MAGE A4-specific T cell receptor fusion protein

Abstract

The present disclosure provides a GVYDGREHTV (SEQ ID NO: 34) HLA-A antibody covalently linked to a T cell engaging domain and an antibody Fc domain that binds to a protein expressed on the cell surface of a T cell. * T cell receptor (TCR) fusion proteins containing a TCR that binds to the O2 complex, as well as related polynucleotides, vectors, kits, host cells, pharmaceutical compositions, methods, and uses, are provided. T cell receptors (TCRs) are naturally expressed by CD4+ and CD8+ T cells.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 399,095, filed August 18, 2022, the entire contents of which are incorporated herein by reference.

[0002] Electronic Sequence Listing Reference The contents of the electronic sequence listing (146392054140seqlist.xml; size: 35,722 bytes; and creation date: August 9, 2023) are incorporated herein by reference in their entirety.

[0003] Field The present disclosure provides a GVYDGREHTV (SEQ ID NO: 34) HLA-A antibody covalently linked to an antigen binding domain and an antibody Fc domain that binds to a protein expressed on the cell surface of a T cell. * The present invention relates to T cell receptor (TCR) fusion proteins comprising a TCR that binds to the .O2 complex, as well as related polynucleotides, vectors, kits, host cells, pharmaceutical compositions, methods, and uses. [Background technology]

[0004] background T cell receptors (TCRs) are naturally expressed by CD4+ T cells and CD8+ T cells. TCRs are designed to recognize short peptide antigens displayed on the surface of antigen-presenting cells in complex with major histocompatibility complex (MHC) molecules (in humans, MHC molecules are also known as human leukocyte antigens, or HLA) (Davis, et al., (1998), Annu Rev Immunol 16:523-544.). CD8+ T cells, also known as cytotoxic T cells, specifically recognize peptides bound to MHC class I and are generally involved in detecting and mediating the destruction of diseased cells. CD8+ T cells can destroy cancerous cells and virus-infected cells; however, the affinity of TCRs expressed by cancer-specific T cells in the natural repertoire is typically low as a result of thymic selection, meaning that cancerous cells frequently escape detection and destruction. Novel immunotherapeutic approaches aimed at promoting cancer recognition by T cells offer highly promising strategies for the development of effective anticancer treatments.

[0005] MAGE A4 belongs to the MAGE family of germline-encoded cancer antigens (De Plaen, et al., (1994), Immunogenetics 40(5):360-369) and has UniProt accession number P43358. Such antigens have been found to be frequently expressed in a wide variety of cancers, while their expression in normal tissues is restricted to other immune-privileged sites, including the adult testis and placenta. The cancer-specific nature of these genes makes them ideal targets for anti-cancer therapy. The exact function of MAGE A4 remains unknown, but it is thought to play a role in embryonic development. High levels of MAGE A4 expression have been reported in several types of tumors, including melanoma, esophageal, head and neck, lung, breast, and bladder cancers (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 stimulates cytotoxic T cells to target MAGE A4-positive, HLA-A * It has been shown that GVYDGREHTV (SEQ ID NO: 34) HLA-A induces lysis of HLA-A positive tumor cells (Duffour, et al., (1999), Eur J Immunol 29(10):3329-3337 and WO 2000020445). * The O2 conjugate provides a useful target antigen for immunotherapeutic intervention. GVYDGREHTV (SEQ ID NO: 34) HLA-A * Soluble TCRs and TCR fusion proteins that bind to the MAGE A4 peptide:HLA complex have been described, for example, in U.S. Patent Application Publication No. 20190092834 and WO 2017175006. 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. All references cited herein, including patent applications and patent publications, are incorporated by reference in their entirety. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2000020445 [Patent Document 2] US Patent Application Publication No. 2019 / 0092834 [Patent Document 3] International Publication No. 2017175006 [Non-patent literature]

[0007] [Non-Patent Document 1] Davis, et al., (1998), Annu Rev Immunol 16:523-544. [Non-patent document 2] De Plaen, et al., (1994), Immunogenetics40(5):360-369 [Non-patent document 3] Bergeron,(2009),Int J Cancer125(6):1365-1371 [Non-patent document 4] Cabezon,et al.,(2013),Mol Cell Proteomics12(2):381-394 [Non-patent document 5] Cuffel,et al.,(2011),Int J Cancer128(11):2625-2634 [Non-patent document 6] Forghanifard,et al.,(2011),Cancer Biol Ther12(3):191-197 [Non-Patent Document 7] Karimi,et al.,(2012),Clin Lung Cancer13(3):214-219 [Non-patent document 8] Svobodova,et al.,(2011),Eur J Cancer47(3):460-469 [Non-Patent Document 9] Duffour, et al., (1999), Eur J Immunol29(10):3329-3337 Summary of the Invention [Means for solving the problem]

[0008] overview In one particular embodiment, GVYDGREHTV (SEQ ID NO: 34) HLA-A *A T cell receptor (TCR) fusion protein comprising a TCR that binds to a TCR-02 complex, the TCR being 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; the TCR comprising: (a) a TCR alpha chain comprising an alpha chain variable region, the alpha chain variable region comprising: (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, the beta chain variable region comprising (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, the N-linked glycosylation site being at residue N18 of the alpha chain variable region numbered according to SEQ ID NO: 7.

[0009] In some embodiments according to any of the embodiments described herein, the TCR comprises an amino acid substitution at every potential N-glycosylation site other than residue N18. In some embodiments, the TCR comprises an amino acid substitution at (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 substitution is N→Q. In some embodiments, the TCR comprises the following amino acid substitutions: (a) N24Q in the alpha chain variable region numbered according to SEQ ID NO: 32; (b) N33Q, N67Q, and N78Q in the alpha chain constant region numbered according to SEQ ID NO: 10; (c) N84Q in the beta chain variable region numbered according to SEQ ID NO: 33; and (d) N70Q in the beta chain constant region numbered according to SEQ ID NO: 15.

[0010] In some embodiments according to any of the embodiments described herein, the TCR comprises one or more engineered cysteine ​​residues in the alpha and / or beta chain constant regions to form non-native disulfide bonds between the alpha and beta chains, hi some embodiments, the TCR comprises a cysteine ​​residue at position 57 of the beta chain constant region numbered according to SEQ ID NO: 15.

[0011] In some embodiments according to any of the embodiments described herein, the alpha chain variable region comprises 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 SEQ ID NO: 7. In some embodiments, the beta chain variable region comprises 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 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, wherein the alpha chain constant region comprises 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 SEQ ID NO: 9. In some embodiments, the TCR beta chain further comprises a beta chain constant region, wherein the beta chain constant region comprises 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 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 alpha chain comprises 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 SEQ ID NO:11.In some embodiments, the beta chain comprises 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 SEQ ID NO: 16. In some embodiments, the alpha chain comprises the amino acid sequence of SEQ ID NO: 11 and the beta chain comprises the amino acid sequence of SEQ ID NO: 16.

[0012] 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 attenuate an effector function of the Fc domain. In some embodiments, the antibody Fc domain is a human IgG1 Fc domain comprising a mutation at residue N297, numbered according to the EU index. In some embodiments, the antibody Fc domain is a human IgG1 Fc domain comprising an N297G substitution, numbered according to the EU index. In some embodiments, the antibody Fc domain is a human IgG1 Fc domain comprising one or more mutation(s) at residue(s) E233, L234, L235, and / or G236, numbered according to the EU index. In some embodiments, the antibody Fc domain is a human IgG1 Fc domain comprising a deletion at substitutions N297G, E233P, L234V, L235A, and G236, numbered according to the EU index. In some embodiments, the antibody Fc domain is a human IgG1 Fc domain comprising one or more mutation(s) at residue(s) L234, L235, and P329, numbered according to the EU index. In some embodiments, the antibody Fc domain is a human IgG1 Fc domain comprising substitutions L234A, L235A, and P329G, numbered according to the EU index. 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 that is associated with the first antibody Fc domain via: (1) one or more covalent bonds; and / or (2) one or more amino acid substitutions in one or both of the antibody Fc domains that promote heterodimerization.

[0013] In some embodiments, the first and second antibody Fc domains both comprise antibody CH2 and CH3 domains. In some embodiments, the first antibody Fc domain is fused to the TCR via a first hinge sequence, and the second hinge sequence is linked to the N-terminus of the second antibody Fc domain. In some embodiments, the first and second hinge sequences are linked via one or more interchain disulfide bonds between the first and second hinge sequences. In some embodiments, both the first and second hinge sequences comprise the amino acid sequence of DKTHTCPP (SEQ ID NO: 31) or DKTHTCPPC (SEQ ID NO: 36). In some embodiments, to promote heterodimerization of the antibody Fc domains, 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. 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, numbered according to 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.

[0014] In some embodiments according to any of the embodiments described herein, the T cell engaging domain binds to human CD3 expressed on the cell surface of a T cell. In some embodiments, the T cell engaging domain comprises an antibody antigen-binding domain. In some embodiments, the T cell engaging 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 engaging domain is covalently linked to the TCR via a linker. In some embodiments, the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 18-25. In some embodiments, the C-terminus of the T cell engaging domain is covalently linked to the N-terminus of the TCR beta chain variable domain. In some embodiments, the C-terminus of the T cell engaging domain is covalently linked to the N-terminus of the TCR beta chain variable domain via a linker. In some embodiments, the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 18-25. 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. 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 TCR fusion comprises three polypeptides: (a) a first polypeptide comprising, from N-terminus to C-terminus, a TCR alpha chain variable region, an alpha chain constant region, a first hinge sequence, and a 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 a T cell, a linker, a beta chain variable region, and a beta 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. In some embodiments, the first and second polypeptides are linked via one or more disulfide bonds between the alpha chain constant region and the beta chain constant region.In some embodiments, the first and third polypeptides are linked via (1) one or more interchain disulfide bonds between the first and second hinge sequences; and / or (2) one or more corresponding knob- and hole-forming mutations on the antibody Fc domain. In some embodiments, the first polypeptide comprises the amino acid sequence of SEQ ID NO:29, the second polypeptide comprises the amino acid sequence of SEQ ID NO:30, and the third polypeptide comprises the amino acid sequence of SEQ ID NO:28.

[0015] In another aspect, 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.

[0016] In another aspect, provided herein is a polynucleotide encoding a TCR fusion protein according to any one of the above embodiments. Further provided herein is a kit of polynucleotides comprising a first polynucleotide encoding a first polypeptide according to any one of the above embodiments, a second polynucleotide encoding a second polypeptide according to any one of the above embodiments, and a third polynucleotide encoding a third polypeptide according to any one of the above embodiments. In another aspect, provided herein is a vector comprising a polynucleotide(s) according to any one of the above embodiments. Further provided herein is a kit of vectors comprising a first vector encoding a first polypeptide according to any one of the above embodiments, a second vector encoding a second polypeptide according to any one of the above embodiments, and a third vector encoding a third polypeptide according to any one of the above embodiments. In some embodiments, the vector(s) are expression vector(s).

[0017] In another aspect, provided herein is a host cell comprising a polynucleotide(s), a kit of polynucleotides, a vector(s), or a kit of 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.

[0018] In another aspect, provided herein is a method of producing a TCR fusion protein, the method comprising culturing a host cell according to any one of the above embodiments under conditions suitable for production of the TCR fusion protein. In some embodiments, the method further comprises recovering the TCR fusion protein from the host cell. Further provided herein is a TCR fusion protein produced by a method according to any one of the above embodiments.

[0019] In another aspect, provided herein is a pharmaceutical composition comprising a TCR fusion protein according to any one of the above embodiments and a pharmaceutically acceptable carrier.

[0020] In another aspect, provided herein is a method for treating cancer, comprising administering to an individual 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. Further provided herein is a TCR fusion protein according to any one of the above embodiments for use in medicine, preferably in a human subject. Further provided herein is a TCR fusion protein according to any one of the above embodiments for use in treating cancer, preferably in a human subject. Further provided herein is the use of a TCR fusion protein according to any one of the above embodiments in the manufacture of a medicament for treating cancer.

[0021] In some embodiments according to any of the embodiments described herein, the individual is a human. In some embodiments, the individual has a cancer that expresses MAGE-A4. In some embodiments, the individual has an HLA-A *The individual has a tumor of the 02 subtype. In some embodiments, the TCR fusion protein or composition is administered by intravenous or intratumoral injection. In some embodiments, the method further comprises administering to the individual a second anti-cancer agent.

[0022] It should be understood that one, some, or all of the features of the various embodiments described herein may be combined to form other embodiments of the present invention. These and other aspects of the present invention will become apparent to those skilled in the art. These and other embodiments of the present invention are further described in the detailed description that follows. [Brief explanation of the drawings]

[0023] [Figure 1] 1A and 1B show schematic diagrams of T cell receptor (TCR):anti-CD3 fusion molecules according to some embodiments. In the example shown in Fig. 1A, effector function is provided by an anti-CD3 single-chain variable fragment (scFv) fused to the N-terminus of the TCR beta 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 an N297G mutation) fused to the C-terminus of the TCR alpha chain. Figure 1B shows more detailed features, including knob-into-hole (KIH) mutations to provide Fc heterodimerization (in this example, T366W on one chain and T366S / L368A / Y407V on the other chain), disulfide bonds in the hinge region, engineered disulfide bonds between the alpha and beta chains of the TCR, the TCR constant regions (Cα and Cβ on the alpha and beta chains, respectively), and the TCR variable regions (Vα and Vβ on the alpha and beta chains, respectively).

[0024] [Figure 2-1]Figures 2A and 2B show the N-glycosylation of TCR chains. Figure 2A shows a schematic of all seven N-glycosylation sites on the TCR (left) and the occupancy of each N-glycosylation site (right). Figure 2B shows variant TCRs with a set of substitution mutations (e.g., N→Q) at various glycosylation sites, resulting in aglycosylated (left) or monoglycosylated (right) TCRs. The arrow indicates the single remaining N-glycosylation site.

[0025] [Figure 2-2] Figures 2C and 2D show the effect of deglycosylation on the yield of TCR:anti-CD3 fusion molecules. Figure 2C shows that removal of N-glycosylation sites from the TCR variable region (using an N→Q substitution) significantly reduced yield, whereas removal of N-glycosylation sites from the TCR constant region had no effect on yield. Figure 2D shows that preservation of N-glycosylation at residue N18 of the alpha chain variable region was the most important site for increasing yield.

[0026] [Figure 3-1] Figures 3A-3E show the in vivo pharmacokinetic properties of TCR:anti-CD3 fusion molecules. Figure 3A shows the in vivo pharmacokinetic properties of various formats of TCR:anti-CD3 fusion molecules with or without an Fc domain, as indicated. Figure 3B shows the serum concentrations over time of N297G control, aglycosylated, or monoglycosylated TCR:anti-CD3 fusion molecules with the indicated Fc formats in a SCID mouse model. Figures 3C and 3D show the serum concentrations over time of monoglycosylated or aglycosylated (respectively) TCR:anti-CD3 fusion molecules shown in Figure 3B in a SCID mouse model. Figure 3E shows the half-life and clearance of aglycosylated or monoglycosylated TCR:anti-CD3 fusion molecules shown in Figure 3B administered at the indicated dose levels. [Figure 3-2] Same as above. [Figure 3-3] Same as above.

[0027] [Figure 4-1] Figures 4A-4D show the potency and selectivity of aglycosylated or monoglycosylated TCR:anti-CD3 fusion molecules. Figure 4A shows the name and type of each cell line, the average copy number and mRNA expression of MAGE-A4 for each cell line, the HLA-A2 expression for each cell line, and the EC50 for cell killing or IFNγ release observed upon treatment with monoglycosylated ("mono") or aglycosylated ("aglyc") TCR:anti-CD3 fusion molecules. ** indicates that the data were averaged from three different PBMC donors. Figures 4B-4D show the percentage of cell lysis over time for NCI-H1755 (Figure 4B), SCaBER (Figure 4C), or NCI-H441 (Figure 4D) cell lines treated with aglycosylated or monoglycosylated TCR:anti-CD3 fusion molecules in the presence of effector cells. The values ​​shown in Figures 4B-4D refer to the lowest concentration of TCR:anti-CD3 fusion molecule observed to produce a killing response. [Figure 4-2] Same as above.

[0028] [Figure 5-1] Figures 5A-5C show the loss of potency upon infusion of Fc, which was partially offset by the use of a variant anti-CD3 scFv. Figures 5A and 5B show the efficacy of cell killing against NCI-H1755 (Figure 5A) or A375 (Figure 5B) cell lines mediated by TCR:anti-CD3 fusion molecules without Fc, TCR:anti-CD3 fusion molecules with Fc, and TCR:anti-CD3 fusion molecules with Fc and a variant anti-CD3 scFv. Figure 5C shows the efficacy of cell killing against MAGE-A4+NCI-H1755 cells versus MAGE-A4-MEL202A2B2M cells, demonstrating the window between on-target and off-target activity. [Figure 5-2] Same as above.

[0029] [Figure 6]Figures 6A and 6B show the results of in vitro safety assays testing TCR:anti-CD3 fusion molecules. Figure 6A 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 6B shows the results of testing a variant anti-CD3 scFv and a TCR:anti-CD3 fusion molecule with an Fc domain against the same molecule without the Fc domain or variant scFv, demonstrating that the therapeutic window against on- and off-target cells was maintained. DETAILED DESCRIPTION OF THE INVENTION

[0030] I. General techniques The techniques and procedures described or referenced herein are widely understood and commonly used by those skilled in the art using conventional methodology (e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (F.M.A.usubel, et al. eds., (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988); Antibodies, A Laboratory Manual, and Animal Cell Culture (R.I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Manual, Humana Press, NY; Notebook(JECellis,ed.,1998)Academic Press;Animal Cell Culture(RIFreshney),ed.,1987);Introduction to Cell and Tissue Culture(JPMather and PERoberts,1998)Plenum Press;Cell and Tissue Culture:Laboratory Procedures(A.Doyle,JBGriffiths,and DGNewell,eds.,1993-8)J.Wiley and Sons; Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.);Gene Transfer Vectors for Mammalian Cells(JMMiller and MPCalos,eds.,1987);PCR: The Polymerase Chain Reaction,(Mullis et al.,eds.,1994);Current Protocols in Immunology(JEColigan et al.,eds.,1991);Short Protocols in Molecular Biology(Wiley and Sons, 1999); Immunobiology (CA Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); 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 Publishers, 1995); and Cancer: Principles and Practice of Oncology (VT DeVita et al., eds., J.B. Lippincott Company, 1993), among other widely used methodologies.

[0031] II. Definition The TCR sequences defined herein are described with reference to the IMGT nomenclature, which is widely known and available to those working in the TCR field. See, e.g., LeFranc and LeFranc, (2001). "T cell Receptor Factsbook," Academic Press; Lefranc, (201 1), Cold Spring Harb Protoc 201 1(6):595-603; Lefranc, (2001), Curr Protoc Immunol Appendix 1:Appendix 100; and Lefranc, (2003), Leukemia 17(1):260-266. Briefly, TCRs consist of two disulfide-linked chains. Each chain (alpha and beta) is generally considered to have two domains: a variable domain and a constant domain. A short junction region connects the variable and constant domains and is typically considered part of the alpha variable region. In addition, the beta chain usually contains a short diversity region adjacent to the joining region, which is also typically considered part of the beta variable region.

[0032] The variable domain of each chain is located at the N-terminus and contains three complementarity-determining regions (CDRs) embedded in a framework sequence. The CDRs contain the recognition sites for peptide-MHC binding. There are several genes encoding alpha chain variable (Va) regions and several genes encoding beta chain variable (Vβ) regions, which are distinguished by the framework, CDR1 and CDR2 sequences, and by a partially defined CDR3 sequence. Va and Vβ genes are referred to by the prefixes TRAV and TRBV, respectively, in the IMGT nomenclature (Folch and Lefranc, (2000), Exp Clin Immunogenet 17(1):42-54; Scaviner and Lefranc, (2000), Exp Clin Immunogenet 17(2):83-96; LeFranc and LeFranc, (2001), "T cell receptor facts book", Academic Press). Similarly, there are several joining genes, or J genes, designated TRAJ or TRBJ for the alpha and beta chains, respectively, and diversity genes, or D genes, designated TRBD for the beta chain (Folch and Lefranc, (2000), Exp Clin Immunogenet 17(2):107-114; Scaviner 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 results from combinatorial rearrangements and junctional 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 1 14(19):4099-4107).The constant (i.e., C) regions of the alpha and beta chains of the TCR are referred to as TRAC and TRBC, respectively (Lefranc, (2001), Curr Protoc Immunol Appendix 1:Appendix 10).

[0033] "Engineered TCR" and "mutant TCR" are used interchangeably herein to refer to a TCR that has one or more mutations introduced, particularly in its alpha and / or beta chain variable domains, compared to the native MAGE A4 TCR. The mutation(s) typically comprise the GVYDGREHTV (SEQ ID NO: 34) HLA-A * The mutations improve the binding affinity of the TCR to the HLA-A 02 complex, but may additionally or alternatively confer other advantages, such as improved stability in isolated form and improved specificity. Mutations at one or more positions may additionally or alternatively affect the interaction with the cognate pMHC complex at an adjacent position, for example, by allowing a more favorable angle for the interaction. GVYDGREHTV (SEQ ID NO: 34) HLA-A * Preferably, mutations are made within one or more of the CDR regions to improve binding of the TCR to the O2 complex.

[0034] Phenotypically silent variants of any TCR disclosed herein are within the scope of this disclosure. As used herein, the term "phenotypically silent variant" is understood to refer to a TCR that incorporates one or more additional amino acid changes (including substitutions, insertions, and deletions) in addition to the changes described above, which TCR has a similar phenotype to a corresponding TCR that does not have said change(s). For purposes of this application, TCR phenotype is defined as the antigen binding affinity (K D and / or binding half-life) and antigen specificity. Phenotypically silent variants have a measured K of the corresponding TCR without said alteration(s) when measured under identical conditions (e.g., 25°C and / or on the same SPR chip). Dand / or GVYDGREHTV (SEQ ID NO: 34) HLA-A within 50%, or more preferably within 20%, of the binding half-life * K for O2 complex D and / or may have a binding half-life. As known to those skilled in the art, GVYDGREHTV (SEQ ID NO: 34) HLA-A * It may be possible to generate TCRs that incorporate changes in their variable domains compared to the TCRs detailed above without altering the affinity of their interaction with the O2 complex. In particular, such silent mutations may be incorporated within portions of the sequence known not to be directly involved in antigen binding (e.g., CDRs, or portions of CDRs that do not contact the peptide antigen). Such obvious variants are included within the scope of the present disclosure.

[0035] A phenotypically silent variant may contain one or more conservative substitutions and / or one or more tolerated substitutions. The tolerated substitutions and conservative substitutions are those that are higher than the measured K of the corresponding TCR without the conservative and / or tolerated substitution(s) when measured under the same conditions (e.g., 25°C and / or the same SPR chip). D and / or GVYDGREHTV (SEQ ID NO: 34) HLA-A within 50%, or more preferably within 20%, and even more preferably within 10% of the binding half-life * K for O2 complex D and / or binding half-life may vary, provided that K D provided that the change in affinity does not result in an affinity lower (i.e., weaker) than 200 μM. By acceptable substitutions we mean substitutions that do not fall within the definition of conservative provided below, but which are nevertheless phenotypically silent.

[0036] The TCRs of the present disclosure may contain one or more conservative substitutions that have a similar amino acid sequence and / or retain the same function (i.e., are phenotypically silent as defined above). Those skilled in the art will recognize 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 substituted with one or more other such amino acids without eliminating the desired activity of said protein, polypeptide, or peptide.

[0037] Therefore, the amino acids glycine, alanine, valine, leucine, and isoleucine can often be substituted for each other (amino acids with aliphatic side chains).Among these possible substitutions, it is preferable to use glycine and alanine to substitute for each other (because they have relatively short side chains), and valine, leucine, and isoleucine to substitute for each other (because they have larger aliphatic side chains that are hydrophobic).Other amino acids that can often be substituted for each other 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 recognized that amino acid substitutions within the scope of the present disclosure can be made using naturally occurring or non-naturally occurring amino acids. For example, it is contemplated herein that the methyl group on alanine may be replaced with an ethyl group and / or the peptide backbone may be slightly altered. Whether natural or synthetic amino acids are used, it is preferred that only L-amino acids be present.

[0038] "Identity," as known in the art, is the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, identity also refers to the degree of relatedness between polypeptide sequences or polynucleotide sequences, sometimes determined by the match between strings of such sequences. There are several methods for measuring the identity between two polypeptide sequences or two polynucleotide sequences, but the commonly used methods for determining identity are codified in computer programs. Preferred computer programs for determining the identity between two sequences include, but are not limited to, the GCG program package (Devereux, et al., Nucleic Acids Research, 12, 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul et al., J. Molec. Biol. 215, 403 (1990)).

[0039] A program such as the CLUSTAL program can be used to compare amino acid sequences. This program compares amino acid sequences and finds the optimal alignment by inserting spaces in either sequence as needed. It is possible to calculate the amino acid identity or similarity (identity + conservation of amino acid type) for the optimal alignment. A program such as BLASTx aligns the longest stretch of similar sequences and assigns a value for the degree of match. Thus, a comparison can find several similar regions, each with a different score. Both types of identity analysis are contemplated in this disclosure.

[0040] The identity percentage of two amino acid sequences or two nucleic acid sequences is determined by aligning the sequences for optimal comparison (for example, gaps can be introduced into the first sequence for the best alignment with this sequence), and then comparing the amino acid residues or nucleotides at corresponding positions.The "best alignment" is the alignment of two sequences that produces the highest identity percentage.The identity percentage is determined by the number of identical amino acid residues or nucleotides in the sequences being compared (i.e., identity%=number of identical positions / total number of positions×100).

[0041] The determination of percent identity between two sequences can be performed using a mathematical algorithm 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, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. The NBLAST and XBLAST programs of Altschul, et al. (1990) J. Mol. Biol. 215:403-410 incorporate such an algorithm. BLAST nucleotide searches can be performed using the NBLAST program, score=100, word length=12, to obtain nucleotide sequences homologous to nucleic acid molecules. BLAST protein searches can be performed using the XBLAST program, score=50, word length=3, to obtain amino acid sequences homologous to protein molecules used in the present disclosure. To obtain gapped alignments for comparison, Gapped BLAST can be used as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast can be used to perform an iterated search that detects distant relationships between molecules (ibid.). 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 utilized for sequence comparison is the algorithm of Myers and Miller, CABIOS (1989). The ALIGN program (version 2.0), which is part of the CGC sequence alignment software package, incorporates such an algorithm.Other algorithms for sequence analysis known in the art include ADVANCE and ADAM, described in Torellis and Robotti (1994) Comput. Appl. Biosci., 10:3-5; and FASTA, described in Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-8. Within FASTA, ktup is a control option that sets the sensitivity and speed of the search.

[0042] Mutations, including conservative and tolerated substitutions, insertions, and substitutions, can be introduced into the provided sequences using any suitable method, including, but not limited to, methods based on polymerase chain reaction (PCR), restriction enzyme-based cloning, or ligation-independent cloning (LIC) procedures, which are detailed in many standard molecular biology textbooks.

[0043] The TCR of the present disclosure may be an αβ heterodimer. The TCR of the present disclosure may be in a single-chain format. Single-chain formats include, but are not limited to, αβ TCR polypeptides of the Vα-L-Vβ, Vβ-L-Vα, Vα-Cα-L-Vβ, Vα-L-Vβ-Cβ, or Vα-Cα-L-Vβ-Cβ types, where Vα and Vβ are TCR α and β variable regions, respectively, Cα and Cβ are TCR α and β constant regions, respectively, and L is a linker sequence (Weidanz et al., (1998) J Immunol Methods. December; 221(1-2):59-76; Epel et al., (2002), Cancer Immunol Immunother. November; 51(10):565-73; WO 2004 / 033685; WO 9918129). One or both of the constant domains may be full-length, truncated as described above, and / or contain mutations. The alpha chain extracellular constant region may have an asparagine (N) or lysine (K) residue at position 4 due to natural polymorphism. In certain embodiments, the single-chain TCRs of the present disclosure may have a disulfide bond introduced between residues of each constant domain, as described in WO 2004 / 033685. Single-chain TCRs are further described in WO 2004 / 033685; WO 98 / 39482; WO 01 / 62908; Weidanz et al. (1998) J Immunol Methods 221(1-2):59-76; Hoo et al. (1992) Proc Natl Acad Sci USA 89(10):4759-4763; Schodin (1996) Mol Immunol 33(9):819-829).

[0044] The term "antibody" includes monoclonal antibodies (including full-length antibodies having an immunoglobulin Fc domain), antibody compositions with polyepitopic specificity, multispecific antibodies (e.g., bispecific antibodies, diabodies, and single-chain molecules), and antibody fragments (e.g., Fab, F(ab'), and Fv). The term "immunoglobulin" (Ig) is used interchangeably with "antibody" herein.

[0045] The basic four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies consist of five basic heterotetrameric units along with an additional polypeptide called the J chain and contain 10 antigen-binding sites, while IgA antibodies contain two to five basic four-chain units, which can polymerize to form multivalent aggregates with the J chain. In the case of IgG, the four-chain unit is generally approximately 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds, depending on the H chain isotype. Each H and L chain also has equally spaced intrachain disulfide bridges. Each H chain consists of an N-terminal variable domain (V H ), and three constant domains (C H ), and four C for μ and ε isotypes H Each L chain has an N-terminal, variable domain (V L ) followed at the other end by a constant domain. L is V H It is aligned with C L is the first constant domain of the heavy chain (C H 1). Particular amino acid residues are thought to form an interface between the light-chain variable domain and the heavy-chain variable domain. V H and V Lpair together to form 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. Light chains from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequence of their constant domains. Depending on the amino acid sequence of the constant domain (CH) of the heavy chain, 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 gamma and alpha classes are further divided into subclasses based on relatively minor differences in CH sequence and function (e.g., humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgA2).

[0046] 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 domains of the heavy and light chains may be referred to as "VH" and "VL," respectively. These domains are generally the most variable parts of an antibody (compared to other antibodies of the same class) and contain the antigen-binding site.

[0047] The term "variable" refers to the fact that the sequences of certain segments of the variable domains vary greatly among antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, variability is not uniformly distributed throughout the variable domains. Instead, variability is concentrated in three segments called hypervariable regions (HVRs) in both the light- and heavy-chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). Native heavy- and light-chain variable domains each contain four FR regions, which primarily adopt a beta-sheet configuration and are connected by three HVRs that form loops that connect to and, in some cases, form part of the beta-sheet structure. The HVRs in each chain are held together in close proximity by the FR regions and, together with HVRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Immunological Interest, Fifth Edition, National Institutes 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.

[0048] As used herein, the term "monoclonal antibody" 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 minor naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation)). Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by a hybridoma culture, uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous antibody population, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies for use in accordance with the present disclosure can be produced using a wide variety of techniques, including hybridoma techniques (e.g., 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, 2002) nded. 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. 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 technology for producing human or human-like antibodies in animals that have some or all of the 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. al.,Proc.Natl.Acad.Sci.USA90:2551(1993);Jakobovits et al.,Nature362:255-258(1993);Bruggemann et al.,Year in Immunol. 7:33 (1993); U.S. Patent No. 5,545,807; U.S. Patent No. 5,545,806; U.S. Patent No. 5,569,825; al.,Bio / Technology10:779-783(1992);Lonberg et al.,Nature368:856-859(1994);Morrison,Nature368:812-813(1994);Fishwild et al. al.,Nature Biotechnol.14:845-851 (1996); Neuberger, Nature Biotechnol. 14:826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13:65-93 (1995)).

[0049] The term "naked antibody" refers to an antibody that is not conjugated to a cytotoxic moiety or radiolabel.

[0050] The terms "full length antibody," "intact antibody," or "whole antibody" are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antibody fragment. Specifically, a whole antibody includes an antibody having a heavy chain and a light chain, including 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, an intact antibody may have one or more effector functions.

[0051] An "antibody fragment" comprises a portion of an intact antibody, preferably the antigen-binding and / or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (see Example 2 of U.S. Pat. No. 5,641,870; Zapata et al., Protein Eng. 8(10):1057-1062

[1995] ); single-chain antibody molecules formed from antibody fragments, and multispecific antibodies. Papain digestion of an antibody produces two identical antigen-binding fragments called "Fab" fragments, and a residual "Fc" fragment (a name reflecting the ability to crystallize readily). Fab fragments comprise the variable region domain of the heavy chain (V H ) and the first constant domain of one heavy chain (C HFab fragments consist of an entire L chain together with a C(ab')2 fragment. Each Fab fragment is monovalent with respect to antigen binding (i.e., has a single antigen-binding site). Pepsin treatment of an antibody produces a single large F(ab')2 fragment, which roughly corresponds to two disulfide-linked Fab fragments with different antigen-binding activities and is still capable of cross-linking antigen. The Fab' fragment contains one or more cysteines from the hinge region, C(ab')2, and C(ab')2. H F(ab')2 antibody fragments differ from Fab fragments by having several additional residues at the carboxy terminus of one domain. 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 which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0052] The Fc fragment contains the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of an antibody are determined by sequences in the Fc domain, the region also recognized by Fc receptors (FcRs) found on certain cell types.

[0053] An "Fv" is the minimum antibody fragment that contains a complete antigen-recognition and antigen-binding site. This fragment consists of a dimer of one heavy-chain and one light-chain variable region domain in tight, non-covalent association. The folding of these two domains results in six hypervariable loops (three loops each from the H and L chains) that provide the amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific HVRs) has the ability to recognize and bind to antigen, albeit with lower affinity than the entire binding site.

[0054] "Single-chain Fv," also abbreviated as "sFv" or "scFv," is a VFv fragment that is linked to a single polypeptide chain. H and V L Preferably, the sFv polypeptide is an antibody fragment containing the V HDomains and V L The sFv further comprises a polypeptide linker between the domains that enables the sFv to form the desired structure for antigen binding. For a review of sFvs, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0055] "Functional fragments" of antibodies of the present invention comprise a portion of an intact antibody (generally comprising the antigen-binding or variable region of the intact antibody) or the Fc domain of an antibody that retains or has altered FcR binding ability. Examples of antibody fragments include linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0056] The term "diabody" refers to a diabody formed by combining V domains using a short linker (about 5-10 residues) such that inter-chain but not intra-chain pairing of the V domains occurs, thereby resulting in a bivalent fragment (i.e., a fragment with two antigen-binding sites). H Domains and V L Bispecific diabodies refer to small antibody fragments prepared by constructing an sFv fragment (see previous paragraph) between the V domains of two antibodies. H Domains and V L Diabodies are heterodimers of two "crossover" sFv fragments whose 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).

[0057] The monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, so long as the desired biological activity is exhibited, and also include fragments of such antibodies (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies of interest herein include PRIMATIZED® antibodies in which the antigen-binding region of the antibody is derived from, for example, an antibody produced by immunizing macaque monkeys with an antigen of interest. As used herein, "humanized antibody" is used as a subset of "chimeric antibody."

[0058] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In one embodiment, humanized antibodies are human immunoglobulins (recipient antibodies) in which residues from an HVR (defined below) of the recipient are replaced by residues from an HVR (donor antibody) of a non-human species, such as mouse, rat, rabbit, or non-human primate, having the desired specificity, affinity, and / or capacity. In some instances, framework ("FR") residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications may be made to further refine antibody performance, such as binding affinity. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which 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 the 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 FRs will typically be no more than six in the H chain and no more than three in the L chain. The humanized antibody will also optionally comprise at least a portion of an immunoglobulin constant region (Fc), typically that 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). See, e.g., 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. Patent Nos. 6,982,321 and 7,087,409.

[0059] A "human antibody" is an antibody that possesses an amino acid sequence corresponding to that of an antibody produced by a human and / or that has been produced using any of the techniques for producing human antibodies disclosed herein. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. Human antibodies can be produced using a variety of 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). Human monoclonal antibodies can also be prepared using 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 prepared by administering antigen to transgenic animals (e.g., immunized xenogeneic mice) that have been engineered to produce such antibodies in response to antigen challenge, but whose endogenous gene loci have been disabled (see, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584 regarding XENOMOUSE™ technology). See also, e.g., Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006) regarding human antibodies generated via human B cell hybridoma technology.

[0060] The terms "hypervariable region," "HVR," or "HV" as used herein refer to the region of an antibody variable domain that exhibits sequence hypervariability and / or forms structurally defined loops. Antibodies generally contain six HVRs: three in the VH (H1, H2, and H3) and three in the VL (L1, L2, and L3). In native antibodies, H3 and L3 exhibit the highest diversity among the six HVRs, and H3, in particular, is thought to play a unique role in conferring exquisite 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). In fact, naturally occurring camelid antibodies consisting only of heavy chains are functional and stable in the absence of light chains. See, e.g., Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

[0061] Several HVR delineations are in use and are included herein. Kabat complementarity-determining regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Chothia instead refers to the location of structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). AbM HVRs represent intermediate positions between Kabat HVRs and Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software. "Contact" HVRs are based on analysis of available complex crystal structures. Residues from each of these HVRs are shown below. [Table 1]

[0062] HVRs may include the following "extended HVRs": 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. The variable domain residues are numbered according to Kabat et al., supra, for each of these definitions.

[0063] The phrases "Kabat variable domain residue numbering" or "Kabat amino acid position numbering," and variations thereof, refer to the numbering system used for the heavy or light chain variable domains of 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 a shortening of, or insertion into, the FR or HVR of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insert 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). The Kabat numbering of residues may be determined for a given antibody by alignment with the "standard" Kabat-numbered sequence at the homologous regions of the antibody sequence.

[0064] "Framework" or "FR" residues are those variable domain residues other than the HVR residues as herein defined.

[0065] A "human consensus framework" or "acceptor human framework" is a framework that represents the amino acid residues most commonly found in a selection of human immunoglobulin VL or VH framework sequences. Generally, the human immunoglobulin VL or VH sequences are selected from a subgroup of variable domain sequences. Generally, the subgroup of sequences is selected from the subgroups of variable domain sequences described in Kabat et al., Sequences of Proteins of Immunological Interest, 5 th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991). Exemplary subgroups for VL can be subgroup kappa I, kappa II, kappa III, or kappa IV as in Kabat et al., supra. Additionally, for VH, the subgroup can be subgroup I, subgroup II, or subgroup III as in Kabat et al., supra. Alternatively, a human consensus framework can be derived from the above at specific residues (e.g., when human framework residues are selected based on their homology with the donor framework by aligning the donor framework sequence with a variety of different 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 or can contain changes in an existing amino acid sequence. 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.

[0066] For example, an "amino acid modification" at a specified position in 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 means an insertion within 1 to 2 residues thereof. The insertion may be at the N-terminus or C-terminus of the specified residue. A preferred amino acid modification herein is a substitution.

[0067] An "affinity matured" antibody is an antibody with one or more alterations in one or more HVRs thereof, resulting in improved affinity of the antibody for antigen compared to a parent antibody lacking the alteration(s). In one embodiment, the affinity matured antibody has nanomolar or even picomolar affinity for the target antibody. Affinity matured antibodies are produced by procedures known in the art. For example, Marks et al., Bio / Technology 10:779-783 (1992) describe affinity maturation by VH- and VL-domain shuffling. Random mutagenesis of HVR and / or framework residues is described, for example, in 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).

[0068] As used herein, the terms "specifically bind to" or "specific for" refer to a measurable and reproducible interaction, such as binding between a target and an antibody, that determines 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 an antibody that binds to this target with higher affinity, avidity, more readily, and / or for a longer duration than it binds to other targets. In one embodiment, the extent to which an antibody binds to an unrelated target is less than about 10% of the binding of the antibody to the target as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that specifically binds to a target has a dissociation constant (Kd) of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, or 0.1 nM or less. In certain embodiments, an antibody specifically binds to an epitope on a protein that is conserved among proteins from different species. In another embodiment, specific binding can include, but is not required to include, exclusive binding.

[0069] The term "Fc domain" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including native-sequence and variant Fc domains. Although the boundaries of an immunoglobulin heavy chain Fc domain can vary, the human IgG heavy chain Fc domain is usually defined as extending from the amino acid residue at Cys226, or from Pro230, to its carboxyl terminus. The C-terminal lysine of the Fc domain (residue 447 according to the EU numbering system) can be removed, for example, during antibody production or purification, or by recombinantly engineering the nucleic acid encoding the antibody heavy chain. Thus, intact antibody compositions can include antibody populations in which all K447 residues have been removed, antibody populations in which the K447 residue has not been removed, and antibody populations containing a mixture of antibodies with and without the K447 residue. Native-sequence Fc domains suitable for use in the antibodies of the present invention include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0070] "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. Further, a preferred FcR is one that binds IgG antibodies (gamma receptors), including receptors of the FcγRI, FcγRII, and FcγRIII subclasses (including allelic variants and alternatively spliced ​​forms of these receptors). FcγRII receptors include FcγRIIA ("activating receptors") and FcγRIIB ("inhibiting receptors"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (see M. Daeoron, 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.

[0071] The term "Fc receptor" or "FcR" also includes the neonatal receptor FcRn, which is responsible for the transfer 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.)). The in vivo FcRn binding and half-life of human FcRn high-affinity binding polypeptides can be assayed, for example, in transgenic mice or transfected human cell lines expressing human FcRn, or in primates administered with polypeptides having variant Fc domains. International Publication No. 2004 / 42072 (Presta) describes antibody variants with improved or reduced FcR binding. See, for example, Shields et al., J.Biol.Chem.9(2):6591-6604(2001).

[0072] The phrases "substantially reduced" or "substantially different," as used herein, refer to a difference between two numerical values ​​(generally one associated with a molecule and the other associated with a reference / comparator molecule) that is sufficiently high such that one skilled 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 values ​​(e.g., Kd values). 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%, depending on the value for the reference / comparator molecule.

[0073] The terms "substantially similar" or "substantially identical," as used herein, refer to a sufficiently high degree of similarity between two numerical values ​​(e.g., one value associated with an antibody of the invention and the other value associated with a reference / comparator antibody) such that one of skill in the art would consider the difference between the two values ​​to have little or no biological and / or statistical significance within the context of the biological characteristic measured by said values ​​(e.g., Kd values). 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%, depending on the reference / comparator value.

[0074] As used herein, the term "carrier" refers to a pharmaceutically acceptable carrier, excipient, or stabilizer that is nontoxic to cells or mammals exposed to the dosage and concentration used. Often, physiologically acceptable carriers are pH-buffered aqueous solutions. Examples of physiologically acceptable carriers include buffers (such as phosphate, citrate, and other organic acid buffers); 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 dextrin); 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®, polyethylene glycol (PEG), and PLURONICS®).

[0075] "Package insert" refers to instructions customarily included in commercial packaging of a pharmaceutical product containing information regarding the indications, including information regarding indications, usage, dosage, administration, contraindications, other medications with which the packaged product may be combined, and / or warnings regarding the use of such medications.

[0076] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include the plural unless the context clearly indicates otherwise. Thus, for example, reference to a "molecule" includes combinations of two or more such molecules, as appropriate.

[0077] The term "about" as used herein refers to a normal error range for each value, which is readily known to one skilled in the art. A reference to "about" a value or parameter herein includes (and describes) embodiments relating to said value or parameter itself.

[0078] It is understood that aspects and embodiments of the invention described herein include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

[0079] III. T Cell Receptors and Fusion Proteins In one embodiment, GVYDGREHTV (SEQ ID NO: 34) HLA-A * Provided herein are TCR fusion proteins comprising a TCR that binds to a 02 complex. In some embodiments, the TCR is a soluble TCR. In some embodiments, the TCR fusion protein comprises a T cell engaging domain that binds to a protein expressed on the cell surface of a T cell, and a TCR of the present disclosure (e.g., a soluble monoclonal TCR) fused or linked (e.g., covalently linked) to an antibody Fc domain. In some embodiments, the TCR is glycosylated, e.g., at a single N-linked glycosylation site. In some embodiments, the N-linked glycosylation site is, e.g., [ka] The alpha chain variable region is at residue N18, numbered according to

[0080] In some embodiments, the TCR comprises a TCR alpha chain comprising an alpha chain variable region and a TCR beta chain comprising a beta chain variable region. In some embodiments, 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 / or 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).

[0081] As is well known in the art, TCRs can be subject to post-translational modifications. Glycosylation is one such modification, involving the covalent attachment of oligosaccharide moieties to defined amino acids in the TCR chain. For example, asparagine residues or serine / threonine residues are well-known sites for oligosaccharide attachment. The glycosylation status of a particular protein depends on several factors, including protein sequence, protein conformation, and the availability of certain enzymes. Furthermore, the glycosylation status (i.e., oligosaccharide type, covalent linkages, and total number of linkages) can affect protein function. Therefore, when producing recombinant proteins, it is often desirable to control glycosylation. Glycosylation control has been used to improve antibody-based therapeutics. (Jefferis et al., (2009) Nat Rev Drug Discov Mar;8(3):226-34.) For the soluble TCRs of the present disclosure, glycosylation can be controlled in vivo, for example, by the use of specific cell lines, or in vitro by chemical modification. Such modifications are desirable because glycosylation can improve pharmacokinetics, reduce immunogenicity, and more closely mimic native human proteins (Sinclair and Elliott, (2005) Pharm Sci. Aug;94(8):1626-35).

[0082] In some embodiments, the TCR or TCR fusion protein of the disclosure is glycosylated, for example, at a single N-linked glycosylation site. In some embodiments, the N-linked glycosylation site is, for example, [ka] In some embodiments, the TCR fusion proteins of the present disclosure comprise a TCR glycosylated at a single N-linked glycosylation site, wherein the N-linked glycosylation site is at residue N18 of the alpha chain variable region numbered according to SEQ ID NO: 7. Advantageously, the present disclosure demonstrates that TCR fusion proteins with this single glycosylation site have superior manufacturability (e.g., protein production yield, resistance to heat stress and aggregation) and in vivo pharmacokinetics (e.g., half-life), in addition to retention of peptide:MHC binding affinity and target cell killing potency, compared to other glycosylation and / or aglycosylation variants.

[0083] In some embodiments, the TCR or TCR fusion proteins of the disclosure comprise an amino acid substitution at every potential N-glycosylation site other than residue N18. For example, in some embodiments, the TCR or TCR fusion proteins of the disclosure comprise an amino acid substitution(s) at one or more of residue N24 of the alpha chain variable region numbered according to SEQ ID NO:32; residues N33, N67, and N78 of the alpha chain constant region numbered according to SEQ ID NO:10; residue N84 of the beta chain variable region numbered according to SEQ ID NO:33; and residue N70 of the beta chain constant region numbered according to SEQ ID NO:15. In some embodiments, the TCR or TCR fusion proteins of the disclosure comprise amino acid substitution(s) at all of residue N24 of the alpha chain variable region numbered according to SEQ ID NO:32; residues N33, N67, and N78 of the alpha chain constant region numbered according to SEQ ID NO:10; residue N84 of the beta chain variable region numbered according to SEQ ID NO:33; and residue N70 of the beta chain constant region numbered according to SEQ ID NO:15.

[0084] In some embodiments, the amino acid substitution is an asparagine to a non-glycosylated amino acid, hi some embodiments, the amino acid substitution is an asparagine to glutamine (N→Q).

[0085] In some embodiments, a TCR or TCR fusion protein of the disclosure comprises one or more of the following amino acid substitutions: N24Q in the alpha chain variable region numbered according to SEQ ID NO: 32; N33Q, N67Q, and N78Q in the alpha chain constant region numbered according to SEQ ID NO: 10; N84Q in the beta chain variable region numbered according to SEQ ID NO: 33; and N70Q in the beta chain constant region numbered according to SEQ ID NO: 15. In some embodiments, a TCR or TCR fusion protein of the disclosure comprises all of the following amino acid substitutions: N24Q in the alpha chain variable region numbered according to SEQ ID NO: 32; N33Q, N67Q, and N78Q in the alpha chain constant region numbered according to SEQ ID NO: 10; N84Q in the beta chain variable region numbered according to SEQ ID NO: 33; and N70Q in the beta chain constant region numbered according to SEQ ID NO: 15.

[0086] The alpha-beta heterodimeric TCRs of the present disclosure typically comprise a TRAC constant domain sequence in the alpha chain and / or a TRBC1 or TRBC2 constant domain sequence in the beta chain. The alpha and beta chain constant domain sequences may be modified by truncation or substitution to eliminate the native disulfide bond between Cys4 in exon 2 of TRAC and Cys2 in exon 2 of TRBC1 or TRBC2. The alpha and / or beta chain constant domain sequence(s) may be modified by substitution of a cysteine ​​residue for Thr48 of TRAC and Ser57 of TRBC1 or TRBC2, which forms a disulfide bond between the alpha and beta constant domains of the TCR. TRBC1 or TRBC2 may 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 domains may additionally or alternatively contain further mutations, substitutions, or deletions compared to the native TRAC and / or TRBC1 / 2 sequences. The terms TRAC and TRBC1 / 2 include naturally occurring polymorphic variants (e.g., N to K at position 4 of TRAC) (Bragado et al. Int Immunol. 1994 Feb;6(2):223-30).

[0087] As will be apparent to those skilled in the art, it may be possible to shorten the provided sequences by 1, 2, 3, 4, 5, or more residues at the C-terminus and / or N-terminus without substantially affecting the binding properties of the TCR, and all such obvious variants are encompassed by the present disclosure.

[0088] The constant domains of wild-type or insoluble TCRs can be full-length or can be shortened and / or mutated to produce soluble TCRs. In either case, cysteine ​​substitutions can be introduced into the TRAC and TRBC regions to allow non-native interchain disulfide bond formation. Suitable locations for the cysteine ​​substitutions are described in WO 03020763.

[0089] In some embodiments, the TCR or TCR fusion protein of the disclosure comprises one or more engineered cysteine ​​residues in the constant regions of the alpha and / or beta chains to form a non-native disulfide bond between the alpha and beta chains. In certain embodiments, the single-chain TCRs of the disclosure may have disulfide bonds introduced between residues of the respective constant domains as described in WO 2004 / 033685. Single chain TCRs are further described in WO 2004 / 033685; WO 98 / 39482; WO 01 / 62908; Weidanz et al. (1998) J Immunol Methods 221(1-2):59-76; Hoo et al. (1992) Proc Natl Acad Sci USA 89(10):4759-4763; Schodin (1996) Mol Immunol 33(9):819-829). In some embodiments, the TCR or TCR fusion protein of the disclosure comprises a cysteine ​​residue at position 57 of the beta chain constant region, numbered according to SEQ ID NO: 15.

[0090] In some embodiments, the TCR or TCR fusion protein of the disclosure comprises: [ka] In some embodiments, the TCR or TCR fusion protein of the present disclosure comprises an alpha chain variable 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 SEQ ID NO:7.

[0091] In some embodiments, the TCR or TCR fusion protein of the disclosure comprises: [ka] [ka] In some embodiments, the TCR or TCR fusion protein of the disclosure comprises a beta chain variable 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 SEQ ID NO: 13. In some embodiments, the TCR or TCR fusion protein of the 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.

[0092] In some embodiments, the TCR or TCR fusion protein of the present disclosure comprises a TCR alpha chain and an alpha chain constant region comprising an alpha chain variable region of the present disclosure. [ka] In some embodiments, the TCR or TCR fusion protein of the present disclosure comprises an alpha 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 SEQ ID NO: 9.

[0093] In some embodiments, the TCR or TCR fusion protein of the present disclosure comprises a TCR beta chain and a beta chain constant region comprising a beta chain variable region of the present disclosure. [ka] In some embodiments, the TCR or TCR fusion protein of the disclosure comprises a beta 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 SEQ ID NO: 14. In some embodiments, the TCR or TCR fusion protein of the disclosure comprises an alpha chain constant region comprising the amino acid sequence of SEQ ID NO: 9 and a beta chain constant region comprising the amino acid sequence of SEQ ID NO: 14.

[0094] In some embodiments, a TCR or TCR fusion protein of the disclosure comprises an 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; and a beta chain comprising 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.

[0095] In some embodiments, the TCR or TCR fusion protein of the disclosure comprises: [ka] In some embodiments, the TCR or TCR fusion protein of the present disclosure comprises an alpha chain 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 SEQ ID NO: 11.

[0096] In some embodiments, the TCR or TCR fusion protein of the disclosure comprises: [ka] In some embodiments, the TCR or TCR fusion protein of the 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. In some embodiments, the TCR or TCR fusion protein of the 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.

[0097] In some embodiments, the TCR fusion proteins of the present disclosure comprise a T cell engaging domain that binds to a protein expressed on the cell surface of a T cell. In some embodiments, the T cell engaging domain binds to a protein expressed on the cell surface of a T cell and binds to the GVYDGREHTV (SEQ ID NO: 34) HLA-A * In some embodiments, the T cell engaging domain binds to a protein expressed on the cell surface of the T cell, e.g., activates the T cell via binding to the 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.

[0098] 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 part of or comprises a single chain variable fragment (scFv). In some embodiments, the scFv is an anti-CD3 scFv. Other single chain antibody fragment formats are known in the art.

[0099] In some embodiments, the scFv is a U28 variant anti-CD3 scFv. [ka] In some embodiments, the scFv comprises the amino acid sequence of: [ka] It contains the amino acid sequence of

[0100] In some embodiments, a T cell engaging domain (e.g., an 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 the group consisting of SEQ ID NOs: 18-25.

[0101] In some embodiments, the C-terminus of a 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 N-terminus of a TCR beta chain variable domain via a linker of the present disclosure.

[0102] 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, hi some embodiments, the hinge sequence comprises the amino acid sequence of DKTHTCPP (SEQ ID NO: 31) or DKTHTCPPC (SEQ ID NO: 36).

[0103] 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 an Fc domain of human IgG1, human IgG2, or human IgG4. Advantageously, the present disclosure demonstrates that TCR fusion proteins with an Fc domain fused to the alpha or beta chain have been found to have substantially improved pharmacokinetics compared to similar molecules without the Fc expressed from E. coli or CHO cells.

[0104] In some embodiments, the antibody Fc domain comprises one or more mutations that attenuate the effector function of the Fc domain. Exemplary effector functions include, but are not limited to, complement-dependent cytotoxicity (CDC) and / or antibody-dependent cellular cytotoxicity (ADCC). In exemplary embodiments, the modification to attenuate effector function is a modification that alters the glycosylation pattern of the Fc domain (e.g., a modification that results in an aglycosylated Fc domain). In exemplary embodiments, the modification to attenuate effector function is a modification that does not alter the glycosylation pattern of the Fc domain. In certain embodiments, the modification to attenuate 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 exemplary embodiments, the Fc variants described herein comprise an N297G or N297A modification in the Fc domain of human IgG1. In exemplary embodiments, the Fc variants described herein comprise the following modifications: L234A, L235A, and P329G in the Fc domain of human IgG1, which result in attenuated effector function. In some embodiments, the antibody Fc domain is a human IgG1 Fc domain comprising a mutation at residue N297, numbered according to the EU index. For example, in some embodiments, the mutation is an N297G substitution. Other suitable mutations (e.g., at residue N297) will be known to those of skill in the art.

[0105] In various embodiments, an Fc variant with reduced effector function refers to an Fc variant that reduces effector function (e.g., activities such as CDC, ADCC, and / or binding to FcR) 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 the 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 variant in which all detectable effector function is abolished compared to the wild-type Fc domain. Assays for measuring effector function are known in the art and are described below.

[0106] In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / depleted CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to confirm that an Fc domain or fusion protein lacks FcγR binding (and thus may lack ADCC activity) but retains FcRn binding ability. NK cells, the primary cells mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No. 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays may be used (see, e.g., the ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA); and the CytoTox96® 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 additionally, ADCC activity of the molecule of interest may 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)). C1q binding assays may also be performed to confirm that the antibody is unable to bind C1q and therefore lacks CDC activity.See, for example, C1q and C3c binding ELISA in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, CDC assays can be performed (see, for example, 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 determinations can also be performed using methods known in the art (see, for example, 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).

[0107] In some embodiments, the antibody Fc domain is a human IgG1 Fc domain that comprises one or more mutation(s) at residue(s) E233, L234, L235, and / or G236, numbered according to the EU index. For example, in some embodiments, the antibody Fc domain is a human IgG1 Fc domain that comprises the substitutions N297G, E233P, L234V, L235A, and a deletion at G236, numbered according to the EU index.

[0108] In some embodiments, the antibody Fc domain is a human IgG1 Fc domain comprising one or more mutation(s) at residue(s) L234, L235, and P329, numbered according to the EU index. For example, in some embodiments, the antibody Fc domain is a human IgG1 Fc domain comprising substitutions L234A, L235A, and P329G, numbered according to the EU index.

[0109] In some embodiments, the antibody Fc domain is fused to a TCR of the present disclosure via a hinge sequence, hi 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 two antibody Fc domains. In some embodiments, one of the two antibody Fc domains is fused or linked (e.g., covalently linked) to a TCR of the present disclosure. Figure 1B discloses a configuration in which one of the two antibody Fc domains is linked to the TCR alpha chain (e.g., via the alpha chain constant domain) according to some embodiments. In some embodiments, the two antibody Fc domains associate with each other, for example, via one or more covalent bonds and / or one or more amino acid substitutions in one or both of the antibody Fc domains that promote heterodimerization.

[0111] In some embodiments, the first antibody Fc domain is fused to the TCR via a first hinge sequence, and the second hinge sequence is linked to the N-terminus of the second antibody Fc domain. In some embodiments, the first and second hinge sequences are linked via one or more interchain disulfide bonds between the first hinge sequence and the second hinge sequence. In some embodiments, both the first and second antibody Fc domains further comprise a hinge sequence, and the first and second hinge sequences are linked via one or more interchain disulfide bonds between the first hinge sequence and the second hinge sequence. In some embodiments, both the first and second hinge sequences 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 (e.g., a sequence derived from an antibody hinge region).

[0112] 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.

[0113] 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, where a first antibody Fc domain contains amino acid modifications that form a convex portion in its CH3 domain, and a second antibody Fc domain contains amino acid modifications that form a concave portion in its CH3 domain. The convex portion can be positioned into the concave portion, thereby forming a TCR fusion protein upon assembly.

[0114] In this approach, two polypeptides comprising antibody Fc domains each contain an interface. The interface of one polypeptide interacts with a corresponding interface on the other polypeptide, allowing the two polypeptides to associate. These interfaces can be engineered so that a "knob" or "protrusion" (these terms may be used interchangeably herein) located in the interface of one polypeptide corresponds to a "hole" or "depression" (these terms may be used interchangeably herein) located in the interface of the other polypeptide. In some embodiments, the holes are the same size as or similar to the knobs and are suitably positioned so that when the two interfaces interact, the knobs of one interface can be positioned into the corresponding holes of the other interface. Without wishing to be bound by theory, this is believed to stabilize heteromultimers, favoring the formation of heteromultimers over other species (e.g., homomultimers). In some embodiments, this approach can be used to promote heteromultimerization of two different polypeptides, thereby promoting the association of two antibody Fc domains.

[0115] In some embodiments, knobs can be constructed by replacing small amino acid side chains with larger side chains. In some embodiments, holes can be constructed by replacing large amino acid side chains with smaller side chains. Knobs or holes can be present in the original interface or can be synthetically introduced. For example, knobs or holes can be synthetically introduced 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 small side chain volume (e.g., alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine), and the introduced residue for knob formation is a naturally occurring amino acid, which can include arginine, phenylalanine, tyrosine, and tryptophan. In some embodiments, the original residue has a large side chain volume (e.g., arginine, phenylalanine, tyrosine, and tryptophan), and the introduced residue for hole formation is a naturally occurring amino acid, which may include alanine, serine, threonine, and valine. [Table B]

[0116] 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 16 residues on each domain arranged on four antiparallel β-strands. Without wishing to be bound by theory, the mutated residues are preferably arranged on the two central antiparallel β-strands to minimize the risk that the knob may be accommodated in the surrounding solvent rather than in the compensatory hole in the partner CH3 domain. In some embodiments, the mutations that form corresponding knobs and holes in the two immunoglobulin polypeptides correspond to one or more pairs provided in the table below. [Table C]

[0117] 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.

[0118] 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, numbered according to the EU index.

[0119] In some embodiments, one of the antibody Fc domains comprises the amino acid sequence of SEQ ID NO: 27, and the other antibody Fc domain comprises the amino acid sequence of SEQ ID NO: 26. In some embodiments, a first antibody Fc domain is covalently linked to the TCR and comprises the amino acid sequence of SEQ ID NO: 27, and a second antibody Fc domain comprises the amino acid sequence of SEQ ID NO: 26.

[0120] After the DNA mutations discussed above, the polynucleotide encoding the modified antibody Fc domain with one or more corresponding knob or hole-forming mutations can be expressed and purified using standard recombinant technology and cell systems known in the art.See, for example, U.S. Patent Nos. 5,731,168; 5,807,706; 5,821,333; 7,642,228; 7,695,936; 8,216,805; U.S. Patent Application Publication No. 2013 / 0089553; and Spiess et al., Nature Biotechnology 31:753-758, 2013.The polypeptides comprising the antibody Fc domain with corresponding knob and hole can be expressed in host cells in co-culture and purified together as heteromultimers, or can be expressed in a single culture, purified separately, and assembled in vitro. Standard techniques known in the art that can measure the abundance of homomultimeric versus heteromultimeric species can include size-exclusion chromatography. In some embodiments, each modified polypeptide can be individually expressed using standard recombinant techniques and assembled together in vitro. For example, each modified polypeptide can be purified, mixed and incubated together in equal masses, disulfide reduced (e.g., by treatment with dithiothreitol), concentrated, and assembled by reoxidizing the polypeptides. The formed TCR fusion protein 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, polypeptides comprising modified antibody Fc domains can be individually expressed in CHO cells and assembled in vitro using the methods described above.

[0121] In some embodiments, the TCR fusion proteins of the present disclosure comprise: a first polypeptide comprising, from N-terminal to C-terminal, a TCR alpha chain variable region, an alpha chain constant region, a first hinge sequence, and a first antibody Fc domain; a second polypeptide comprising, from N-terminal to C-terminal, a single-chain variable fragment (scFv) that binds to human CD3 expressed on the cell surface of a T cell, a linker, a beta chain variable region, and a beta chain constant region; and a third polypeptide comprising, from N-terminal to C-terminal, a second hinge sequence and a second antibody Fc domain. Advantageously, the present disclosure demonstrates that this TCR fusion protein format (see, e.g., Figure 1B) had the most favorable pharmacokinetic properties and the highest activity (i.e., potency and selectivity) of the formats tested.

[0122] In some embodiments, the first and second polypeptides are linked via one or more disulfide bonds between the alpha chain constant region and the beta chain constant region, e.g., as described herein.

[0123] In some embodiments, the first and third polypeptides are linked via one or more interchain disulfide bonds between the first and second hinge sequences and / or one or more corresponding knob- and hole-forming mutations on the antibody Fc domain.

[0124] In some embodiments, a 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.

[0125] Further provided herein are polynucleotides encoding any of the TCRs and TCR fusion proteins disclosed herein. For example, in some embodiments, the present disclosure provides a polynucleotide kit comprising one, two, or three polynucleotides encoding one, two, or three polypeptides of the present disclosure. In some embodiments, the present disclosure provides a polynucleotide kit comprising a first polynucleotide encoding a first polypeptide comprising the amino acid sequence of SEQ ID NO:29, a second polynucleotide encoding a second polypeptide comprising the amino acid sequence of SEQ ID NO:30, and a third polynucleotide encoding a third polypeptide comprising the amino acid sequence of SEQ ID NO:28.

[0126] Further provided herein are vectors (e.g., expression vectors) comprising any of the polynucleotides of the present disclosure. In some embodiments, the vectors of the present disclosure comprise polynucleotides encoding one, two, or three (e.g., all) polypeptides of a TCR fusion protein of the present disclosure. For example, in some embodiments, the vector comprises a first polynucleotide encoding a first polypeptide comprising the amino acid sequence of SEQ ID NO:29, a second polynucleotide encoding a second polypeptide comprising the amino acid sequence of SEQ ID NO:30, and a third polynucleotide encoding a third polypeptide comprising the amino acid sequence of SEQ ID NO:28. In some embodiments, the vector encodes 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. Further provided herein is a kit of vectors (e.g., expression vectors) comprising a first vector comprising a first polynucleotide encoding a first polypeptide comprising the amino acid sequence of SEQ ID NO:29, a second vector comprising a second polynucleotide encoding a second polypeptide comprising the amino acid sequence of SEQ ID NO:30, and a third vector comprising a third polynucleotide encoding a third polypeptide comprising the amino acid sequence of SEQ ID NO:28.

[0127] For recombinant production of a TCR fusion protein, a nucleic acid encoding the TCR fusion protein is isolated, e.g., as described above, and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the polypeptide chains of the TCR fusion protein), produced by recombinant methods, or obtained by chemical synthesis.

[0128] Further provided herein are host cells containing any of the polynucleotides and / or vectors of the present disclosure. Suitable host cells for cloning or expressing the polynucleotides and / or vectors of the present disclosure are known in the art. Suitable host cells for expressing (glycosylated) proteins are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures can also be used as hosts. See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe the PLANTIBODIES™ technology for producing antibodies in transgenic plants). Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to growth in suspension may be useful. Other examples of useful mammalian host cell lines are the SV40-transformed monkey kidney CV1 line (COS-7); human embryonic kidney lines (e.g., 293 or 293T cells described in Graham, F. et al., J. Gen. Virol. 36 (1977) 59-74); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells described in Mather, J.P., Biol. Reprod. 23 (1980) 243-252); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat hepatocytes (BRL3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT060562); TRI cells (e.g., Mather, J.P. et al., Annals NY Acad. Sci. 383 (1982) 44-68); MRC5 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, e.g., Yazaki, P. and Wu, A.M., Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268. In one embodiment, 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)).

[0129] Further provided herein are methods for producing any of the TCR fusion proteins disclosed herein. In some embodiments, the methods comprise culturing a host cell of the present disclosure under conditions suitable for producing the TCR fusion protein. In some embodiments, the methods further comprise recovering the TCR fusion protein from the host cell.

[0130] IV. Methods and Uses Certain aspects of the present disclosure relate to methods for treating cancer. In some embodiments, the method comprises 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 a human.

[0131] In some embodiments, the individual has a cancer that expresses MAGE-A4.

[0132] For example, the cancer or tumor can be a breast, esophageal, gastric (e.g., gastric cancer), head and neck, lung, ovarian, or bladder cancer or tumor. 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 squamous cell histology (i.e., is a squamous cell carcinoma or tumor).

[0133] In some embodiments, the individual has HLA-A * This is an individual of the 02 subtype.

[0134] The present disclosure further includes pharmaceutical compositions comprising the 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 may be provided in a pharmaceutical composition together with one or more pharmaceutically acceptable carriers or excipients. Therapeutic or imaging TCRs or cells according to the present disclosure are typically supplied as part of a sterile pharmaceutical composition, usually including a pharmaceutically acceptable carrier. The pharmaceutical composition may be in any suitable form (depending on its desired method of administration to a patient). The pharmaceutical composition may be provided in unit dosage form, generally provided in a hermetically sealed container, and may be provided as part of a kit. Such a kit will usually (but not necessarily) include instructions for use. The kit may include a plurality of such unit dosage forms.

[0135] In some embodiments, the TCR fusion protein or pharmaceutical composition is administered by intravenous or intratumoral injection. The TCR fusion protein or pharmaceutical composition may be adapted for administration by any suitable route, such as parenteral (including subcutaneous, intramuscular, or intravenous), enteral (including oral or rectal), inhalation, or intranasal routes. Such compositions may be prepared by any method known in the pharmaceutical arts (e.g., mixing the active ingredient with the carrier or excipient(s) under sterile conditions).

[0136] The dosage of the substances of the present disclosure can vary within a wide range depending on the disease or disorder being treated, the age and condition of the individual being treated, etc. A suitable dosage range for the soluble TCR of the present disclosure in association 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.

[0137] The TCRs, pharmaceutical compositions, vectors, nucleic acids, and cells of the present disclosure may 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).

[0138] In some embodiments, the methods of the present disclosure further comprise administering to the individual a second anti-cancer agent.

[0139] V. Kit or Product In another aspect, an article of manufacture containing materials useful for the treatment and / or prevention of the above-mentioned disorders is provided. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, intravenous infusion bags, etc. The container can be formed from a variety of materials, such as glass or plastic. The container holds the composition alone or in combination with another composition effective for the treatment, prevention, and / or diagnosis of a condition and can have a sterile access port (e.g., the container can be an intravenous infusion bag or vial with a stopper pierceable by a hypodermic needle). At least one active ingredient in the composition is an antibody of the present invention. The label or package insert indicates that the composition is used for treating the selected condition. Furthermore, the article of manufacture can include (a) a first container containing a composition, where the composition comprises a TCR fusion protein of the present disclosure; and (b) a second container containing a composition, where the composition comprises an additional cytotoxic or other therapeutic agent. The article of manufacture in this aspect of the invention may further include a package insert indicating that the composition can be used to treat a particular condition. Alternatively, or in addition, the article of manufacture may further include a second (or third) container containing a pharmaceutically acceptable buffer (such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution). The article of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0140] The specification is considered to be 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 become 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 hereby incorporated by reference in their entirety for all purposes. [Example]

[0141] Example The present invention will be more fully understood by reference to the following examples. However, the examples should not be understood to limit the scope of the present invention. The examples and embodiments described herein are for illustrative purposes only, and it is understood that various modifications or alterations in light of the examples and embodiments will be suggested to those skilled in the art and are within the spirit and scope of the present application and the scope of the appended claims.

[0142] Example 1: Engineering glycosylation on TCR:anti-CD3 fusion molecules Fusion proteins containing a soluble TCR that specifically and with high affinity binds to the germline cancer antigen MAGE-A4 and a T cell-binding antibody fragment (e.g., anti-CD3 scFv) have been described as potential immunotherapeutics. See, e.g., WO 2017175006. Further engineering was undertaken to provide TCR:anti-CD3 fusion molecules with favorable properties (e.g., potency and in vivo pharmacokinetics).

[0143] material and method TCR: anti-CD3 fusion molecule The monoglycosylated TCR:anti-CD3 fusion molecule contained three polypeptide chains corresponding to SEQ ID NOs: 28-30 expressed in CHO cells. The three polypeptide chains included: (1) a free Fc domain 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 containing an alpha chain variable region containing the amino acid sequence of SEQ ID NO: 7 and an alpha chain constant region containing the amino acid sequence of SEQ ID NO: 9 with an Fc domain with a hole mutation (SEQ ID NO: 27) linked to the C-terminus of the TCR alpha chain via a hinge sequence (SEQ ID NO: 31); and (3) an anti-CD3 scFv variant containing the amino acid sequence of SEQ ID NO: 17 linked to the N-terminus of the TCR beta chain via a linker (SEQ ID NO: 18), wherein the beta chain contained a beta chain variable region containing the amino acid sequence of SEQ ID NO: 13 and a beta chain constant region containing the amino acid sequence of SEQ ID NO: 14. Compared to the parental anti-CD3 scFv, the variant contained a T164A mutation in CDR1 and an I201F mutation in FR3 (numbered according to SEQ ID NO: 17).

[0144] The aglycosylated TCR:anti-CD3 fusion molecule contained the same subcomponents as the monoglycosylated form, except that the alpha chain variable region contained the amino acid sequence of SEQ ID NO:8 (hence, the full-length alpha chain contained the amino acid sequence of SEQ ID NO:12). Both the aglycosylated and monoglycosylated forms had N→Q substitutions at the three N-glycosylation sites of the parent alpha chain constant region (SEQ ID NO:10). Both the aglycosylated and monoglycosylated forms had N→Q substitutions at the N-glycosylation sites of the parent beta chain constant region (SEQ ID NO:15).

[0145] Pharmacokinetics Male SCID mice were used (N=4). Aglycosylated or monoglycosylated TCR:anti-CD3 fusion molecules were injected at 0.665 mg / kg, 0.0665 mg / kg, or 0.00665 mg / kg via a single IV bolus injection. The control N297G TCR:anti-CD3 fusion molecule was injected at 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. Serum samples were detected by electrochemiluminescence immunoassay using biotinylated MAGEA4 peptide-HLA capture and sulfo-tagged anti-scFv antibody detection.

[0146] Analysis of overall N-linked glycan composition Ten micrograms of protein were denatured with 8 M guanidine HCl in a 1:1 volume ratio and reduced with 100 mM dithiothreitol at 95°C for 10 minutes. The samples were diluted with 100 mM Tris HCl (pH 7.5) to a final concentration of 2 M guanidine HCl, followed by N-linked deglycosylation using 2 μl of PNGase F (P0705S, New England BioLabs) overnight at 37°C. After deglycosylation, 150 ng of each sample was injected onto 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 as a gradient from 2% to 32% solvent B over 6 min and held for 1.5 min. Solvent B was then stepped to 85% over 0.5 min and held for 1 min to clean the column. Finally, solvent B was stepped to 2% for re-equilibration and held for 3 min.

[0147] The separated glycans were analyzed online via nanospray ionization into a Q-TOF mass spectrometer (Agilent 6520) using the following parameters for data acquisition: spray voltage of 1.9 kV; gas temperature of 325 °C; drying gas flow of 5 L / min; fragmentor voltage of 160 V; skimmer voltage of 65 V; oct1 RF Vpp voltage of 750 V; scan range of 400-3,000 m / z; positive polarity; MS1 centiloid data acquisition using extended dynamic range (2 GHz) instrument mode; 3 spectra / s; 333.3 ms / spectrum; 3243 transients / spectrum; and CE settings of 0. The acquired mass spectral data were searched against a glycan library in Agilent MassHunter Qualitative Analysis software. This software algorithm utilized a combination of accurate mass with a mass tolerance of 10 ppm and predicted retention time for glucan identification. Each N-linked glycan was quantified label-free relative to the sum of all identified N-linked glycans within each sample by integrating the AUC of each extracted glycan chromatogram.

[0148] Mapping of N-linked glycosylation sites Twenty micrograms of protein were denatured with 8 M guanidine HCl in a 1:1 volume ratio and reduced with 100 mM dithiothreitol at 95°C for 10 minutes. The sample was diluted with 100 mM Tris HCl (pH 7.5) to a final concentration of 2 M guanidine HCl, followed by deglycosylation of N-linked glycosylation with 4 μl of PNGase F (P0705S, New England BioLabs) overnight at 37°C. After deglycosylation, the sample was alkylated with 40 mM iodoacetamide at room temperature for 30 minutes. The sample was split in half for separate enzymatic digestions with 0.1 μg trypsin (Promega) and 0.1 μg chymotrypsin (Thermo Fisher Scientific) overnight at 37°C. The digest was quenched with 0.1% TFA and subjected to cleanup on a C18 stage tip using an elution step of 59.9% water + 40% acetonitrile containing 0.1% TFA. After cleanup, peptides were dried and reconstituted with 50 μl of 0.1% TFA. 1 μl was then injected via an autosampler into 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 to 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 as a gradient from 2% to 25% solvent B over 35 min. The solvent was stepped to 50% solvent B over 2 minutes, then held at 90% for 6 minutes to clean the column. Finally, the solvent was stepped to 2% solvent B for re-equilibration and held for 7 minutes.The separated peptides were analyzed online via nanospray ionization into an Orbitrap Elite Hybrid Ion Trap-Orbitrap mass spectrometer (Thermo Fisher Scientific) using the following parameters for data acquisition: resolution 60,000; scan range 375–1,600 m / z; positive polarity; centroid mode; 1 m / z isolation width with an activation Q of 0.25 and an activation time of 10 ms; CID activation; and CE setting 35. Data were collected in data-dependent mode, precursor ions were analyzed in the FTMS, and the top 15 abundant ions were selected for fragmentation and analysis in the ITMS.

[0149] The acquired mass spectral data were searched against protein sequences using Byonic software (Protein Metrics Inc.) with the following parameters: a combination of a 20 ppm precursor mass tolerance and a 0.5 Da fragment mass tolerance; strict specificity for arginine and lysine with up to one missed cleavage by trypsin; strict specificity for leucine, phenylalanine, tryptophan, and tyrosine with up to two missed cleavage by chymotrypsin; fixed carbamidomethylation for cysteine; variable oxidation for methionine; and variable deamidation for asparagine. The results of the Byonic search were analyzed with Byologic software (Protein Metrics Inc.) and filtered using a minimum MS2 score cutoff of 200. Peptide identification was confirmed by MS2 peptide fragmentation. Deamidated peptides containing N-linked sites were quantified label-free relative to their fixed forms by AUC integration of their extracted ion chromatograms in Thermo Xcalibur Qual Browser software (Thermo Fisher Scientific). The deamidation change was used to quantify glycosylation.

[0150] binding affinity Anti-hIgG1 antibody was immobilized on a Biacore CM5 capture chip using a standard EDC / NHS immobilization protocol. ImmTAC molecules were prepared at 1 μg / ml in HBS-EP buffer and captured on the anti-hIgG1-immobilized CM5 chip. Subsequently, antigen (CD3 epsilon delta heterodimer Fc fusion) was flowed over the ImmTAC capture flow cell at a flow rate of 100 μl / min with a contact time of 210 seconds and a dissociation time of 300 seconds. The Biacore was run at 37°C, and the analyte (human CD3 epsilon delta heterodimer Fc fusion) concentration series was 0, 0.5, 2.5, 12.5, 50, and 150 nM.

[0151] result Figures 1A and 1B show the anti-CD3 scFv, the soluble monoclonal high-affinity TCR, and the TCR:anti-CD3 fusion molecule containing an antibody Fc domain for extended in vivo half-life. As shown in Figure 2A, the soluble monoclonal TCR has seven N-glycosylation sites when produced in CHO cells. All glycosylation sites were found to be removable without affecting activity or selectivity. The aglycosylation and monoglycosylation variants were selected for further study (Figure 2B). In the monoglycosylation 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 in the alpha chain variable region and N84Q substitution in the beta chain variable region). The aglycosylation variant contained an additional N18Q substitution in the alpha chain variable region to remove the final N-glycosylation site.

[0152] 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 2C). A series of double and single N-glycosylation site variants were constructed to test the effect of removing individual N-glycosylation sites from the TCR variable region (Figure 2D). Results demonstrated a stepwise decrease in yield with reduced glycosylation, with fully aglycosylated molecules showing significantly reduced yield. However, preserving N-glycosylation of the N18 residue was found to increase yield more than any single site.

[0153] These TCR:anti-CD3 fusion molecules contained an antibody Fc domain, which was found to substantially improve in vivo pharmacokinetic properties in the SCID mouse model described above. Formats with the Fc domain fused to the alpha or beta chain were found to have substantially improved pharmacokinetics compared with similar molecules without the Fc expressed from E. coli or CHO cells (Figure 3A and Table A). All N-glycosylation sites for these molecules were intact. The format shown in Figure 1B (with an scFv at the N-terminus of the TCR beta chain and an Fc at the C-terminus of the alpha chain) had the most favorable pharmacokinetic properties. Fusion of the Fc to the C-terminus of the alpha chain also yielded a molecule with the highest activity (i.e., potency and selectivity) among the various formats tested. This format was selected for further testing. [Table A]

[0154] Different linker sequences were also tested in this format. No differences in production yield were observed when using different linker sequences. Furthermore, molecules with different linkers exhibited similar potency and levels of off-target activity. Finally, molecules with different linkers exhibited comparable stability in serum. These results demonstrate that the linker sequence did not affect activity, yield, or serum stability.

[0155] To further improve pharmacokinetics, aglycosylated and monoglycosylated variants of the same TCR:anti-CD3 fusion molecule (as shown in Figure 2B) were generated and tested for in vivo pharmacokinetic properties in a SCID mouse model. As shown in Figures 3B-3E, the monoglycosylated variant exhibited better pharmacokinetic properties (including slower elimination, half-life, and clearance) compared to the aglycosylated form. The monoglycosylated form exhibited substantially slower elimination (Figures 3B and 3E), while pharmacokinetics were close to linear over a 100-fold dose range for both formats (Figures 3C and 3D). Thus, the monoglycosylated form, which has a single N-linked glycosylation site at N18 in the alpha chain variable region, exhibited improved half-life compared to the aglycosylated form.

[0156] Both variants were also subjected to stability testing. For heat stress testing, aglycosylated and monoglycosylated variants of the same TCR:anti-CD3 fusion molecule (as shown in Figure 2B) were exposed to heat stress at 30°C for 4 weeks at 1 mg / mL in 20 mM His-Acetate and 240 mM sucrose (pH 5.5). After incubation, the monoglycosylated form showed a 0.8% increase in monomer loss by SEC, whereas the aglycosylated form showed a significantly higher 11.3% monomer loss (+6.1% vHMW form and 4.8% dimer). Both forms demonstrated stability at all potential deamidation / isomerization sites.

[0157] Both forms were also subjected to heat stress at 37°C for 2 weeks at 1 mg / mL in PBS (pH 7.4). After incubation, the aglycosylated molecule showed a 7.3% increase (4.1% vHMW form, 1.7% dimer), while the monoglycosylated monomer loss was only 1.4%. Both forms demonstrated little change at all potential deamidation / isomerization sites.

[0158] In summary, the aglycosylated and monoglycosylated forms were found to have substantially different stabilities. These results demonstrated that the monoglycosylated form has better thermal stress resistance than the aglycosylated form. Both forms also demonstrated stability at each potential oxidation site on the anti-CD3 scFv in the AAPH oxidation assay. Collectively, these results demonstrate that the glycan at position N18 in the alpha chain variable domain is important for preventing aggregation and suggest that the monoglycosylated form had a more favorable manufacturability profile than the aglycosylated form.

[0159] The binding affinity for the aglycosylated and monoglycosylated variants was also measured. Both forms had similar affinity for the MAGEA4 peptide via the TCR as measured by BIACORE, with the monoglycosylated variant having a K of 0.17 nM. D and the aglycosylated variant has a K of 0.18 nM. D had the following characteristics:

[0160] The affinity of the anti-CD3scFv U28 variant and the original UCHT1v9-based aglycosylated and monoglycosylated molecules was measured for human CD3ε via Biacore. Both U28-based molecules had similar affinities for CD3ε (14 nM K for the aglycosylated variant). D ; 15 nM K for monoglycosylation D ) and UCHT1v9-based molecules had similar affinity for CD3 (17 nM K for aglycosylation).D ; 18 nM K for monoglycosylation D While the equilibrium affinity of the U28-based molecules was similar to that of the original UCHT1v9-based molecules, the anti-CD3 scFvs were characterized by slightly different binding kinetics (U28 had approximately two-fold faster on- and off-rates).

[0161] In summary, pMHC binding affinity was not altered by glycosylation.

[0162] Example 2: Potency and selectivity of monoglycosylated or aglycosylated TCR:anti-CD3 fusion molecules The aglycosylated and monoglycosylated TCR:anti-CD3 fusion molecules described in Example 1 were tested for potency and selectivity of target cell killing.

[0163] material and method cell killing For Figures 4B-4D, assays were performed using the xCELLigence platform (Agilent). Effector cells were used at an effector-target cell ratio of 10:1. Percent cell lysis was determined using normalized cell index (impedance measurements). In all cases, assays were performed in triplicate every 2 hours for 96 hours. EC50 values ​​were obtained from percent cell lysis curves at 72 hours. Curve fitting was performed in PRISM.

[0164] For Figures 5A and 5B, assays were performed using the Opera Phenix high-content screening system (Perkin Elmer). Effector cells were used at a 5:1 effector-target cell ratio, and AUC values ​​were obtained and curve fitting was performed in PRISM. EC50 values ​​were calculated from the curves.

[0165] For Figure 5C, the assay was performed using a human IFN-γ ELISpot kit (BD Biosciences). Target cells were cultured at 1 × 10 in assay medium. 6Cells were prepared at a density of 1000 / ml and plated at 50,000 cells per well in a volume of 50 μl. PBMCs isolated from fresh donor blood were used as effector cells. Effector cells were used at an effector-target cell ratio of 1:1 for antigen-positive cells and 0.8:1 for antigen-negative cells. Samples were detected using AEC chromogen. Spot counting was performed using a CTL analyzer (Cellular Technology Limited) equipped with Immunospot software. Curve fitting was performed in PRISM, and EC50 values ​​were calculated.

[0166] The copy number of the MAGEA4 antigen was determined by quantitative mass spectrometry.

[0167] result A wide variety of cancer cell lines were used, displaying a range of target MAGE-A4 antigen expression. All cell lines expressed HLA-A2. EC50 values ​​for cell lysis against all cell lines are shown in Figure 4A and were calculated from data obtained at 72 h. Killing curves and the lowest concentrations of TCR:anti-CD3 fusion molecules that produced a killing response are shown in Figures 4B–4D. Both aglycosylated and monoglycosylated TCR:anti-CD3 fusion molecules demonstrated low pM cell-killing activity and selectivity. For example, cell killing against NCI-H1755 lung adenocarcinoma cells (which had the highest MAGE-A4 expression) was observed at a TCR:anti-CD3 fusion molecule concentration of 0.17 pM for both molecules (Figure 4B). Cell killing against SCaBER bladder urothelial carcinoma cells (which have a moderate level of MAGE-A4 expression) was observed at a TCR:anti-CD3 fusion molecule concentration of 14 pM for both molecules (Figure 4C). In contrast, cell killing of NCI-H441 cells (MAGE-A4 negative but HLA-A2 positive) was observed at a TCR:anti-CD3 fusion molecule concentration of 3 nM (Figure 4D), indicating that selectivity was maintained down to the nM level. Thus, killing activity was related to target (pMHC) copy number. These results demonstrate high potency and selectivity for both molecules.

[0168] The TCR:anti-CD3 fusion molecules with the Fc fusion described above were found to have reduced cell-killing potency against target cells expressing the MAGE-A4 antigen:HLA complex (Figures 5A and 5B). However, as shown in Figure 3A, the inclusion of an Fc domain was desirable to improve in vivo pharmacokinetics. As such, a variant anti-CD3 scFv was tested. As described above, this variant contained point mutations in CDR-H1 and FR3. The loss of potency observed with the Fc fusion was found to be offset by the use of the variant anti-CD3 scFv (Figures 5A and 5B). Against antigen-positive cancer cells, the EC50 values ​​of the TCR:anti-CD3 fusion molecules with the Fc fusion and variant anti-CD3 scFv were within 3-10-fold of those without the Fc domain.

[0169] All three molecules showed reduced reactivity to antigen-negative cells, which paralleled their reduced potency on antigen-positive cells (Figure 5C). These results demonstrate that the window between on- and off-target activity was maintained among all three TCR:anti-CD3 fusion molecules tested.

[0170] Example 3: In vitro safety testing of monoglycosylated TCR:anti-CD3 fusion molecules The monoglycosylated TCR:anti-CD3 fusion molecule described in Example 1 was tested in vitro for safety on normal cell lines.

[0171] material and method In vitro safety testing Reactivity was determined by ELISpot assay to detect the release of IFNγ and granzyme B against normal cells. The lowest concentration of TCR-anti-CD3 fusion molecule at which each cytokine was detected was recorded. Alloreactivity (i.e., binding to alternative HLA) was assessed using the five most common HLA-A (HLA-A) antibodies in the world population. *Reactivity was assessed using an IFNγ ELISpot assay against a six-cell lot panel covering all HLA-B, HLA-C, and HLA-C antigens (excluding 02:01). Whole blood assays were performed using the Proinflammatory Panel 1 (Human) Kit (Meso Scale Discovery) for the detection of TNFα, IL-2, IL-6, IL-1β, and IFNγ in whole blood from three donors. TCR-anti-CD3 fusions were applied at various concentrations between 0.01 and 10 nM.

[0172] result The monoglycosylated TCR:anti-CD3 fusion molecule was tested on a panel of normal cell lines covering high-risk tissue types and on cell types for which reactivity to the anti-MAGE-A4 TCR had been demonstrated. Using IFNγ as the readout, no detectable reactivity to normal cells was observed in a six-cell panel covering the 15 most frequently occurring HLA types (Figure 6A). In whole blood assays, no cytokine release was observed at sub-nM concentrations. Taken together, no reactivity to normal cell lines was detected at sub-nM concentrations, demonstrating safety in this in vitro assay.

[0173] Comparison of the TCR:anti-CD3 fusion molecule with the variant anti-CD3 scFv to the Fc-less TCR:anti-CD3 fusion molecule again showed that the window of efficacy between on-target and off-target activity against normal cells was maintained (Figure 6B), demonstrating that the TCR:anti-CD3 fusion molecule with the Fc domain and variant anti-CD3 scFv maintained a therapeutic index equivalent to the form without the Fc fusion. [ka] [ka] [ka] [ka]

Claims

1. GVYDGREHTV (Sequence ID 34) HLA-A ＊ A T cell receptor (TCR) fusion protein comprising a TCR that binds to the O2 complex, wherein the TCR is a soluble TCR covalently bound 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, where the T cell engaging domain is a single-chain variable fragment (scFv); and the TCR is (a) A TCR alpha chain comprising an alpha chain variable region and an alpha chain constant region, wherein the alpha chain variable region comprises (i) CDR1 comprising the amino acid sequence of VSPFSN (SEQ ID NO: 1), (ii) CDR2 comprising the amino acid sequence of LTFSENT (SEQ ID NO: 2), and (iii) CDR3 comprising the amino acid sequence of VVNSAQGLYIPTF (SEQ ID NO: 3); and (b) A TCR beta chain comprising a beta-chain variable region and a beta-chain constant region, wherein the beta-chain variable region comprises (i) CDR1 comprising the amino acid sequence of LDHEN (SEQ ID NO: 4), (ii) CDR2 comprising the amino acid sequence of SRFATG (SEQ ID NO: 5), and (iii) CDR3 comprising the amino acid sequence of ASSSDQNSGDPYEQYF (SEQ ID NO: 6); Includes, The TCR is glycosylated at a single N-linked glycosylation site, and the N-linked glycosylation site is located at residue N18 of the alpha-chain variable region, which is numbered according to Sequence ID No.

7. The aforementioned TCR fusion protein, (a) A first polypeptide comprising, in the direction from the N-terminus to the C-terminus, the alpha chain variable region, the alpha chain constant region, a first hinge sequence, and a first antibody Fc domain; (b) A second polypeptide comprising the scFv, linker, beta-chain variable region, and beta-chain constant region in the direction from the N-terminus to the C-terminus; and (c) A third polypeptide comprising a second hinge sequence and a second antibody Fc domain in the direction from the N-terminus to the C-terminus. A T cell receptor (TCR) fusion protein containing [the specified component].

2. The TCR fusion protein according to claim 1, wherein the TCR includes amino acid substitutions at any potential N-glycosylation site other than residue N18.

3. The TCR fusion protein according to claim 2, wherein the amino acid substitution is N→Q.

4. (i) The alpha chain variable region comprises 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 with the amino acid sequence of SEQ ID NO: 7; and / or (ii) The beta-chain variable region includes an amino acid sequence that has 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 with the amino acid sequence of SEQ ID NO:

13. The TCR fusion protein according to claim 1.

5. (i) The alpha chain constant region comprises 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 with the amino acid sequence of SEQ ID NO: 9; and / or (ii) The beta chain constant region includes 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 with the amino acid sequence of SEQ ID NO:

14. The TCR fusion protein according to claim 1.

6. (i) The TCR alpha chain comprises 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 with the amino acid sequence of SEQ ID NO: 11; and / or (ii) The TCR beta chain contains an amino acid sequence that has 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 with the amino acid sequence of SEQ ID NO:

16. The TCR fusion protein according to claim 1.

7. The TCR fusion protein according to claim 1, wherein the TCR alpha chain comprises the amino acid sequence of SEQ ID NO: 11 and the TCR beta chain comprises the amino acid sequence of SEQ ID NO:

16.

8. The TCR fusion protein according to claim 1, wherein the antibody Fc domain is a human Fc domain, and the antibody Fc domain is a human IgG1 Fc domain, a human IgG2 Fc domain, or a human IgG4 Fc domain.

9. The antibody Fc domain is i) Mutation to residue N297, which is numbered according to the EU index; ii) One or more mutations in the residue(s) E233, L234, L235, and / or G236 numbered according to the EU index; and / or iii) One or more mutations in the residue(s) L234, L235, and P329, numbered according to the EU index. The TCR fusion protein according to claim 8, which is a human IgG1 Fc domain containing

10. The antibody Fc domain is i) Replacement of N297G numbered according to the EU index; ii) Deletions in substitutions numbered according to the EU index N297G, E233P, L234V, L235A, and G236; and / or iii) Substitutions L234A, L235A, and P329G numbered according to the EU index The TCR fusion protein according to claim 8, which is a human IgG1 Fc domain containing

11. The TCR fusion protein according to claim 1, wherein the second antibody Fc domain associates with the first antibody Fc domain via (1) one or more covalent bonds; and / or (2) one or more amino acid substitutions in one or both of the antibody Fc domains that promote heterodimerization.

12. The TCR fusion protein according to claim 11, wherein both the first and second antibody Fc domains include the CH2 and CH3 domains of the antibody.

13. i) The first and second hinge arrays are linked by one or more interchain disulfide bonds between the first and second hinge arrays; and / or ii) Both the first and second hinge sequences contain the amino acid sequence of DKTHTCPP (SEQ ID NO: 31), The TCR fusion protein according to claim 1.

14. The TCR fusion protein according to claim 1, 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, in order to promote heterodimerization of the antibody Fc domain.

15. i) 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; or ii) The first antibody Fc domain 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, The TCR fusion protein according to claim 14.

16. The TCR fusion protein according to claim 1, wherein the T cell engaging domain binds to human CD3 expressed on the cell surface of a T cell.

17. The T cell engagement domain is a single-chain variable fragment (scFv), i) The scFv contains the amino acid sequence of SEQ ID NO: 17; or ii) The scFv contains the amino acid sequence of SEQ ID NO: 35, The TCR fusion protein according to claim 1.

18. The TCR fusion protein according to claim 1, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 29, the second polypeptide comprises the amino acid sequence of SEQ ID NO: 30, and the third polypeptide comprises the amino acid sequence of SEQ ID NO:

28.

19. The TCR fusion protein according to claim 1, wherein the first and third polypeptides are linked by one or more disulfide bonds.

20. The TCR fusion protein according to claim 1, wherein the first and second polypeptides are linked by one or more disulfide bonds.

21. One or more polynucleotides encoding the TCR fusion protein according to any one of claims 1 to 20.

22. A pharmaceutical composition comprising a TCR fusion protein according to any one of claims 1 to 20 and a pharmaceutically acceptable carrier.

23. A composition comprising the TCR fusion protein according to any one of claims 1 to 20 for use in the treatment of cancer, or a pharmaceutical composition comprising the TCR fusion protein according to any one of claims 1 to 20 and a pharmaceutically acceptable carrier.

24. The composition or pharmaceutical composition according to claim 23, wherein the subject is a human subject having cancer that expresses MAGE-A4.

25. The composition or pharmaceutical composition according to claim 23, wherein the subject is a subject of the HLA-A*02 subtype.

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