human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., WO 1998 / 24893; WO 1996 / 34096; WO 1996 / 33735; WO 1991 / 10741; Jakobovits et al., Proc. Natl.Acad. Sci. USA 90:2551 (1993); Jakobovits et al., Nature 362:255-258 (1993); Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425 and 5,661,016; Marks et al., Bio / Technology 10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature 368:812-813 (1994); Fishwild et al., Nature Biotechnol. 14:845-851 (1996); Neuberger, Nature Biotechnol. 14:826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13:65-93 (1995)). .

[0053] The term "naked antibody" refers to an antibody that is not conjugated to a cytotoxic moiety or radiolabel.

[0054] The terms "full-length antibody," "intact antibody," or "whole antibody" are used interchangeably to refer to an antibody in substantially its entire form, as opposed to an antibody fragment. Specifically, whole antibodies include those having heavy and light chains and comprising an Fc domain. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. In some cases, intact antibodies may have one or more effector functions.

[0055] "Antibody fragments" comprise a portion of an intact antibody, preferably the antigen-binding and / or variable regions of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (see U.S. Patent No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10) :1057-1062

[1995] ); single-chain antibody molecules and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments (called "Fab" fragments) and a residual "Fc" fragment (the name reflects the ability to crystallize easily). The Fab fragment consists of the entire L chain and the variable region domain (V H ) and the first constant domain of one heavy chain (C H1) Composition. Each Fab fragment is monovalent for antigen binding, that is, it has a single antigen binding site. Pepsin treatment of an antibody produces a single F(ab')2 fragment, which roughly corresponds to two disulfide-linked Fab fragments with different antigen binding activities and still capable of cross-linking antigen. The Fab' fragment differs from the Fab fragment in that the C H The constant domains have additional residues at the carboxyl terminus, including one or more cysteines from the hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0056] The Fc fragment comprises the carboxyl-terminal portions of two H chains held together by disulfide bonds.The effector functions of an antibody are determined by sequences in the Fc domain, the region that is also recognized by Fc receptors (FcRs) present on certain cell types.

[0057] "Fv" is the smallest antibody fragment containing a complete antigen recognition and antigen binding site. This fragment consists of a dimer of a heavy chain variable region domain and a light chain variable region domain that are tightly non-covalently associated. The folding of these two domains produces six hypervariable loops (3 loops each from the H chain and the L chain) that contribute to the amino acid residues for antigen binding and confer antibody antigen binding specificity to the antibody. However, even a single variable domain (or only half an Fv comprising three HVRs specific for an antigen) still has the ability to recognize and bind to an antigen, although the affinity is lower than that of the entire binding site.

[0058] "Single-chain Fv" is also abbreviated as "sFv" or "scFv" and is a fragment comprising V sequences linked into a single polypeptide chain. H Antibody domains and V L Preferably, the sFv polypeptide chain further comprises a V H domain and V L A polypeptide linker between the domains that enables the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0059] The "functional fragment" of an antibody of the present invention comprises a portion of an intact antibody, generally comprising the antigen-binding region or variable region of an intact antibody or the Fc domain of an antibody that retains or has modified FcR binding ability. Examples of antibody fragments include linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0060] The term "diabodies" refers to small antibody fragments prepared by constructing sFv fragments (see previous paragraph) that H domain and V L There are short linkers (about 5-10 residues) between the domains to enable interchain pairing of the V domains (but not intrachain pairing), thereby forming a bivalent fragment (i.e., a fragment with two antigen-binding sites). Bispecific diabodies are heterodimers of two "crossover" sFv fragments, in which the V domains of the two antibodies are bound to each other. H domain and V L The domains are present on different polypeptide chains. Diabodies are described in more detail in, for example, EP 404,097; WO 93 / 11161; Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993).

[0061] The monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical or homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass (as well as fragments of such antibodies, so long as they exhibit the desired biological activity) (U.S. Patent No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies of interest herein include An antibody, wherein the antigen binding region of the antibody is derived from an antibody produced by, for example, immunizing macaques with an antigen of interest. As used herein, "humanized antibodies" are used as a subset of "chimeric antibodies."

[0062] In some embodiments, the humanized antibody is a chimeric antibody containing a minimal sequence derived from a non-human immunoglobulin (HIG). In one embodiment, a humanized antibody is a human immunoglobulin (HIG) in which the residues from the HVR (defined hereinafter) of a receptor antibody are replaced with residues from the HVR of a non-human species (donor antibody, such as mouse, rat, rabbit, or non-human primate) and have the desired specificity, affinity, or ability. In some cases, the framework ("FR") residues of the human immunoglobulin are replaced with corresponding non-human residues. In addition, the humanized antibody may include residues that are not present in either the receptor or the donor antibody. These modifications may be performed to further optimize antibody performance, such as binding affinity. In general, a humanized antibody will comprise substantially all of at least one (usually two) variable domains, wherein all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin sequence, and all or substantially all of the FR regions are those of a human immunoglobulin sequence, although these FR regions may contain one or more individual FR residue substitutions that improve antibody performance (such as binding affinity, isomerization, immunogenicity, etc.). The number of these amino acid substitutions in the FR is generally no more than 6 in the H chain and no more than 3 in the L chain. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically the Fc of a human immunoglobulin. For further details, see, e.g., Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992); Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994); and U.S. Pat. Nos. 6,982,321 and 7,087,409.

[0063] "Human antibody" refers to an antibody having an amino acid sequence that corresponds to an amino acid sequence produced by a human and / or that has been produced using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes humanized antibodies that comprise non-human antigen-binding residues. Human antibodies can be produced using various techniques known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Also useful for the preparation of human monoclonal antibodies are the methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.77 (1985); Boerner et al., J. Immunol., 147(1):86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5:368-74 (2001). Human antibodies can be produced by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been inactivated, e.g., an immunized xenogeneic mouse (referred to as XENOMOUSE). TM For human antibodies produced by human B cell hybridoma technology, see, for example, Li et al., Proc. Natl. Acad. Sci. USA, 103: 3557-3562 (2006).

[0064] As used herein, the term "hypervariable region", "HVR" or "HV" refers to a region in an antibody variable domain that is highly variable in sequence and / or forms a structurally defined loop. Typically, an antibody comprises six HVRs; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). In natural antibodies, H3 and L3 exhibit the highest diversity of the six HVRs, and H3 is believed to play a unique role, in particular, in conferring fine specificity to antibodies. See, e.g., Xu et al., Immunity 13:37-45 (2000); Johnson and Wu, in Methods in Molecular Biology 248:1-25 (Lo ed., Human Press, Totowa, NJ, 2003). Indeed, in the absence of light chains, naturally occurring camelid antibodies consisting only of heavy chains still have functionality and stability. See, eg, Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

[0065] The delineation of various HVRs is currently in use and is encompassed herein. The Kabat complementarity determining region (CDR) is based on sequence variability and is the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Chothia refers to the positioning of structural loops (Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987)). AbM HVR represents a compromise between Kabat HVR and Chothia structural loops and is used by Oxford Molecular's AbM antibody modeling software. The "contact" HVR is based on an analysis of available complex crystal structures. The residues from each of these HVRs are mentioned below.

[0066]

[0067]

[0068] An HVR may comprise an "extended HVR" as follows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in VL; and 26-35 (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in VH. For each of these definitions, the variable domain residues are numbered according to Kabat et al. (supra).

[0069] The expression "variable domain residues according to Kabat numbering" or "amino acid positions according to Kabat numbering" and variations thereof refer to the numbering system for heavy chain variable domains or light chain variable domains used in the compilation of antibodies in Kabat et al. (supra). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to shortening of, or insertions into, a FR or HVR of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat), and inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c, etc. according to Kabat). For a given antibody, the Kabat numbering of the residues can be determined by aligning the sequence of the antibody with the "standard" Kabat numbering sequence at a region of homology.

[0070] "Framework" or "FR" residues are those variable domain residues other than the HVR residues as herein defined.

[0071] A "human consensus framework" or "acceptor human framework" is a framework that represents the most frequently occurring amino acid residues in a selection of human immunoglobulin VL framework sequences or VH framework sequences. Typically, the selection of human immunoglobulin VL sequences or VH sequences is from a subset of variable domain sequences. Typically, the subset of sequences is a subset as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991). For VL, the subgroup may be subgroup κI, κII, κIII, or κIV as in Kabat et al. (supra). Additionally, for VH, the subgroup may be subgroup I, subgroup II, or subgroup III as in Kabat et al. (supra). Alternatively, the human consensus framework may be derived from the above subgroups, wherein the selection of specific residues (e.g., human framework residues) is based on their homology to the donor framework by aligning the donor framework sequence with a collection of various human framework sequences. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework can comprise the same amino acid sequence therefrom, or it can contain pre-existing amino acid sequence changes. In some embodiments, the number of pre-existing amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less.

[0072] An "amino acid modification" at a specified position, such as an amino acid modification of an Fc domain, refers to a substitution or deletion of the specified residue, or an insertion of at least one amino acid residue adjacent to the specified residue. An insertion "adjacent" to a specified residue refers to an insertion within one to two residues thereof. The insertion may be at the N-terminus or C-terminus of the specified residue. Preferred amino acid modifications herein are substitutions.

[0073] "Affinity matured" antibodies are antibodies that have alterations in one or more HVRs that result in an increase in the affinity of the antibody for the antigen compared to a parent antibody that does not have these alterations. In one embodiment, affinity matured antibodies have nanomolar or even picomolar affinities for the target antigen. Affinity matured antibodies are produced by methods known in the art. For example, Marks et al., Bio / Technology 10:779-783 (1992) describe affinity maturation by shuffling VH and VL domains. Random mutagenesis of HVR and / or framework residues is described by, e.g., Barbas et al., Proc Nat. Acad. Sci. USA 91:3809-3813 (1994); Schier et al., Gene 169:147-155 (1995); Yelton et al., J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al., J. Mol. Biol. 226:889-896 (1992).

[0074] As used herein, the term "specifically binds to ..." or "is specific for ..." refers to a measurable and repeatable interaction (e.g., binding) between a target and an antibody that can determine the presence of the target in the presence of a heterogeneous population of molecules (including biomolecules). For example, an antibody that specifically binds to a target (which may be an epitope) is one that binds to this target with greater affinity, avidity, more readily, and for a longer period of time than it binds to other targets. In one embodiment, the degree of binding of an antibody to an unrelated target is less than about 10% of the binding of the antibody to the target, as measured by, for example, a radioimmunoassay (RIA). In certain embodiments, the dissociation constant (Kd) of an antibody that specifically binds to a target is ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, or ≤0.1 nM. In certain embodiments, an antibody specifically binds to an epitope on the protein that is conserved between proteins from different species. In another embodiment, specific binding can include exclusive binding, but exclusive binding is not required.

[0075] " Fc domain " is used to define the C-terminal region of immunoglobulin heavy chain herein, including native sequence Fc domain and variant Fc domain. Although the boundaries of the Fc domain of immunoglobulin heavy chain may vary, human IgG heavy chain Fc domain is generally defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc domain can be, for example, removed during the production or purification process of the antibody, or removed by the nucleic acid of the heavy chain of the recombinantly engineered antibody. Therefore, the composition of the complete antibody can include an antibody population that removes all K447 residues, an antibody population that does not remove the K447 residue, and an antibody population with and without a mixture of antibodies with the K447 residue. Suitable native sequence Fc domains for antibodies of the present invention include human IgG1, IgG2 (IgG2A, IgG2B), IgG3 and IgG4.

[0076] "Fc receptor" or "FcR" describes a receptor that binds to the Fc domain of an antibody. A preferred FcR is a native sequence human FcR. Furthermore, a preferred FcR is one that binds to IgG antibodies (gamma receptors), and includes receptors of the FcγRI subclass, FcγRII subclass, and FcγRIII subclass, including allelic variants and alternatively spliced forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic region. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic region. (See M. Annu. Rev. Immunol. 15:203-234 (1997). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs (including those identified in the future) are also encompassed by the term "FcR" herein.

[0077] The term "Fc receptor" or "FcR" also includes the neonatal receptor FcRn, which is responsible for the transport of maternal IgG to the fetus. Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994). Methods for measuring binding to FcRn are known (see, e.g., Ghetie and Ward, Immunol. Today 18(12):592-8 (1997); Ghetie et al., Nature Biotechnology 15(7):637-40 (1997); Hinton et al., J. Biol. Chem. 279(8):6213-6 (2004); WO 2004 / 92219 (Hinton et al.). Binding to FcRn in vivo and serum half-life of high-affinity human FcRn binding polypeptides can be determined, for example, in transgenic mice or transfected human cell lines expressing human FcRn, or in primates administered polypeptides having variant Fc domains. WO 2004 / 42072 (Presta) describes antibody variants that have enhanced or diminished binding to FcRs. See also, eg, Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).

[0078] As used herein, the phrase "substantially reduced" or "substantially different" refers to a sufficiently high degree of difference between two values (generally, one associated with a molecule and the other associated with a reference / comparator molecule) that one of skill in the art would consider the difference between the two values to be statistically significant within the context of the biological characteristic measured by the value (e.g., Kd value). The difference between the two values is, for example, greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, and / or greater than about 50%, as a function of the value of the reference / comparator molecule.

[0079] As used herein, the term "substantially similar" or "substantially identical" means that there is a sufficiently high degree of similarity between two values (e.g., one associated with an antibody of the invention and the other associated with a reference / comparator antibody) that one of skill in the art would consider the difference between the two values to be of little or no biological and / or statistical significance within the context of the biological characteristic measured by the value (e.g., Kd value). The difference between the two values is, for example, less than about 50%, less than about 40%, less than about 30%, less than about 20%, and / or less than about 10%, as the value of the reference / comparator varies.

[0080] As used herein, "carrier" includes a pharmaceutically acceptable carrier, excipient, or stabilizer that is nontoxic to the cells or mammals to which it is exposed at the dosages and concentrations employed. Physiologically acceptable carriers are often aqueous pH buffered solutions. Examples of physiologically acceptable carriers include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextran; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN®. TM , polyethylene glycol (PEG) and PLURONICS TM .

[0081] “Package insert” means instructions customarily included in commercial packages of drugs that contain information customarily included in commercial packages of drugs concerning the indications, usage, dosage, administration, contraindications, other drugs to be combined with the packaged product, and / or warnings concerning the use of such drugs.

[0082] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a molecule" optionally includes a combination of two or more such molecules, and the like.

[0083] As used herein, the term "about" refers to the usual error range for each numerical value known to those skilled in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments for that value or parameter itself.

[0084] It is to be understood that aspects and embodiments of the present invention described herein include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

[0085] III. T cell receptors and fusion proteins

[0086] On the one hand, provided herein is a TCR fusion protein comprising a TCR that binds to a GVYDGREHTV (SEQ ID NO: 34) HLA-A*02 complex. In some embodiments, the TCR is a soluble TCR. In some embodiments, the TCR fusion protein comprises a T cell engagement domain and an antibody Fc domain fused or connected (e.g., covalently linked) to a protein expressed on the cell surface of a T cell. In some embodiments, the TCR is glycosylated, for example, glycosylated at a single N-linked glycosylation site. In some embodiments, for example, according to ANQVEQSPQSLIILEGKNVTLQCQYTVSPFSNLRWYKQDTGRGPV SLTILTFSENTKSNGRYTATLDADTKQSSLHITASQLSDSASYICVV NSAQGLYIPTFGRGTSLIVHP (SEQ ID NO: 7) numbering, the N-linked glycosylation site is at residue N18 of the α chain variable region.

[0087] In some embodiments, the TCR comprises a TCR α chain comprising an α chain variable region and a TCR β chain comprising a β chain variable region. In some embodiments, the α chain variable region comprises (i) a CDR1 comprising the amino acid sequence of VSPFSN (SEQ ID NO: 1), (ii) a CDR2 comprising the amino acid sequence of LTFSENT (SEQ ID NO: 2), and (iii) a CDR3 comprising the amino acid sequence of VVNSAQGLYIPTF (SEQ ID NO: 3), and / or the β chain variable region comprises (i) a CDR1 comprising the amino acid sequence of LDHEN (SEQ ID NO: 4), (ii) a CDR2 comprising the amino acid sequence of SRFATG (SEQ ID NO: 5), and (iii) a CDR3 comprising the amino acid sequence of ASSSDQNSGDPYEQYF (SEQ ID NO: 6).

[0088] As is well known in the art, TCRs can be post-translationally modified. Glycosylation is a modification that involves covalently attaching oligosaccharide moieties to defined amino acids in the TCR chain. For example, asparagine residues or serine / threonine residues are well-known locations for oligosaccharide attachment. The glycosylation state of a particular protein depends on a variety of factors, including protein sequence, protein conformation, and the availability of certain enzymes. In addition, the glycosylation state (i.e., oligosaccharide type, covalent linkage, and total number of attachments) can affect protein function. Therefore, when producing recombinant proteins, controlling glycosylation is generally desired. Controlled glycosylation has been used to improve antibody-based therapies. (Jefferis et al., (2009) Nat Rev Drug Discov March; 8(3): 226-34.). For the soluble TCRs of the present disclosure, glycosylation can be controlled in vivo, for example, by using specific cell lines, or in vitro by chemical modification. Such modifications are desirable because glycosylation can improve pharmacokinetics, reduce immunogenicity, and more closely mimic the native human protein (Sinclair and Elliott, (2005) Pharm Sci. Aug;94(8):1626-35).

[0089] In some embodiments, the TCR or TCR fusion protein of the present disclosure is glycosylated at a single N-linked glycosylation site. In some embodiments, the N-linked glycosylation site is at residue N18 of the α chain variable region, for example, as numbered according to ANQVEQSPQSLIILEGKNVTLQCQYTVSPFSNLRWYKQDTGRGPV SLTILTFSENTKSNGRYTATLDADTKQSSLHITASQLSDSASYICVV NSAQGLYIPTFGRGTSLIVHP (SEQ ID NO: 7). In some embodiments, the TCR fusion protein of the present disclosure comprises a TCR that is glycosylated at a single N-linked glycosylation site, wherein the N-linked glycosylation site is at residue N18 of the α chain variable region, as numbered according to SEQ ID NO: 7. Advantageously, the present disclosure demonstrates that TCR fusion proteins with this single glycosylation site have better manufacturability (e.g., protein production yield, tolerance to heat stress and aggregation) and in vivo pharmacokinetics (e.g., half-life) compared to other glycosylated or unglycosylated variants, in addition to retaining affinity for peptide:MHC binding and potency for target cell killing.

[0090] In some embodiments, the TCR or TCR fusion protein of the present disclosure comprises an amino acid replacement at each potential N-glycosylation site except residue N18. For example, in some embodiments, the TCR or TCR fusion protein of the present disclosure comprises an amino acid replacement at one or more of the following residues: residue N24 of the α chain variable region, numbered according to SEQ ID NO: 32; residues N33, N67, and N78 of the α chain constant region, numbered according to SEQ ID NO: 10; residue N84 of the β chain variable region, numbered according to SEQ ID NO: 33; and residue N70 of the β chain constant region, numbered according to SEQ ID NO: 15. In some embodiments, the TCR or TCR fusion protein of the present disclosure comprises an amino acid replacement at all of the following residues: residue N24 of the α chain variable region, numbered according to SEQ ID NO: 32; residues N33, N67, and N78 of the α chain constant region, numbered according to SEQ ID NO: 10; residue N84 of the β chain variable region, numbered according to SEQ ID NO: 33; and residue N70 of the β chain constant region, numbered according to SEQ ID NO: 15.

[0091] In some embodiments, the amino acid substitutions are asparagine to a non-glycosylated amino acid. In some embodiments, the amino acid substitutions are asparagine to glutamine (N→Q).

[0092] In some embodiments, the TCR or TCR fusion protein of the present disclosure comprises one or more of the following amino acid substitutions: N24Q in the α chain variable region according to SEQ ID NO: 32; N33Q, N67Q, and N78Q in the α chain constant region according to SEQ ID NO: 10; N84Q in the β chain variable region according to SEQ ID NO: 33; and N70Q in the β chain constant region according to SEQ ID NO: 15. In some embodiments, the TCR or TCR fusion protein of the present disclosure comprises all of the following amino acid substitutions: N24Q in the α chain variable region according to SEQ ID NO: 32; N33Q, N67Q, and N78Q in the α chain constant region according to SEQ ID NO: 10; N84Q in the β chain variable region according to SEQ ID NO: 33; and N70Q in the β chain constant region according to SEQ ID NO: 15.

[0093] The αβ heterodimeric TCRs of the present disclosure typically comprise an α chain TRAC constant domain sequence and / or a β chain TRBC1 or TRBC2 constant domain sequence. The α chain constant domain sequence and the β chain constant domain sequence can be modified by truncation or substitution to delete the native disulfide bond between Cys4 of exon 2 of TRAC and Cys2 of exon 2 of TRBC1 or TRBC2. The α chain constant domain sequence and / or the β chain constant domain sequence can be modified by replacing Thr48 of TRAC and Ser57 of TRBC1 or TRBC2 with cysteine residues, which form a disulfide bond between the α constant domain and the β constant domain of the TCR. TRBC1 or TRBC2 can additionally comprise a cysteine to alanine mutation at position 75 of the constant domain and an asparagine to aspartic acid mutation at position 89 of the constant domain. The constant domain can additionally or alternatively contain further mutations, substitutions, or deletions relative to the native TRAC and / or TRBC1 / 2 sequence. The terms TRAC and TRBC1 / 2 encompass naturally occurring polymorphic variations, such as N to K at position 4 of TRAC (Bragado et al. Int Immunol. 1994 Feb;6(2):223-30).

[0094] As will be apparent to those skilled in the art, the provided sequences can be truncated at the C-terminus and / or N-terminus by 1, 2, 3, 4, 5 or more residues without substantially affecting the binding characteristics of the TCR. All such minor variations are encompassed in the present disclosure.

[0095] The constant domains of wild-type or insoluble TCRs can be full-length, or can be truncated and / or mutated to generate soluble TCRs. In either case, cysteine substitutions can be introduced into the TRAC and TRBC regions to allow for the formation of non-native interchain disulfide bonds. Suitable locations for the positioning of such cysteine substitutions are described in WO03020763.

[0096] In some embodiments, TCR or TCR fusion protein of the present disclosure are included in one or more engineered cysteine residues in the α chain constant region and / or the β chain constant region to form a non-natural disulfide bond between the α chain and the β chain. In certain embodiments, the single-chain TCR of the present disclosure may have an introduced disulfide bond between the residues of each constant domain, as described in WO2004 / 033685. Single-chain TCR is described in WO2004 / 033685; WO98 / 39482; WO01 / 62908; Weidanz et al. (1998) J Immunol Methods 221(1-2):59-76; Hoo et al. (1992) Proc Natl Acad Sci US A89(10):4759-4763; Schodin (1996) Mol Immunol 33(9):819-829. In some embodiments, a TCR or TCR fusion protein of the present disclosure comprises a cysteine residue at position 57 of the beta chain constant region, according to SEQ ID NO: 15.

[0097] In some embodiments, a TCR or TCR fusion protein of the present disclosure comprises an alpha chain variable region comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of ANQVEQSPQSLIILEGKNVTLQCQYTVSPFSNLRWYKQDTGRGPV SLTILTFSENTKSNGRYTATLDADTKQSSLHITASQLSDSASYICVVNSAQGLYIPTFGRGTSLIVHP (SEQ ID NO: 7). In some embodiments, a TCR or TCR fusion protein of the present disclosure comprises an alpha chain variable region comprising the amino acid sequence of SEQ ID NO: 7.

[0098] In some embodiments, a TCR or TCR fusion protein of the present disclosure comprises a beta chain variable region comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of DVKVTQSSRYLVKRTGEKVFLECVQDLDHENMFWYRQDPGLGLR LIYFSRFATGKEKGDIPEGYSVSREKKERFSLILESASTQQTSMYLCASSSDQNSGDPYEQYFGPGTRLTVT (SEQ ID NO: 13). In some embodiments, a TCR or TCR fusion protein of the present disclosure comprises a beta chain variable region comprising the amino acid sequence of SEQ ID NO: 13. In some embodiments, a TCR or TCR fusion protein of the present disclosure comprises an alpha chain variable region comprising the amino acid sequence of SEQ ID NO: 7 and a beta chain variable region comprising the amino acid sequence of SEQ ID NO: 13.

[0099] In some embodiments, the TCR or TCR fusion protein of the present disclosure comprises a TCR α chain comprising an α chain variable region and an α chain constant region of the present disclosure. In some embodiments, the TCR or TCR fusion protein of the present disclosure comprises an α chain constant region comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence of YIQKPDPAVYQLRDSKSSDKSVCLFTDFDSQTQVSQSKDSDVYITD KCVLDMRSMDFKSNSAVAWSQKSDFACANAFQNSIIPEDT (SEQ ID NO: 9). In some embodiments, the TCR or TCR fusion protein of the present disclosure comprises an α chain constant region comprising an amino acid sequence of SEQ ID NO: 9.

[0100] In some embodiments, the TCR or TCR fusion protein of the present disclosure comprises a TCR β chain comprising a β chain variable region and a β chain constant region of the present disclosure. In some embodiments, the TCR or TCR fusion protein of the present disclosure comprises a β chain constant region comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence of EDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALQDSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRAD (SEQ ID NO: 14). In some embodiments, the TCR or TCR fusion protein of the present disclosure comprises a β chain constant region comprising an amino acid sequence of SEQ ID NO: 14. In some embodiments, a TCR or TCR fusion protein of the present disclosure comprises an α chain constant region comprising the amino acid sequence of SEQ ID NO:9 and a β chain constant region comprising the amino acid sequence of SEQ ID NO:14.

[0101] In some embodiments, the TCR or TCR fusion protein of the present disclosure comprises: an α chain comprising an α chain variable region comprising the amino acid sequence of SEQ ID NO: 7 and an α chain constant region comprising the amino acid sequence of SEQ ID NO: 9; and a β chain comprising a β chain variable region comprising the amino acid sequence of SEQ ID NO: 13 and a β chain constant region comprising the amino acid sequence of SEQ ID NO: 14.

[0102] In some embodiments, a TCR or TCR fusion protein of the present disclosure comprises an alpha chain comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of ANQVEQSPQSLIILEGKNVTLQCQYTVSPFSNLRWYKQDTGRGPVSLTILTFSENTKSNGRYTATLDADTKQSSLHITASQLSDSASYICVVNSAQGLYIPTFGRGTSLIVHPYIQKPDPAVYQLRDSKSSDKSVCLFTDFDSQTQVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSQKSDFACANAFQNSIIPEDT (SEQ ID NO: 11). In some embodiments, a TCR or TCR fusion protein of the present disclosure comprises an alpha chain comprising the amino acid sequence of SEQ ID NO: 11.

[0103] In some embodiments, a TCR or TCR fusion protein of the present disclosure comprises a beta chain comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of DVKVTQSSRYLVKRTGEKVFLECVQDLDHENMFWYRQDPGLGLRLIYFSRFATGKEKGDIPEGYSVSREKKERFSLILESASTQQTSMYLCASSSDQNSGDPYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALQDSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRAD (SEQ ID NO: 16). In some embodiments, the TCR or TCR fusion protein of the present disclosure comprises a beta chain comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, the TCR or TCR fusion protein of the present disclosure comprises an alpha chain comprising the amino acid sequence of SEQ ID NO: 11 and a beta chain comprising the amino acid sequence of SEQ ID NO: 16.

[0104] In some embodiments, the TCR fusion protein of the present disclosure comprises a T cell engagement domain that binds to a protein expressed on the cell surface of a T cell. In some embodiments, the T cell engagement domain binds to a protein expressed on the cell surface of a T cell and recruits the T cell to a cell expressing the GVYDGREHTV (SEQ ID NO: 34) HLA-A*02 complex. In some embodiments, the T cell engagement domain binds to a protein expressed on the cell surface of a T cell and activates the T cell, for example, by binding to a protein expressed on the cell surface. In some embodiments, the protein expressed on the cell surface of the T cell is a cell surface receptor. In some embodiments, the protein expressed on the cell surface of the T cell is a human CD3 polypeptide.

[0105] In some embodiments, the T cell engaging domain comprises an antibody antigen binding domain. In some embodiments, the antibody antigen binding domain binds to a cell surface receptor expressed by a T cell. In some embodiments, the antibody antigen binding domain binds to a cell surface receptor expressed by a T cell and causes activation of the T cell. In some embodiments, the T cell engaging domain is a portion of a single chain variable fragment (scFv) or comprises an scFv. In some embodiments, the scFv is an anti-CD3 scFv. Other single chain antibody fragment formats are known in the art.

[0106] In some embodiments, the scFv is a U28 variant anti-CD3 scFv. In some embodiments, the scFv comprises the amino acid sequence of AIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYAMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTFSVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSS (SEQ ID NO: 17). In some embodiments, the scFv comprises the amino acid sequence of AIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSS (SEQ ID NO: 35).

[0107] In some embodiments, the T cell engaging domain (e.g., scFv) that binds to a protein expressed on the cell surface of a T cell is covalently linked to the TCR via a linker. In some embodiments, the linker is a Gly-Ser linker. In some embodiments, the linker comprises an amino acid sequence selected from SEQ ID NOs: 18-25.

[0108] In some embodiments, the C-terminus of the T cell engaging domain (e.g., the scFv) that binds to a protein expressed on the cell surface of a T cell is covalently linked to the N-terminus of the TCR β chain variable domain via a linker of the present disclosure.

[0109] In some embodiments, the N-terminus of the antibody Fc domain is covalently linked to the C-terminus of the TCR alpha chain constant domain via a hinge sequence. In some embodiments, the hinge sequence comprises the amino acid sequence of DKTHTCPP (SEQ ID NO: 31) or DKTHTCPPC (SEQ ID NO: 36).

[0110] In some embodiments, the TCR fusion proteins of the present disclosure comprise an antibody Fc domain, e.g., a human antibody Fc domain. In some embodiments, the TCR fusion proteins of the present disclosure comprise human IgG1, human IgG2, or human IgG4 Fc domains. Advantageously, the present disclosure describes TCR fusion proteins having an Fc domain fused to an α chain or β chain that have substantially improved pharmacokinetics compared to similar molecules without Fc expressed from E. coli or CHO cells.

[0111] In some embodiments, the antibody Fc domain comprises one or more mutations that weaken the effector function of the Fc domain. Exemplary effector functions include, but are not limited to, complement dependent cytotoxicity (CDC) and / or antibody dependent cellular cytotoxicity (ADCC). In an exemplary embodiment, the modification that weakens the effector function is a modification that changes the glycosylation pattern of the Fc domain, for example, a modification that produces an unglycosylated Fc domain. In an exemplary embodiment, the modification that weakens the effector function is a modification that does not change the glycosylation pattern of the Fc domain. In certain embodiments, the modification that weakens the effector function reduces or eliminates binding to human effector cells, binding to one or more Fc receptors, and / or binding to cells expressing Fc receptors. In an exemplary embodiment, the Fc variants described herein comprise N297G or N297A modifications in the Fc domain of human IgG1. In an exemplary embodiment, the Fc variants described herein comprise the following modifications: L234A, L235A, and P329G modifications in the Fc domain of human IgG1 that result in weakened effector function. In some embodiments, the antibody Fc domain is a human IgG1 Fc domain comprising a mutation at residue N297 according to the EU index. For example, in some embodiments, the mutation is an N297G substitution. Other suitable mutations (e.g., at residue N297) are known to those skilled in the art.

[0112] In various embodiments, an Fc variant with reduced effector function refers to an Fc variant that has an effector function (e.g., CDC, ADCC, and / or binding activity to FcR, etc.) reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or more compared to the effector function achieved by a wild-type Fc domain (e.g., an Fc domain that does not have a mutation that reduces effector function, although it may have other mutations). In certain embodiments, an Fc variant with reduced effector function refers to an Fc domain that eliminates all detectable effector function compared to a wild-type Fc domain. Assays for measuring effector function are known in the art and are described below.

[0113] In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / depletion of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the Fc domain or fusion protein lacks FcγR binding (and therefore may lack ADCC activity), but retains FcRn binding ability. The main cells (NK cells) used to mediate ADCC express only FcγRIII, while monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-492 (1991). Non-limiting examples of in vitro assays for evaluating ADCC activity of a molecule of interest are described in U.S. Pat. Nos. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays can be employed (see, e.g., ACTI for flow cytometry). TM Non-radioactive cytotoxicity assay (Cell Technology, Inc., Mountain View, CA) and CytoTox Non-radioactive cytotoxicity assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or in addition, ADCC activity of the molecule of interest can be assessed in vivo, for example, in an animal model such as that disclosed in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay can also be performed to confirm that the antibody is unable to bind to C1q and therefore lacks CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996); Cragg, MS et al., Blood 101: 1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103: 2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determination can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12): 1759-1769 (2006)). In certain embodiments, the Fc variants described herein comprise modifications to the Fc domain that reduce effector function, as described in Strohl, Current Opinion in Biotechnology, 20; 685-691 (2009).

[0114] In some embodiments, the antibody Fc domain is a human IgG1 Fc domain comprising one or more mutations at residues E233, L234, L235, and / or G236, according to the EU index numbering. For example, in some embodiments, the antibody Fc domain is a human IgG1 Fc domain comprising substitutions N297G, E233P, L234V, L235A, and a deletion at G236, according to the EU index numbering.

[0115] In some embodiments, the antibody Fc domain is a human IgG1 Fc domain comprising one or more mutations at residues L234, L235, and P329, according to the EU index numbering. For example, in some embodiments, the antibody Fc domain is a human IgG1 Fc domain comprising substitutions L234A, L235A, and P329G, according to the EU index numbering.

[0116] In some embodiments, the antibody Fc domain is fused to the TCR of the present disclosure via a hinge sequence. In some embodiments, the hinge sequence comprises the amino acid sequence of DKTHTCPP (SEQ ID NO: 31) or DKTHTCPPC (SEQ ID NO: 36).

[0117] In some embodiments, the TCR fusion proteins of the present disclosure comprise two antibody Fc domains. In some embodiments, one of the two antibody Fc domains is fused or linked (e.g., covalently linked) to the TCR of the present disclosure. Figure 1 B discloses a conformation wherein, according to some embodiments, one of the two antibody Fc domains is connected to a TCR α chain (e.g., via an α chain constant domain). In some embodiments, the two antibody Fc domains are associated with each other, e.g., by one or more covalent linkages and / or one or more amino acid substitutions on one or both of these antibody Fc domains that promote heterodimerization.

[0118] In some embodiments, the first antibody Fc domain is fused to the TCR via a first hinge sequence, and the second hinge sequence is connected to the N-terminus of the second antibody Fc domain. In some embodiments, the first hinge sequence and the second hinge sequence are connected via one or more interchain disulfide bonds between the first hinge sequence and the second hinge sequence. In some embodiments, the first antibody Fc domain and the second antibody Fc domain further comprise a hinge sequence, and the first hinge sequence and the second hinge sequence are connected via one or more interchain disulfide bonds between the first hinge sequence and the second hinge sequence. In some embodiments, the first hinge sequence and the second hinge sequence both comprise the amino acid sequence of DKTHTCPP (SEQ ID NO: 31) or DKTHTCPPC (SEQ ID NO: 36). In some embodiments, the hinge sequence of the present disclosure is an antibody hinge sequence, for example, a sequence from an antibody hinge region.

[0119] In some embodiments, one of the first and second antibody Fc domains comprises one or more knob-forming mutations, and the other of the first and second antibody Fc domains comprises one or more corresponding hole-forming mutations, to promote heterodimerization of the antibody Fc domains.

[0120] In some embodiments, heterodimerization of two antibody Fc domains is facilitated by "knob-in-hole" engineering. For example, two polypeptides comprising antibody Fc domains can be assembled in vitro into a TCR fusion protein, wherein the first antibody Fc domain comprises amino acid modifications in its CH3 domain that form a protrusion, and the second antibody Fc domain comprises amino acid modifications in its CH3 domain that form a cavity. The protrusion can be positioned in the cavity, thereby forming the TCR fusion protein upon assembly.

[0121] In this method, two polypeptides comprising an antibody Fc domain each comprise an interface. The interface of one polypeptide interacts with the corresponding interface of another polypeptide, thereby allowing the two polypeptides to associate. These interfaces can be engineered so that a "knob" or "protrusion" (these terms can be used interchangeably herein) located in the interface of one polypeptide corresponds to a "hole" or "cavity" (these terms can be used interchangeably herein) located in the interface of another polypeptide. In some embodiments, the hole is the same or similar in size to the knob and is suitably positioned so that when the two interfaces interact, the knob of one interface can be positioned in the corresponding hole of the other interface. Without wishing to be bound by theory, this is believed to stabilize heteromultimers and facilitate the formation of heteromultimers rather than other species (e.g., homomultimers). In some embodiments, this method can be used to promote heteromultimerization of two different polypeptides, promoting the association of the two antibody Fc domains.

[0122] In some embodiments, a knob can be constructed by replacing a small amino acid side chain with a larger side chain. In some embodiments, a hole can be constructed by replacing a large amino acid side chain with a smaller side chain. The knob or hole can be present in the original interface, or they can be introduced synthetically. For example, the knob or hole can be introduced synthetically by altering the nucleic acid sequence encoding the interface to replace at least one "original" amino acid residue with at least one "import" amino acid residue. Methods for altering nucleic acid sequences can include standard molecular biology techniques well known in the art. The side chain volumes of various amino acid residues are shown in the table below. In some embodiments, the original residue has a smaller side chain volume (e.g., alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine), while the import residue used to form the knob is a naturally occurring amino acid and can include arginine, phenylalanine, tyrosine, and tryptophan. In some embodiments, the original residue has a larger side chain volume (e.g., arginine, phenylalanine, tyrosine, and tryptophan), while the import residue used to form the hole is a naturally occurring amino acid and can include alanine, serine, threonine, and valine.

[0123] Table B. Properties of amino acid residues

[0124]

[0125]

[0126] a The molecular weight of the amino acid minus the molecular weight of water. Values are from Handbook of Chemistry and Physics, 43rd edition. Cleveland, Chemical Rubber Publishing Co., 1961.

[0127] b Values are from AA Zamyatnin, Prog. Biophys. Mol. Biol. 24: 107-123, 1972.

[0128] c The values are from C. Chothia, J. Mol. Biol. 105: 1-14, 1975. The accessible surface area in this reference Figure 6-Figure 2 0.

[0129] In some embodiments, the original residues for forming the knob or hole are identified based on the three-dimensional structure of the heteromultimer. Techniques known in the art for obtaining three-dimensional structures may include X-ray crystallography and NMR. In some embodiments, the interface is the CH3 domain of an immunoglobulin constant domain. In these embodiments, the CH3 / CH3 interface of human IgG1 involves sixteen residues located on four antiparallel beta strands on each domain. Without wishing to be bound by theory, the mutated residues are preferably located on the two central antiparallel beta strands to minimize the risk that the knob can be accommodated by the surrounding solvent rather than the compensatory hole in the partner CH3 domain. In some embodiments, the mutations that form the corresponding knob and hole in the two immunoglobulin polypeptides correspond to one or more pairings provided in the following table.

[0130] Table C. Exemplary groups of corresponding knob-forming mutations and hole-forming mutations

[0131]

[0132]

[0133] Mutations are named as follows: the original residue, followed by the position using the Kabat numbering system, and then the input residue (all residues are given in single-letter amino acid code). Multiple mutations are separated by colons.

[0134] In some embodiments, the antibody Fc domain comprises a CH3 domain comprising one or more amino acid substitutions listed in Table C above. In some embodiments, the TCR fusion protein comprises a first antibody Fc domain comprising a CH3 domain comprising one or more amino acid substitutions listed in the left column of Table C, and a second antibody Fc domain comprising a CH3 domain comprising one or more corresponding amino acid substitutions listed in the right column of Table C.

[0135] For example, in some embodiments, one of the first and second antibody Fc domains comprises a T366W substitution, and the other of the first and second antibody Fc domains comprises a T366S, L368A, Y407V substitution, as numbered by the EU index.

[0136] In some embodiments, one of the antibody Fc domains comprises the amino acid sequence of SEQ ID NO: 27, and the other of the antibody Fc domains comprises the amino acid sequence of SEQ ID NO: 26. In some embodiments, the first antibody Fc domain is covalently linked to the TCR and comprises the amino acid sequence of SEQ ID NO: 27, and the second antibody Fc domain comprises the amino acid sequence of SEQ ID NO: 26.

[0137] Following mutation of the DNA as discussed above, polynucleotides encoding modified antibody Fc domains having one or more corresponding knob-forming mutations or hole-forming mutations can be expressed and purified by standard recombinant techniques and cell systems known in the art. See, e.g., U.S. Patent Nos. 5,731,168; 5,807,706; 5,821,333; 7,642,228; 7,695,936; 8,216,805; U.S. Publication No. 2013 / 0089553; and Spiess et al., Nature Biotechnology 31:753-758, 2013. Polypeptides containing antibody Fc domains with corresponding knobs and holes can be expressed in co-cultured host cells and purified together as heteromultimers, or they can be expressed in a single culture, purified separately, and assembled in vitro. Standard techniques known in the art that allow for the measurement of the abundance of homo- and heteromultimeric species can include size exclusion chromatography. In some embodiments, the polypeptide of each modification is expressed separately using standard recombinant technology, and they can be assembled together in vitro.Assembling can be for example realized by following: purifying the polypeptide of every kind of modification, mixing and hatching together with equal mass, reducing disulfide bonds (for example, by treating with dithiothreitol), concentrating, and reoxidizing these polypeptides.The TCR fusion protein formed can be purified using standard techniques (including cation exchange chromatography) and measured using standard techniques (including size exclusion chromatography).For a more detailed description of these methods, see Speiss et al., Nat Biotechnol 31:753-8,2013.In some embodiments, the polypeptide comprising the modified antibody Fc domains can be expressed separately in CHO cells, and assembled in vitro using the above method.

[0138] In some embodiments, the TCR fusion protein of the present disclosure comprises: a first polypeptide comprising, from N-terminus to C-terminus: the TCR α chain variable region, the α chain constant region, the first hinge sequence, and the first antibody Fc domain; a second polypeptide comprising, from N-terminus to C-terminus: a single-chain variable fragment (scFv) that binds to human CD3 expressed on the cell surface of T cells, a linker, the β chain variable region, and the β chain constant region; and a third polypeptide comprising, from N-terminus to C-terminus: a second hinge sequence and a second antibody Fc domain. Advantageously, the present disclosure describes this TCR fusion protein format (see, e.g., Figure 1 B) had the most favorable pharmacokinetic properties and the highest activity (ie, potency and selectivity) in the formats tested.

[0139] In some embodiments, the first polypeptide and the second polypeptide are linked by one or more disulfide bonds between the α chain constant region and the β chain constant region, e.g., as described herein.

[0140] In some embodiments, the first polypeptide and the third polypeptide are linked by one or more interchain disulfide bonds between the first and second hinge sequences and / or one or more corresponding knob-forming and hole-forming mutations on the antibody Fc domains.

[0141] In some embodiments, the TCR fusion protein of the present disclosure comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 29, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 30, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 28.

[0142] Further provided herein are polynucleotides encoding any of the TCRs and TCR fusion proteins disclosed herein. For example, in some embodiments, the disclosure provides a polynucleotide kit comprising one, two or three polynucleotides encoding one, two or three polypeptides of the disclosure. In some embodiments, the disclosure provides a polynucleotide kit comprising a first polynucleotide encoding a first polypeptide having an amino acid sequence of SEQ ID NO: 29, a second polynucleotide encoding a second polypeptide having an amino acid sequence of SEQ ID NO: 30, and a third polynucleotide encoding a third polypeptide having an amino acid sequence of SEQ ID NO: 28.

[0143] Further provided herein is a vector (e.g., an expression vector) comprising any one of the polynucleotides of the present disclosure. In some embodiments, the vector of the present disclosure comprises polynucleotides encoding one, two or three (e.g., all) polypeptides of the TCR fusion protein of the present disclosure. For example, in some embodiments, the vector comprises a first polynucleotide encoding a first polypeptide containing an amino acid sequence of SEQ ID NO: 29, a second polynucleotide encoding a second polypeptide containing an amino acid sequence of SEQ ID NO: 30, and a third polynucleotide encoding a third polypeptide containing an amino acid sequence of SEQ ID NO: 28. In some embodiments, the vector encodes a first polypeptide containing an amino acid sequence of SEQ ID NO: 29, a second polypeptide containing an amino acid sequence of SEQ ID NO: 30, and a third polypeptide containing an amino acid sequence of SEQ ID NO: 28. Further provided herein is a kit of a vector (e.g., an expression vector) comprising: a first vector comprising a first polynucleotide encoding a first polypeptide containing an amino acid sequence of SEQ ID NO: 29; a second vector comprising a second polynucleotide encoding a second polypeptide containing an amino acid sequence of SEQ ID NO: 30; and a third vector comprising a third polynucleotide encoding a third polypeptide containing an amino acid sequence of SEQ ID NO: 28.

[0144] For the recombinant production of TCR fusion proteins, nucleic acids encoding the TCR fusion proteins (e.g., as described above) are isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids can be easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the polypeptide chains of the TCR fusion proteins) or produced by recombinant methods or obtained by chemical synthesis.

[0145] Further provided herein is a host cell comprising any one of the polynucleotides and / or vectors disclosed herein. Suitable host cells for cloning or expressing the polynucleotides and / or vectors disclosed herein are known in the art. Suitable host cells for expressing (glycosylated) proteins are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Many baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfecting Spodoptera frugiperda cells. Plant cell cultures can also be used as hosts. See, for example, US 5,959,177, US 6,040,498, US 6,420,548, US 7,125,978 and US 6,417,429 (describing PLANTIBODIES for producing antibodies in transgenic plants). TMTechnology). Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 cell line (COS-7) transformed by SV40; human embryonic kidney cell lines (such as 293 or 293T cells as described in, for example, Graham, FL et al., J. Gen Virol. 36 (1977) 59-74); baby hamster kidney cells (BHK); mouse Sertoli cells (such as TM4 cells as described in, for example, Mather, JP, Biol. Reprod. 23 (1980) 243-252); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (HepG2); mouse mammary tumor (MMT) 060562); TRI cells (as described, for example, in Mather, JP et al., Annals N.Y. Acad. Sci. 383 (1982) 44-68); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980) 4216-4220); and myeloma cell lines, such as Y0, NS0, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268. In one aspect, the host cell is a eukaryotic cell, e.g., a Chinese hamster ovary (CHO) cell or a lymphoid cell (e.g., Y0, NS0, Sp20 cell).

[0146] Further provided herein are methods for producing any of the TCR fusion proteins disclosed herein. In some embodiments, these methods include culturing a host cell of the present disclosure under conditions suitable for producing the TCR fusion protein. In some embodiments, these methods further include recovering the TCR fusion protein from the host cell.

[0147] IV. Methods and Uses

[0148] Certain aspects of the present disclosure relate to methods for treating cancer. In some embodiments, these methods comprise administering an effective amount of a TCR fusion protein or pharmaceutical composition of the present disclosure to an individual. In some embodiments, the individual is human.

[0149] In some embodiments, the individual has a cancer that expresses MAGE-A4.

[0150] For example, the cancer or tumor can be breast cancer, esophageal cancer, stomach cancer (e.g., gastric cancer), head and neck cancer, lung cancer, ovarian cancer, or bladder cancer. The cancer or tumor can express MAGE A4 and / or can be a solid tumor. In some embodiments, the cancer or tumor is a synovial sarcoma. In some embodiments, the cancer or tumor has a squamous cell histology, i.e., is a squamous cell carcinoma or tumor.

[0151] In some embodiments, the individual is of the HLA-A*02 subtype.

[0152] The present disclosure further includes a pharmaceutical composition comprising a TCR or TCR fusion protein of the present disclosure and a pharmaceutically acceptable carrier. For administration to a patient, the TCR and TCR-anti-CD3 fusion molecules of the present disclosure can be provided in a pharmaceutical composition together with one or more pharmaceutically acceptable carriers or excipients. The therapeutic TCR or imaging TCR or its cells according to the present disclosure will typically be supplied as part of a sterile pharmaceutical composition (which will typically include a pharmaceutically acceptable carrier). This pharmaceutical composition can be in any suitable form (depending on the desired method for administering it to the patient). It can be provided in unit dose form, generally in a sealed container, and can be provided as part of a test kit. Such a test kit will typically (although not necessarily) include instructions for use. It can include multiple such unit dose forms.

[0153] In some embodiments, the TCR fusion protein or pharmaceutical composition is administered intravenously or by intratumoral injection. The TCR fusion protein or pharmaceutical composition can be suitable for administration by any suitable route, such as parenteral (including subcutaneous, intramuscular or intravenous), enteral (including oral or rectal), inhalation or intranasal. Such compositions can be prepared by any method known in the pharmaceutical field, for example, by mixing the active ingredient with (one or more) carriers or excipients under aseptic conditions.

[0154] The dosage of the substances of the present disclosure may vary between wide ranges, depending on the disease or disorder to be treated, the age and condition of the individual to be treated, etc. A suitable dosage range for the soluble TCR of the present disclosure associated with an anti-CD3 antibody may be between 25 ng / kg and 50 μg / kg. A physician will ultimately determine the appropriate dosage to be used.

[0155] The TCRs, pharmaceutical compositions, vectors, nucleic acids, and cells of the present disclosure can be provided in a substantially pure form, e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure.

[0156] In some embodiments, the methods of the present disclosure further comprise administering to the individual a second anti-cancer agent.

[0157] V. Kits or Products

[0158] On the other hand, provided herein is a product containing materials that can be used to treat and / or prevent the above-mentioned disorders. The product includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. These containers can be formed from a variety of materials (such as glass or plastic). The composition contained in the container is alone or in combination with another composition that is effective for treating, preventing and / or diagnosing the condition and can have a sterile access port (for example, the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). At least one active agent in the composition is an antibody of the present invention. The label or package insert indicates that the composition is used to treat the selected condition. In addition, the product may include (a) a first container containing a composition, wherein the composition comprises a TCR fusion protein of the present disclosure; and (b) a second container containing a composition, wherein the composition comprises a further cytotoxic agent or other therapeutic agent. The product of this aspect of the invention may further include a package insert indicating that the composition can be used to treat a specific condition. Alternatively or in addition, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer (such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution). It may further comprise other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0159] This description is considered sufficient to enable one skilled in the art to practice the invention. Various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety for all purposes. Example

[0160] By reference to the following examples, the present invention will be more fully understood. However, they should not be construed as limiting the scope of the present invention. It is understood that the embodiments and embodiments described herein are for illustrative purposes only, and various modifications or variations thereof will be suggested to those skilled in the art and will be included within the spirit and scope of the application and the scope of the appended claims.

[0161] Example 1: Engineering glycosylation on a TCR:anti-CD3 fusion molecule

[0162] Fusion proteins comprising a soluble TCR that binds specifically and with high affinity to the germline cancer antigen MAGE-A4 and an antibody fragment that binds to T cells (e.g., an anti-CD3 scFv) have been described as potential immunotherapeutics. See, for example, WO2017175006. Further engineering efforts have been conducted to provide TCR: anti-CD3 fusion molecules with favorable properties (e.g., potency and in vivo pharmacokinetics).

[0163] Materials and methods

[0164] TCR: anti-CD3 fusion molecule

[0165] The monoglycosylated TCR:anti-CD3 fusion molecule comprises three polypeptide chains corresponding to SEQ ID NOs: 28-30 expressed in CHO cells. The three polypeptide chains comprise: (1) a free Fc region with a knob mutation (SEQ ID NO:26) and a hinge sequence (SEQ ID NO:31); (2) a soluble monoclonal high affinity anti-MAGE-A4 TCR alpha chain comprising an alpha chain variable region comprising the amino acid sequence of SEQ ID NO:7 and an alpha chain constant region comprising the amino acid sequence of SEQ ID NO:9, with an Fc domain with a hole mutation (SEQ ID NO:27) connected to the C-terminus of the TCR alpha chain via a hinge sequence (SEQ ID NO:31); and (3) an anti-CD3 scFv variant comprising the amino acid sequence of SEQ ID NO:17 connected to the N-terminus of the TCR beta chain via a linker (SEQ ID NO:18), wherein the beta chain comprises a beta chain variable region comprising the amino acid sequence of SEQ ID NO:13 and a beta chain constant region comprising the amino acid sequence of SEQ ID NO:14. Compared to the parental anti-CD3 scFv, this variant comprises a T164A mutation in CDR1 and an I201F mutation in FR3 (numbering according to SEQ ID NO: 17).

[0166] The unglycosylated TCR: anti-CD3 fusion molecule comprises the same subcomponents as the monoglycosylated form, except that the α chain variable region comprises the amino acid sequence of SEQ ID NO: 8 (thus, the full-length α chain comprises the amino acid sequence of SEQ ID NO: 12). Both the unglycosylated and monoglycosylated forms have N→Q substitutions at the three N-glycosylation sites of the parental α chain constant region (SEQ ID NO: 10). Both the unglycosylated and monoglycosylated forms have N→Q substitutions at the N-glycosylation sites of the parental β chain constant region (SEQ ID NO: 15).

[0167] Pharmacokinetics

[0168] Male SCID mice (N=4) were used. Unglycosylated or monoglycosylated TCR: anti-CD3 fusion molecules were injected with 0.665 mg / kg, 0.0665 mg / kg or 0.00665 mg / kg of single IV infusion. The control N297GTCR: anti-CD3 fusion molecule was injected with 0.665 mg / kg as a control. All TCR: anti-CD3 fusion molecules were provided at 0.133 mg / mL. Serial samples (25 μL blood → 10 μL serum) were collected 5 minutes, 1 hour, 24 hours, 7 days, 10 days, 14 days and 21 days after injection. Samples in serum were detected by electrochemiluminescence immunoassay, captured on biotinylated MAGEA4 peptide-HLA, and detected with sulfo-tagged anti-scFv antibodies.

[0169] Analysis of global N-linked glycan composition

[0170] 10 μg of protein was denatured with 8M guanidine HCl at a volume ratio of 1:1 and reduced with 100 mM dithiothreitol at 95°C for 10 min. The sample was diluted with 100 mM Tris HCl, pH 7.5 to a final concentration of 2 M guanidine HCl, followed by N-linked deglycosylation overnight at 37°C using 2 μl PNGaseF (P0705S, New England BioLabs). After deglycosylation, 150 ng of each sample was injected into an HPLC system (Agilent 1260) via an autosampler. Glycans were enriched and separated on a PGC chip (G4240-64010, Agilent) containing a porous graphitized carbon column. A binary pump was used to deliver solvent A (99.88% water, 0.1% formic acid, and 0.02% trifluoroacetic acid) and solvent B (90% acetonitrile, 9.88% water, 0.1% formic acid, and 0.02% trifluoroacetic acid) at 0.5 μl / min over 6 min as a gradient from 2% to 32% solvent B, which was held for 1.5 min. Solvent B was then gradually increased to 85% over 0.5 min and held for 1 min to clean the column. Finally, solvent B was gradually increased to 2% and held for 3 min to re-equilibrate.

[0171] Separated glycans were analyzed online by nanospray ionization on a Q-TOF mass spectrometer (Agilent 6520) using the following parameters for data acquisition: 1.9 kV spray voltage; 325°C gas temperature; 5 l / min drying gas flow; 160 V fragmentor voltage; 65 V skimmer voltage; 750 V oct 1RF Vpp voltage; 400 to 3000 m / z scan range; positive polarity; MS1 centroid data acquisition using extended dynamic range (2 GHz) instrument mode; 3 spectra / s; 333.3 ms / spectrum; 3243 transients / spectrum; and CE set to 0. The acquired mass spectral data were searched against a glycan library in Agilent MassHunter Qualitative Analysis Software. The software algorithm utilized a combination of accurate mass with a mass tolerance of 10 ppm and the expected retention times for glycan identification. Label-free quantification of each N-linked glycan was performed by integrating the AUC of each extracted glycan chromatogram relative to the sum of all identified N-linked glycans within each sample.

[0172] N-linked glycosylation site mapping

[0173] 20 μg of protein was denatured with 8 M guanidine-HCl at a 1:1 volume ratio and reduced with 100 mM dithiothreitol at 95°C for 10 min. The sample was diluted with 100 mM Tris-HCl, pH 7.5, to a final concentration of 2 M guanidine-HCl, followed by N-linked deglycosylation with 4 μl of PNGaseF (P0705S, New England BioLabs) at 37°C overnight. Following deglycosylation, the sample was alkylated with 40 mM iodoacetamide at room temperature for 30 min. The sample was split in half and enzymatically digested with 0.1 μg trypsin (Promega) and 0.1 μg chymotrypsin (Thermo Fisher Scientific) at 37°C overnight, respectively. The digest was quenched with 0.1% TFA and subjected to a C18 stage-tip cleanup. The elution step was performed with 40% acetonitrile containing 59.9% water and 0.1% TFA. After cleaning, the peptide was dried and redissolved in 50 μl of 0.1% TFA solution, of which 1 μl was injected via an autosampler onto a UPLC system (Waters NanoAcquity) and separated on an Acquity M-Class BEH C18 column (0.1 mm × 100 mm, 1.7 μm resin, Waters) heated at 45°C. A binary gradient pump was used to deliver solvent A (97.9% water, 2% acetonitrile, and 0.1% formic acid) and solvent B (97.9% acetonitrile, 2% water, and 0.1% formic acid) at 1 μl / min over 35 min as a gradient from 2% to 25% solvent B. The solvent was gradually changed to 50% solvent B over 2 min, then maintained at 90% for 6 min to clean the column. Finally, the solvent was gradually changed to 2% solvent B and maintained for 7 min to re-equilibrate. The separated peptides were analyzed online by nanospray ionization on an Orbitrap Elite hybrid ion trap-Orbitrap mass spectrometer (Thermo Fisher Scientific), using the following parameters for data acquisition: 60,000 resolution; 375-1600 m / z scan range; positive polarity; centroid mode; 1 m / z isolation width, activation Q of 0.25, activation time of 10 ms; CID activation; and CE setting of 35. Data were collected in data-dependent mode, precursor ions were analyzed in FTMS, and the top 15 most abundant ions were selected for fragmentation and analysis in ITMS.

[0174] The acquired mass spectrometry data were searched against the protein sequence using Byonic software (Protein Metrics Inc.) using the following parameters: 20 ppm precursor mass tolerance, 0.5 Da fragment mass tolerance; strict specificity for arginine and lysine, with a maximum of one missed cleavage by trypsin; strict specificity for leucine, phenylalanine, tryptophan, and tyrosine, with a maximum of two missed cleavages by chymotrypsin; carbamidomethylation at fixed cysteine; oxidation at variable methionine; and deamidation at variable asparagine. Byonic search results were analyzed in Byologic software (Protein Metrics Inc.) and filtered with a minimum MS2 score cutoff of 200. Peptide identification was confirmed by MS2 peptide fragmentation. Deamidated peptides containing N-linked sites were label-free quantified relative to their unmodified forms by AUC integration of extracted ion chromatograms in Thermo Xcalibur Qual Browser software (Thermo Fisher Scientific). Changes in deamidation were used to quantify glycosylation.

[0175] Binding affinity

[0176] Anti-hIgG1 antibodies were immobilized on a Biacore CM5 capture chip using a standard EDC / NHS immobilization protocol. ImmTAC molecules were prepared at 1 ug / ml in HBS-EP buffer and captured on an anti-hIgG1 immobilized CM5 chip. Subsequently, the antigen (CD3εδ heterodimeric Fc fusion) was flowed through the flow cell capturing the ImmTAC with a contact time of 210 seconds and a dissociation time of 300 seconds at a flow rate of 100 ul / min. Biacore was performed at 37°C, and the analyte (human CD3εδ heterodimeric Fc fusion) concentration series was 0, 0.5, 2.5, 12.5, 50, 150 nM.

[0177] result

[0178] Figure 1 Figures A and B show TCR:anti-CD3 fusion molecules comprising anti-CD3 scFv, a soluble monoclonal high-affinity TCR, and an antibody Fc domain to extend half-life in vivo. Figure 2 As shown in Figure A, when produced in CHO cells, the soluble monoclonal TCR has 7 N-glycosylation sites. All glycosylation sites were found to be removable without affecting activity or selectivity. The unglycosylated variant and the monoglycosylated variant were selected for further study ( Figure 2B). In the monoglycosylated variant, all N-glycosylation sites were removed from the TCR constant region using N→Q substitutions, and two of the three N-glycosylation sites from the variable region were mutated (N24Q substitution on the α chain variable region and N84Q substitution on the β chain variable region). The unglycosylated variant contained a further N18Q substitution on the α chain variable region to remove the final N-glycosylation site.

[0179] The effect of removing N-glycosylation sites from the variable and constant regions on protein yield was also examined. Removal of N-glycosylation sites from the TCR variable region had a significant negative effect on yield, whereas removal of N-glycosylation sites from the TCR constant region had no effect on yield ( Figure 2 C). A series of double and single N-glycosylation site variants were constructed to examine the effect of removing individual N-glycosylation sites from the TCR variable region ( Figure 2 D). The results show that as glycosylation decreases, yield is gradually lost, while the completely unglycosylated molecule shows a significant reduction in yield. However, it was found that retaining N-glycosylation at the N18 residue provided the greatest enhancement to yield at any single site.

[0180] These TCR: anti-CD3 fusion molecules comprise an antibody Fc domain as this was found to substantially improve in vivo pharmacokinetic properties in the SCID mouse model described above. Formats with the Fc domain fused to either the α or β chain were found to have substantially improved pharmacokinetics compared to similar molecules without the Fc expressed from E. coli or CHO cells. Figure 3 A and Table A). All N-glycosylation sites of these molecules are intact. Figure 1 The format shown in Figure B (scFv at the N-terminus of the TCR β chain and Fc at the C-terminus of the α chain) has the most favorable pharmacokinetic properties. Fusion of the Fc to the C-terminus of the α chain also produced a molecule with the highest activity (i.e., potency and selectivity) among the various formats tested. This format was selected for further testing.

[0181] Table A. Pharmacokinetics of TCR:anti-CD3 fusion molecules.

[0182]

[0183] Different linker sequences were also tested in this format. No differences in yield were observed using different linker sequences. In addition, molecules with different linkers showed comparable potency and off-target activity levels. Finally, molecules with different linkers exhibited equivalent stability in serum. These results suggest that linker sequence does not affect activity, yield, or serum stability.

[0184] To further improve pharmacokinetics, unglycosylated and monoglycosylated variants of the same TCR:anti-CD3 fusion molecule were generated (e.g. Figure 2 The pharmacokinetic properties of the drug were tested in a SCID mouse model. Figure 3 The monoglycosylated variant exhibited better pharmacokinetic properties, including slower elimination, half-life, and clearance, compared to the unglycosylated form, as shown by the BE of 4. Figure 3 B and E), while the pharmacokinetics of these two forms are nearly linear across a 100-fold dose range ( Figure 3 C and D). Thus, the monoglycosylated form having a single N-linked glycosylation site at N18 of the α chain variable region showed improved half-life compared to the unglycosylated form.

[0185] The two variants were also subjected to stability testing. For the heat stress test, 1 mg / mL of the same TCR: anti-CD3 fusion molecule, both unglycosylated and monoglycosylated variants (e.g., Figure 2 (B) was exposed to 30°C heat stress for 4 weeks. After incubation, the monoglycosylated form showed an increase of 0.8% monomer loss by SEC, while the unglycosylated form showed a significantly higher 11.3% monomer loss (+6.1% vHMW form and 4.8% dimer). Both forms showed stability at all potential deamidation / isomerization sites.

[0186] Both forms were also heat-stressed at 37°C for 2 weeks at 1 mg / mL in PBS, pH 7.4. After incubation, the unglycosylated molecule showed a 7.3% increase (4.1% vHMW form, 1.7% dimer), while the monomer loss of the monoglycosylated form was only 1.4%. Both forms showed little change at all potential deamidation / isomerization sites.

[0187] In summary, the unglycosylated and monoglycosylated forms were found to have substantially different stabilities. These results suggest that the monoglycosylated form has better tolerance to heat stress than the unglycosylated form. Both forms also demonstrated stability at each potential oxidation site on the anti-CD3 scFv in the AAPH oxidation assay. Taken together, these results demonstrate that the glycan at position N18 in the α chain variable domain is critical for preventing aggregation and suggest that the monoglycosylated form has better manufacturability than the unglycosylated form.

[0188] The binding affinities of the unglycosylated and monoglycosylated variants were also measured. As measured by BIACORE, the two forms had similar affinities for the MAGEA4 peptide via TCR, with the monoglycosylated variant having a K ofD was 0.17 nM, while the K D is 0.18nM.

[0189] The affinity of the unglycosylated and monoglycosylated anti-CD3 scFv U28 variants and the original UCHT1v9 molecules for human CD3ε was measured by Biacore. The two U28-based molecules had comparable affinities for CD3ε (K = 14 nM for the unglycosylated form). D ; 15 nM K for monosaccharides D The UCHT1v9-based molecule also has comparable affinity for CD3 (K of 17 nM for the unglycosylated form). D ; 18 nM K for monosaccharides D Although the equilibrium affinity of the U28-based molecule was similar to that of the original UCHT1v9-based molecule, the anti-CD3 scFv was characterized to have slightly different binding kinetics (the association (on) and dissociation (off) rates of U28 were approximately 2-fold faster).

[0190] In summary, the presence or absence of glycosylation does not alter the affinity of pMHC binding.

[0191] Example 2: Potency and selectivity of monoglycosylated or unglycosylated TCR:anti-CD3 fusion molecules

[0192] The aglycosylated and monoglycosylated TCR:anti-CD3 fusion molecules described in Example 1 were tested for potency and selectivity in target cell killing.

[0193] Materials and methods

[0194] Cell killing

[0195] for Figure 4 BD, using the xCELLigence platform (Agilent). Effector cells were used at a 10:1 effector to target cell ratio. Percent cytolysis was determined using normalized cell index (impedance measurement). In all cases, the assay was performed with triplicate measurements every 2 hours for 96 hours. EC50 values were derived from the 72-hour percentage cytolysis curve. Curve fitting was performed in PRISM.

[0196] for Figure 5 A and B were assayed using the Opera Phenix High Content Screening System (Perkin Elmer). Effector cells were used at a 5:1 effector-to-target cell ratio. AUC values were obtained and curve fitting was performed in PRISM. EC50 values were calculated from the curves.

[0197] for Figure 5 The C of human IFN-γ was measured using a human IFN-γ ELISpot kit (BD Biosciences). 6 Target cells were prepared at a density of 50000 cells / ml and plated at a volume of 50 μl per well. PBMCs isolated from fresh donor blood were used as effector cells. Effector cells were used for antigen-positive cells at an effector-target cell ratio of 1:1 and for antigen-negative cells at an effector-target cell ratio of 0.8:1. Samples were detected using AEC colorimetric reagent. Spots were counted using a CTL analyzer and Immunospot software (Cellular Technology Limited). Curve fitting was performed in PRISM and EC50 values were calculated.

[0198] MAGEA4 antigen copy number was determined by quantitative mass spectrometry.

[0199] result

[0200] A variety of cancer cell lines were used, representing a range of target MAGE-A4 antigen expression. All cell lines expressed HLA-A2. EC50 values for cytolysis against all cell lines are shown in Figure 4 The killing curve and the lowest concentration of TCR: anti-CD3 fusion molecule that resulted in a killing response are shown in Figure A and calculated from the data obtained at the 72 hour time point. Figure 4 Both unglycosylated and monoglycosylated TCR: anti-CD3 fusion molecules exhibited very low pM cell killing activity and selectivity. For example, at a TCR: anti-CD3 fusion molecule concentration of 0.17 pM for both molecules, cell killing was observed against NCI-H1755 lung adenocarcinoma cells (which have the highest expression of MAGE-A4). Figure 4 B). Cell killing was observed against SCaBER bladder urothelial carcinoma cells (which have moderate levels of MAGE-A4 expression) at a TCR: anti-CD3 fusion molecule concentration of 14 pM for both molecules ( Figure 4 In contrast, at a TCR:anti-CD3 fusion molecule concentration of 3 nM, cell killing was observed against NCI-H441 cells (which are MAGE-A4 negative but HLA-A2 positive). Figure 4 D), indicating that selectivity is maintained down to the nM level. Thus, killing activity correlates with target (pMHC) copy number. These results demonstrate the high potency and selectivity of these two molecules.

[0201] It was found that the TCR:anti-CD3 fusion molecule with Fc fusion as described above had reduced cell killing efficacy against target cells expressing MAGE-A4 antigen:HLA complex ( Figure 5A and B). However, if Figure 3 As shown in Figure A, it is desirable to include an Fc domain in order to improve in vivo pharmacokinetics. Therefore, a variant anti-CD3 scFv was tested. As mentioned above, this variant contains point mutations in CDR-H1 and FR3. It was found that the loss of potency when using Fc fusion was offset by using the variant anti-CD3 scFv ( Figure 5 Against antigen-positive cancer cells, the EC50 of cell killing by TCR:anti-CD3 fusion molecules with Fc fusion and variant anti-CD3 scFv was within 3-10 fold of that of molecules without the Fc domain.

[0202] All three molecules showed reduced responsiveness to antigen (-) cells, similar to the reduced potency against antigen (+) cells ( Figure 5 C). These results demonstrate that for all three TCR:anti-CD3 fusion molecules tested, a window between on-target and off-target activity was maintained.

[0203] Example 3: In vitro safety testing of monoglycosylated TCR: anti-CD3 fusion molecules

[0204] The safety of the monoglycosylated TCR:anti-CD3 fusion molecules described in Example 1 was tested in vitro against normal cell lines.

[0205] Materials and methods

[0206] In vitro safety testing

[0207] For testing on normal cells, reactivity was determined by ELISpot assay to detect the release of IFNγ and granzyme b. The lowest concentration of TCR-anti-D3 fusion molecules at which each cytokine was detected was recorded. For alloreactivity (i.e., binding to alternative HLA), reactivity was assessed using the IFNγ ELISpot assay, covering the five most prevalent HLA-A (excluding HLA*02:01), four HLA-B, and six HLA-C species in the global population. Whole blood was assayed using the Proinflammatory Panel 1 (Human) kit (Meso Scale Discovery) to detect TNFα, IL-2, IL-6, IL-1β, and IFNγ in whole blood obtained from three donors. The TCR-anti-CD3 fusion was applied at various concentrations ranging from 0.01 to 10 nM.

[0208] result

[0209] Monoglycosylated TCR: anti-CD3 fusion molecules were tested in a panel of normal cell lines covering high-risk tissue types, as well as cell types that have shown reactivity to anti-MAGE-A4 TCRs. Using IFNγ as a readout, no detectable reactivity was observed against normal cells in a 6-cell panel covering the 15 most common HLA types ( Figure 6 A). In the whole blood assay, no cytokine release was observed at concentrations less than nM. In summary, no reactivity against normal cell lines was detected at concentrations less than nM, demonstrating the safety of this in vitro assay.

[0210] Comparison of TCR:anti-CD3 fusion molecules with variant anti-CD3 scFvs to TCR:anti-CD3 fusion molecules without Fc again showed that the window of potency between on-target and off-target activity on normal cells was maintained ( Figure 6 B). This demonstrates that the TCR:anti-CD3 fusion molecule with an Fc domain and a variant anti-CD3 scFv maintains an equivalent therapeutic index to the format without the Fc fusion.

[0211] sequence

[0212] Anti-MAGE-A4 TCR Vα CDR-1

[0213] VSPFSN (SEQ ID NO: 1)

[0214] Anti-MAGE-A4 TCR Vα CDR-2

[0215] LTFSENT (SEQ ID NO: 2)

[0216] Anti-MAGE-A4 TCR Vα CDR-3

[0217] VVNSAQGLYTPTF (SEQ ID NO: 3)

[0218] Anti-MAGE-A4 TCR VβCDR-1

[0219] LDHEN (SEQ ID NO: 4)

[0220] Anti-MAGE-A4 TCR Vβ CDR-2

[0221] SRFATG (SEQ ID NO: 5)

[0222] Anti-MAGE-A4 TCR Vβ CDR-3

[0223] ASSSDQNSGDPYEQYF (SEQ ID NO: 6)

[0224] Anti-MAGE-A4 TCR α chain variable region (monoglycosylated form)

[0225]

[0226] Anti-MAGE-A4 TCR α chain variable region (unglycosylated form)

[0227]

[0228] Anti-MAGE-A4 TCR α chain constant region (monoglycosylated or unglycosylated form)

[0229]

[0230] Parental anti-MAGE-A4 TCR α chain constant region (fully glycosylated form)

[0231]

[0232] Anti-MAGE-A4 TCRα chain (monoglycosylated form)

[0233]

[0234] Anti-MAGE-A4 TCRα chain (unglycosylated form)

[0235]

[0236] Anti-MAGE-A4 TCR beta chain variable region (monoglycosylated or unglycosylated forms)

[0237]

[0238] Anti-MAGE-A4 TCR beta chain constant region (monoglycosylated or unglycosylated form)

[0239]

[0240] Parental anti-MAGE-A4 TCRβ chain constant region (fully glycosylated form)

[0241]

[0242] Anti-MAGE-A4 TCRβ chain (monoglycosylated or unglycosylated forms)

[0243]

[0244] Anti-CD3 scFv U28 variant

[0245]

[0246] connector

[0247] GGGGS (SEQ ID NO: 18)

[0248] connector

[0249] GGGSG (SEQ ID NO: 19)

[0250] connector

[0251] GGSGG (SEQ ID NO: 20)

[0252] connector

[0253] GSGGG (SEQ ID NO: 21)

[0254] connector

[0255] GSGGGP (SEQ ID NO: 22)

[0256] connector

[0257] GGEPS (SEQ ID NO: 23)

[0258] connector

[0259] GGEGGGP (SEQ ID NO: 24)

[0260] connector

[0261] GGEGGGSEGGGS (SEQ ID NO: 25)

[0262] Fc domain with knob mutation

[0263]

[0264] Fc domain with hole mutation

[0265]

[0266] Monoglycosylated anti-MAGE-A4 TCR: anti-CD3 scFv Fc fusion chain 1 (Fc with knob mutation)

[0267]

[0268] Monoglycosylated anti-MAGE-A4 TCR:anti-CD3 scFv Fc fusion chain 2 (TCR α chain fused to Fc with a hole mutation)

[0269]

[0270] Monoglycosylated anti-MAGE-A4 TCR:anti-CD3 scFv Fc fusion chain 3 (variant anti-CD3 scFv fused to TCR beta chain)

[0271]

[0272] Hinge sequence

[0273] DKTHTCPP(SEQ ID NO:31) or DKTHTCPPC(SEQ ID NO:36)

[0274] Anti-MAGE-A4 TCRα variable chain (fully glycosylated form)

[0275]

[0276] Anti-MAGE-A4 TCRβ variable chain (fully glycosylated form)

[0277]

[0278] MAGE-A4 target antigen

[0279] GVYDGREHTV (SEQ ID NO: 34)

[0280] Anti-CD3 scFv (parental)

[0281]

Claims

1. A T cell receptor (TCR) fusion protein comprising a TCR that binds to the GVYDGREHTV (SEQ ID NO: 34) HLA-A*02 complex, wherein the TCR is a soluble TCR covalently linked to: (1) a T cell engaging domain that binds to a protein expressed on the cell surface of a T cell, and (2) an antibody Fc domain; wherein the TCR comprises: (a) a TCR α chain comprising an α chain variable region, wherein the α chain variable region comprises (i) a CDR1 comprising the amino acid sequence of VSPFSN (SEQ ID NO: 1), (ii) a CDR2 comprising the amino acid sequence of LTFSENT (SEQ ID NO: 2), and (iii) a CDR3 comprising the amino acid sequence of VVNSAQGLYIPTF (SEQ ID NO: 3); and (b) a TCR beta chain comprising a beta chain variable region, wherein the beta chain variable region comprises (i) a CDR1 comprising the amino acid sequence of LDHEN (SEQ ID NO: 4), (ii) a CDR2 comprising the amino acid sequence of SRFATG (SEQ ID NO: 5), and (iii) a CDR3 comprising the amino acid sequence of ASSSDQNSGDPYEQYF (SEQ ID NO: 6); wherein the TCR is glycosylated at a single N-linked glycosylation site, wherein the N-linked glycosylation site is at residue N18 of the α chain variable region according to the numbering of SEQ ID NO:

7.

2. The TCR fusion protein of claim 1, wherein the TCR comprises an amino acid substitution at each potential N-glycosylation site except residue N18.

3. The TCR fusion protein of claim 2, wherein the TCR comprises an amino acid substitution at the following residues: (a) residue N24 of the α chain variable region, numbered according to SEQ ID NO: 32; (b) residues N33, N67, and N78 of the α chain constant region, numbered according to SEQ ID NO: 10; (c) residue N84 of the beta chain variable region, as numbered according to SEQ ID NO: 33; and (d) Residue N70 of the beta chain constant region, according to the numbering of SEQ ID NO:

15.

4. The TCR fusion protein of claim 2 or claim 3, wherein the amino acid substitution is N→Q.

5. The TCR fusion protein of claim 4, wherein the TCR comprises the following amino acid substitutions: (a) N24Q in the α chain variable region according to SEQ ID NO: 32; (b) N33Q, N67Q and N78Q in the α chain constant region according to the numbering of SEQ ID NO: 10; (c) N84Q in the beta chain variable region according to SEQ ID NO: 33; and (d) N70Q in the β chain constant region according to SEQ ID NO:

15.

6. The TCR fusion protein of any one of claims 1-5, wherein the TCR comprises one or more engineered cysteine residues in the α chain and / or β chain constant region to form a non-native disulfide bond between the α chain and β chain.

7. The TCR fusion protein of claim 6, wherein the TCR comprises a cysteine residue at position 57 of the β chain constant region according to SEQ ID NO:

15.

8. The TCR fusion protein of claim 1 , wherein the α chain variable region comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:

7.

9. The TCR fusion protein of claim 1 or claim 8, wherein the beta chain variable region comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:

13.

10. The TCR fusion protein of claim 1, wherein the α chain variable region comprises the amino acid sequence of SEQ ID NO: 7, and the β chain variable region comprises the amino acid sequence of SEQ ID NO:

13.

11. The TCR fusion protein of any one of claims 1 and 8-10, wherein the TCR α chain further comprises an α chain constant region, and wherein the α chain constant region comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:

9.

12. The TCR fusion protein of any one of claims 1 and 8-11, wherein the TCR β chain further comprises a β chain constant region, and wherein the β chain constant region comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:

14.

13. The TCR fusion protein of claim 1 or claim 10, wherein the TCR α chain further comprises an α chain constant region comprising the amino acid sequence of SEQ ID NO: 9, and wherein the TCR β chain further comprises a β chain constant region comprising the amino acid sequence of SEQ ID NO:

14.

14. The TCR fusion protein of claim 1, wherein the α chain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:

11.

15. The TCR fusion protein of claim 1 or claim 14, wherein the beta chain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:

16.

16. The TCR fusion protein of claim 1, wherein the α chain comprises the amino acid sequence of SEQ ID NO: 11, and the β chain comprises the amino acid sequence of SEQ ID NO:

16.

17. The TCR fusion protein of any one of claims 1-16, wherein the antibody Fc domain is a human Fc domain. The TCR fusion protein of claim 17 , wherein the antibody Fc domain is a human IgG1, human IgG2, or human IgG4 Fc domain.

19. The TCR fusion protein of claim 17 or claim 18, wherein the antibody Fc domain comprises one or more mutations that reduce the effector function of the Fc domain.

20. The TCR fusion protein of claim 19, wherein the antibody Fc domain is a human IgG1 Fc domain comprising a mutation at residue N297, as numbered by the EU index.

21. The TCR fusion protein of claim 20, wherein the antibody Fc domain is a human IgG1 Fc domain comprising an N297G substitution according to EU index numbering.

22. The TCR fusion protein of any one of claims 19-21, wherein the antibody Fc domain is a human IgG1 Fc domain comprising one or more mutations at residues E233, L234, L235, and / or G236, as numbered by the EU index.

23. The TCR fusion protein of claim 22, wherein the antibody Fc domain is a human IgG1 Fc domain comprising substitutions N297G, E233P, L234V, L235A, and a deletion at G236, as numbered by the EU index.

24. The TCR fusion protein of claim 19, wherein the antibody Fc domain is a human IgG1 Fc domain comprising one or more mutations at residues L234, L235, and P329, as numbered by the EU index.

25. The TCR fusion protein of claim 24, wherein the antibody Fc domain is a human IgG1 Fc domain comprising the substitutions L234A, L235A, and P329G, as numbered by the EU index.

26. The TCR fusion protein of any one of claims 1-25, wherein the antibody Fc domain is fused to the TCR via a hinge sequence.

27. The TCR of claim 26, wherein the hinge sequence comprises the amino acid sequence of DKTHTCPP (SEQ ID NO: 31).

28. The TCR fusion protein of any one of claims 1-27, further comprising a second antibody Fc domain, wherein the second antibody Fc domain is associated with the first antibody Fc domain by: (1) one or more covalent linkages; and / or (2) one or more amino acid substitutions on one or both of the antibody Fc domains that promote heterodimerization.

29. The TCR fusion protein of claim 28, wherein the first antibody Fc domain and the second antibody Fc domain both comprise an antibody CH2 domain and a CH3 domain.

30. The TCR fusion protein of claim 28 or claim 29, wherein the first antibody Fc domain is fused to the TCR via a first hinge sequence, and wherein a second hinge sequence is linked to the N-terminus of the second antibody Fc domain.

31. The TCR fusion protein of claim 30, wherein the first hinge sequence and the second hinge sequence are linked by one or more interchain disulfide bonds between the first hinge sequence and the second hinge sequence.

32. The TCR fusion protein of claim 30 or claim 31, wherein the first hinge sequence and the second hinge sequence both comprise the amino acid sequence of DKTHTCPP (SEQ ID NO: 31).

33. The TCR fusion protein of any one of claims 28-32, wherein one of the first and second antibody Fc domains comprises one or more knob-forming mutations and the other of the first and second antibody Fc domains comprises one or more corresponding hole-forming mutations to promote heterodimerization of the antibody Fc domains.

34. The TCR fusion protein of claim 33, wherein one of the first and second antibody Fc domains comprises a T366W substitution and the other of the first and second antibody Fc domains comprises a T366S, L368A, Y407V substitution, as numbered by the EU index.

35. The TCR fusion protein of claim 34, wherein one of the first antibody Fc domain and the second antibody Fc domain comprises the amino acid sequence of SEQ ID NO: 27, and the other of the first antibody Fc domain and the second antibody Fc domain comprises the amino acid sequence of SEQ ID NO:

26.

36. The TCR fusion protein of claim 35, wherein the first antibody Fc domain covalently linked to the TCR comprises the amino acid sequence of SEQ ID NO: 27, and the second antibody Fc domain comprises the amino acid sequence of SEQ ID NO:

26.

37. The TCR fusion protein of any one of claims 1-36, wherein the T cell engaging domain binds to human CD3 expressed on the cell surface of T cells.

38. The TCR fusion protein of any one of claims 1-37, wherein the T cell engaging domain comprises an antibody antigen binding domain.

39. The TCR fusion protein of any one of claims 1-38, wherein the T cell engaging domain is a single-chain variable fragment (scFv).

40. The TCR fusion protein of claim 39, wherein the scFv comprises the amino acid sequence of SEQ ID NO:

17.

41. The TCR fusion protein of claim 39, wherein the scFv comprises the amino acid sequence of SEQ ID NO:

35.

42. The TCR fusion protein of any one of claims 1-41, wherein the T cell engaging domain is covalently linked to the TCR via a linker.

43. The TCR fusion protein of claim 42, wherein the linker comprises an amino acid sequence selected from SEQ ID NOs: 18-25.

44. The TCR fusion protein of any one of claims 1-43, wherein the C-terminus of the T cell engaging domain is covalently linked to the N-terminus of the TCR β chain variable domain.

45. The TCR fusion protein of claim 44, wherein the C-terminus of the T cell engagement domain is covalently linked to the N-terminus of the TCR β chain variable domain via a linker.

46. The TCR fusion protein of claim 45, wherein the linker comprises an amino acid sequence selected from SEQ ID NOs: 18-25.

47. The TCR fusion protein of any one of claims 1-46, wherein the N-terminus of the antibody Fc domain is covalently linked to the C-terminus of the TCR alpha chain constant domain.

48. The TCR fusion protein of claim 47, wherein the N-terminus of the antibody Fc domain is covalently linked to the C-terminus of the TCR α chain constant domain via a hinge sequence.

49. The TCR fusion protein of claim 48, wherein the hinge sequence comprises the amino acid sequence of DKTHTCPP (SEQ ID NO: 31).

50. The TCR fusion protein of any one of claims 1-49, wherein the TCR fusion comprises three polypeptides, including: (a) a first polypeptide comprising, from N-terminus to C-terminus, the TCR α chain variable region, the α chain constant region, the first hinge sequence, and the first antibody Fc domain; (b) a second polypeptide comprising, from N-terminus to C-terminus, a single-chain variable fragment (scFv) that binds to human CD3 expressed on the cell surface of T cells, a linker, the β chain variable region, and the β chain constant region; and (c) a third polypeptide comprising, from N-terminus to C-terminus, a second hinge sequence and a second antibody Fc domain.

51. The TCR fusion protein of claim 50, wherein the first polypeptide and the second polypeptide are linked by one or more disulfide bonds between the α chain constant region and the β chain constant region.

52. The TCR fusion protein of claim 51 , wherein the first polypeptide and the third polypeptide are linked by: (1) one or more interchain disulfide bonds between the first hinge sequence and the second hinge sequence; and / or (2) one or more corresponding knob-forming mutations and hole-forming mutations on the Fc domain of the antibody.

53. The TCR fusion protein of any one of claims 50-52, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 29, wherein the second polypeptide comprises the amino acid sequence of SEQ ID NO: 30, and wherein the third polypeptide comprises the amino acid sequence of SEQ ID NO:

28.

54. A T cell receptor (TCR) fusion protein comprising a TCR that binds to the GVYDGREHTV (SEQ ID NO:34) HLA-A*02 complex, wherein the TCR fusion protein comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO:29, a second polypeptide comprising the amino acid sequence of SEQ ID NO:30, and a third polypeptide comprising the amino acid sequence of SEQ ID NO:

28.

55. A polynucleotide encoding the TCR fusion protein according to any one of claims 1-54.

56. A kit for polynucleotides encoding the TCR fusion protein according to any one of claims 50-54, wherein the kit comprises a first polynucleotide encoding the first polypeptide, a second polynucleotide encoding the second polypeptide, and a third polynucleotide encoding the third polypeptide.

57. A vector comprising the polynucleotide according to claim 55.

58. The vector of claim 57, wherein the vector is an expression vector.

59. A kit for vectors encoding the TCR fusion protein according to any one of claims 50-54, wherein the kit comprises a first vector encoding the first polypeptide, a second vector encoding the second polypeptide, and a third vector encoding the third polypeptide.

60. The kit of claim 59, wherein the vector is an expression vector.

61. A host cell comprising the polynucleotide of claim 55, a kit of polynucleotides of claim 56, a vector of claim 57 or claim 58, or a kit of vectors of claim 59 or claim 60.

62. The host cell of claim 61, wherein the host cell is a mammalian cell.

63. The host cell of claim 62, wherein the mammalian cell is a Chinese Hamster Ovary (CHO) cell.

64. A method for producing a TCR fusion protein, comprising culturing the host cell according to any one of claims 61-63 under conditions suitable for production of the TCR fusion protein.

65. The method of claim 64, further comprising recovering the TCR fusion protein from the host cell.

66. A TCR fusion protein produced by the method of claim 64 or claim 65.

67. A pharmaceutical composition comprising the TCR fusion protein according to any one of claims 1-54 and 66 and a pharmaceutically acceptable carrier.

68. A method of treating cancer, comprising administering an effective amount of the TCR fusion protein according to any one of claims 1-54 and 66 or the pharmaceutical composition according to claim 67 to an individual.

69. The method of claim 68, wherein the individual is a human.

70. The method of claim 69, wherein the individual has a cancer that expresses MAGE-A4.

71. The method of claim 69 or claim 70, wherein the individual is of the HLA-A*02 subtype.

72. The method of any one of claims 68-71, wherein the TCR fusion protein or composition is administered intravenously or by intratumoral injection.

73. The method of any one of claims 68-72, further comprising administering to the individual a second anticancer agent.

74. The TCR fusion protein of any one of claims 1-54 and 66 or the pharmaceutical composition of claim 67 for medical use, preferably for use in human subjects.

75. The TCR fusion protein according to any one of claims 1-54 and 66 or the pharmaceutical composition according to claim 67 for use in treating cancer, preferably in a human subject.

76. Use of the TCR fusion protein according to any one of claims 1-54 and 66 or the pharmaceutical composition according to claim 67 for the manufacture of a medicament for treating cancer.

Images