# Patent

## Title
High-affinity DMF5 T cell receptor (TCR) variants

## Abstract

High-affinity variants of the DMF5 TCR, and methods of use thereof for the treatment and imaging of cancer in a patient.

## Background
CLAIM OF PRIORITY
This application claims the benefit of U.S. Provisional Patent Application No. 61/824,831, filed on May 17, 2013. The entire contents of the foregoing are hereby incorporated by reference herein.


FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
This invention was made with Government support under Grant Nos. GM067079, GM084884, RR025761, and GM103773 awarded by the National Institutes of Health. The Government has certain rights in the invention.


TECHNICAL FIELD
The present invention relates to high-affinity variants of the DMF5 TCR, and methods of use thereof for the treatment and imaging of cancer in a patient.
BACKGROUND
T cell receptors are heterodimeric immunoglobulin proteins, with approximately 30,000 on the surface of each clonally unique T cell (Janeway, Immunobiology: the Immune System in Health and Disease. 6th ed. New York: Garland Science; 2005). The specific binding of αβ TCRs to peptides bound by MHC proteins (pMHC) is a critical step in the antigen-specific T cell-mediated immune response. As with antibodies, the antigen-binding Complementarity Determining Region (CDR) loops are variable, with multiple germ-line segments, junctional combinations, and addition or deletion of nucleotides at these junctions used to generate diversity.
SUMMARY
At least in part, the present invention is based on the development of high-affinity variants of the DMF5 TCR. Thus, provided herein are the high-affinity variants themselves, nucleic acids encoding and host cells expressing the variants, and methods of use thereof.
In one aspect, the invention provides a high-affinity DMF5 T cell receptor variant a chain, wherein the amino acid at position 27 is a Y (αD27Y) or W (αD27W), e.g., a variant comprising SEQ ID NO:1, 3, or 5 with a D27Y or D27W mutation. (Herein, the numbering within the alpha chain is with regard to SEQ ID NO:1; the corresponding amino acid in SEQ ID NO:3 is D49, in SEQ ID NO:5, D27).
In another aspect, the invention provides a high-affinity DMF5 T cell receptor variant β chain, wherein the amino acid at position 95 is a W (βL95W), e.g., a variant comprising SEQ ID NO:2, 4, or 6 with an L95W mutation. Herein, the numbering within the beta chain is with regard to SEQ ID NO:2; the corresponding amino acid in SEQ ID NO:4 is L114; in SEQ ID NO:6, L96).
In yet another aspect, the invention provides a high-affinity DMF5 T cell receptor variant comprising: a high-affinity DMF5 T cell receptor variant α chain, wherein the amino acid at position 27 is a Y (αD27Y) or W (αD27W), and a high-affinity DMF5 T cell receptor variant β chain, wherein the amino acid at position 95 is a W (βL95W).
In another aspect, the invention provides a fusion protein comprising:

    a high-affinity DMF5 T cell receptor variant α chain as described herein;
    a high-affinity DMF5 T cell receptor variant β chain as described herein; or
    a high-affinity DMF5 T cell receptor variant as described herein; linked to
    a non-T cell receptor sequence.


In another aspect, the invention provides a therapeutic composition comprising:

    a therapeutic agent linked to one or more of:
    a high-affinity DMF5 T cell receptor variant α chain as described herein;
    a high-affinity DMF5 T cell receptor variant β chain as described herein; or
    a high-affinity DMF5 T cell receptor variant as described herein; or
    a fusion protein as described herein.


In some embodiments the therapeutic agent is a radioisotope; an anticancer drug such as a genotoxin; or a cytotoxic moiety.
In another aspect, the invention provides compositions comprising:

    a detectable moiety linked to one or more of:
    a high-affinity DMF5 T cell receptor variant α chain as described herein;
    a high-affinity DMF5 T cell receptor variant β chain as described herein; or
    a high-affinity DMF5 T cell receptor variant as described herein; or
    a fusion protein as described herein.


In some embodiments the detectable moiety is a contrast agent.
In some embodiments the contrast agent is selected from the group consisting of gold, gadolinium, iron oxides, manganese chelates, barium sulfate, iodinated contrast media, microbubbles, and perfluorocarbons.
In another aspect, the invention provides a nucleic acid encoding:

    a high-affinity DMF5 T cell receptor variant α chain as described herein;
    a high-affinity DMF5 T cell receptor variant β chain as described herein; or
    a high-affinity DMF5 T cell receptor variant as described herein; or

a fusion protein as described herein. In some embodiments the nucleic acid also includes one or more of an N-terminal leader sequence, some stuff after our C-terminus to help it reach the membrane, a transmembrane spanning alpha helix, and a C-terminal sequence inside of the cell, e.g., from a wild type human TCR, e.g., as known in the art, e.g., as described in Johnson et al., Blood, 2009, and US 2012/0071420, incorporated herein by reference.



In another aspect, the invention provides a vector, e.g., a retroviral vector, comprising a nucleic acid as described herein.
In another aspect, the invention provides a host cell expressing:

    a high-affinity DMF5 T cell receptor variant α chain as described herein;
    a high-affinity DMF5 T cell receptor variant β chain as described herein; or
    a high-affinity DMF5 T cell receptor variant as described herein; or
    a fusion protein as described herein.


In another aspect, the invention provides a host cell comprising a nucleic acid or vector as described herein. In some embodiments the host cell is a peripheral blood lymphocyte (PBL).
In another aspect, the invention provides a method of treating a subject who has melanoma, the method comprising administering to the subject a therapeutically effective amount of a therapeutic composition as described herein.
In another aspect, the invention provides a method of treating a subject who has melanoma, the method comprising administering to the subject a therapeutically effective amount of the host cells as described herein. In some embodiments the host cells are autologous to the subject.
In another aspect, the invention provides a method of treating a subject who has melanoma, the method comprising:

    obtaining cells from the subject;
    transducing the cells with a vector expressing:
    a high-affinity DMF5 T cell receptor variant α chain as described herein;
    a high-affinity DMF5 T cell receptor variant β chain as described herein; or
    a high-affinity DMF5 T cell receptor variant as described herein; or
    a fusion protein as described herein;

optionally maintaining the cells in culture for a time sufficient for the cells to express the variant or fusion protein and/or to proliferate; and

administering a therapeutically effective amount of the cells to the subject thereafter.



In some embodiments the cells are PBLs.
In another aspect, the invention provides a method of detecting the presence of a cancer cell in a subject, the method comprising administering to the subject a detectable amount of a composition comprising a detectable moiety as described herein; and
detecting the presence of the composition in the subject;
thereby detecting the presence of a cancer cell in the subject.
In some embodiments the detectable moiety can be selected from the group consisting of a radioactive isotope, a magnetic compound, an x-ray absorber, a chemical compound, a biological tag, and a fluorescent molecule. The therapeutic agent can be, e.g., a cytotoxic moiety or an immunomodulatory moiety (e.g., a compound that enhances the immune response to the tumor, e.g., an inflammatory cytokine such as interleukin-1 (IL-1), and tumor necrosis factor-alpha (TNF-a).
In some embodiments, there is a linker between the first portion and the second portion, e.g., a flexible amino acid sequence, e.g., a photolinker.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.
Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.



DESCRIPTION OF DRAWINGS
FIGS. 1A-C. Hydrogen/deuterium exchange reveals that the A6 TCR undergoes a global reduction in flexibility upon pMHC binding. (A) MALDI-TOF mass spectra for the fragment of the FG loop (sequence 225WTQDRAKPVTQ235) of A6 free (blue) and bound to Tax/HLA-A2 (red) ten minutes after initiation of hydrogen/deuterium exchange. (B) Time courses for the exchange reaction of the FG loop fragment in panel A demonstrates that in both free and bound A6, exchange follows simple kinetics and is complete within 10 minutes. (C) Percent deuteration of each fragment of free and ligand-bound A6 at the 10 minute time point. Fragments including the CDR and the AB, DE, and FG loops in the constant domains are indicated. The dashed blue and red lines indicate the average deuteration of the free and bound protein, respectively.
FIGS. 2A-C. The DMF5 TCR also undergoes a global reduction in flexibility upon pMHC binding. (A) Steady-state binding equilibrium data for ELA/HLA-A2 binding wild type DMF5 and βL95W and αG29P mutants. Solid lines represent a fit to a 1:1 equilibrium binding model. (B) Kinetic titration data for ELA/HLA-A2 binding of the high affinity YW (αD26Y/βL98W) mutant of DMF5. Data are in black in the bottom panel; the grey line is a fit to a 1:1 kinetic titration model with drift. Residuals (difference between data and fitted curve) are shown in the smaller top panel. (C) Percent deuteration of fragments of free and ligand-bound high affinity DMF5 variant at the 10 minute time point. Fragments including the CDR, AB, CC′ and FG loops and the C and F strands are indicated. The dashed blue and red lines indicate the average deuteration of the free and bound protein, respectively.



DETAILED DESCRIPTION
The principles of TCR recognition of pMHC have been studied extensively, with the first crystal structures of TCRs in complex with pMHC published in the mid-90's (Garcia et al., Science. 1996; 274(5285):209-19; Garboczi et al., J. Immunol. 1996; 157(12):5403-10). These structures revealed a generally conserved diagonal binding mode that in most cases places the hypervariable CDR3 loops of the TCR over (or near) the center of the peptide (Rudolph et al., Annu Rev Immunol. 2006; 24:419-66). A number of features are shared among the interfaces of TCRs with pMHCs, including particular inter-residue contacts by evolutionarily conserved residues (Marrack et al., Annu Rev Immunol. 2008; 26:171-203), leading to the concept of distinct germline “codons” influencing the TCR/pMHC binding modes (Garcia et al., Nat Immunol. 2009; 10(2):143-7).
Significant effort is underway to develop TCRs as a novel class of biotherapeutics. Therapeutic interest is largely based on using genetic engineering to direct T cells towards antigens of interest, such as those presented either exclusively or preferentially by virally infected or cancerous cells. In some of the most recent work, T cells from patients have been engineered to express a T cell receptor specific for a tumor antigen and infused back into the patient (Rosenberg et al., Nat Rev Cancer. 2008; 8(4):299-308). This exciting approach has shown promise in treating metastatic melanoma (Morgan et al., Science. 2006; 314(5796):126-9; Johnson et al., Blood. 2009; 114(3):535-46) and synovial cell sarcoma (Robbins et al., J Clin Oncol. 2011; 29(7):917-24). Yet there are limitations to this approach, including limited potency due to weak TCR avidity, which can result from the low to moderate affinity TCRs typically maintain towards their cognate antigen: TCR/pMHC affinities are typically in the range of 1-200 μM when measured in solution (Davis et al., Annual review of immunology. 1998; 16:523-44), with the recognition of tumor antigens biased towards the weaker end (Laugel et al., J Biol Chem. 2007; 282(33):23799-810). As T cell sensitivity and potency is generally correlated with TCR affinity (Holler et al., J Exp Med. 2001; 194(8):1043-52; Varela-Rohena et al., Nat Med. 2008; 14(12):1390-5; Zhao et al., J. Immunol. 2007; 179(9):5845-54), this has led to numerous proposals for enhancing immunotherapy by engineering higher affinity receptors. Indeed, in a recent thematic review series on strategies for controlling metastasis and recurrence of cancers, Zhang and Morgan emphasized that “the most critical property for a TCR used for engineering T cells for clinical application is the requirement for high affinity specific recognition” (Zhang and Morgan, Adv Drug Deliv Rev. 2011; 64(8): 756-762).
Recent work has suggested that even small (3-5-fold) improvements in TCR binding affinity can result in stronger in vivo immune responses (Borbulevych et al. (2011) J Immunol 187 p. 2453). In addition to enhancing avidity and sensitivity, high affinity TCRs are independent of the need for coreceptor (Chervin et al., J. Immunol. 2009; 183(2):1166-78), allowing, for example, the creation of both CD8+ and CD4+ T cells that recognize antigens presented by class I MHC proteins and thus permitting both cytotoxic and helper immune responses against a single antigen (Morgan et al., supra; Robbins et al., supra). High affinity TCRs can also be used to overcome processes associated with cellular escape from immune destruction. For example, Sewell and colleagues described a TCR with high affinity towards an HIV gag epitope that when expressed in T cells is able to direct the killing of cells infected with HIV variants possessing all known HIV gag escape mutants (Varela-Rohena et al., supra).
To overcome their inherent low affinity for peptide/MHC and improve their immunological efficacy, TCRs have been engineered for higher affinity in several studies, primarily via phage display and yeast display (Varela-Rohena et al., supra; Holler et al., Proc Natl Acad Sci USA. 2000; 97(10):5387-92; Weber et al., Proc Natl Acad Sci USA. 2005; 102(52):19033-8; Li et al., Nat Biotechnol. 2005; 23(3):349-54; Chlewicki et al., J Mol Biol. 2005; 346(1):223-39; Dunn et al., Protein Sci. 2006; 15(4):710-21). These have achieved impressive affinity improvements (approximately 1,000,000-fold in one case), and though antigen specificity appears to be conserved for some high affinity variants (Chlewicki et al., supra; Laugel et al., J Biol Chem. 2005; 280(3):1882-92), there is evidence they can induce off-target T cell responses by raising non-targeted ligand affinities above the activation threshold (Zhao et al., supra; Stone et al., Immunology. 2009; 126(2):165-76). Clinically, such non-specific recognition would lead to dangerous autoimmune reactions, substantially weakening efficacy if not preventing use altogether.
Recent work has shown that very high affinity TCRs, while demonstrating stronger cell killing effects in vitro, can yield low avidity T cells that paradoxically require higher ligand concentrations for activation (Schmid et al., J Immunol. 2010; 184(9):4936-46; Thomas et al., Blood. 2011; 118(2):319-29). This effect has also been measured by vaccinating mice using peptides recognized with a range of TCR affinities; only the peptides recognized by TCRs intermediate affinity induced in vivo tumor protection (McMahan et al., J Clin Invest. 2006; 116(9):2543-51; Corse et al., PLoS Biol. 2010; 8(9)). It is unclear from these studies whether the attenuation of high affinity in vivo T cell response is due to loss of specificity from enhanced recognition of MHC, or other factors related to immune memory, cell differentiation, or signaling mechanisms (Corse et al., Journal of immunology. 2011; 186(9):5039-45). These results as well as others (Zhao et al., supra) suggest that TCR design efforts should emphasize the creation of variants with moderate gains in affinity that fully retain antigen specificity. Indeed, the difference in affinity between the human DMF5 and DMF4 TCRs toward the MART-1 27:35 tumor antigen is only 4-fold, yet in cellular assays DMF5 is substantially more potent (Johnson et al., Journal of Immunology. 2006; 177(9):6548-59), and in clinical trials with genetically-engineered TCRs, DMF5 resulted in higher rates of cancer regression than DMF4 (Johnson et al., Blood. 2009; 114(3):535-46). Described herein are high-affinity DMF5 variants with improved TCR affinity and specificity.
High-affinity DMF5 Variants
The DMF5 TCR, a high-avidity CD8-independent TCR, was originally isolated from a MART-1:27-35 (MART-1) melanoma Ag-reactive tumor-infiltrating lymphocyte (TIL) clone obtained from the tumors of a melanoma patient (Johnson et al., J. Immunol. 177: 6548-6559, 206). DMF5 is directed against the immunodominant HLA-A2-restricted peptide epitopes corresponding to amino acids 26-35 and 27-35 of the melanoma Ag MART-1 (Kawakami et al., J. Exp. Med. 180: 347-352, 1994), a gene product over-expressed in 80-90% of fresh, uncultured melanomas as well as cultured melanoma cell lines (Marincola et al., J. Immunother. Emphasis Tumor Immunol. 19: 192-205, 1996) but not in other tumor or normal tissues except melanocytes. The wild-type sequences of the human DMF5 TCRs are as follows:
In some embodiments, the wild type alpha chain sequence comprises a wild type sequence as set forth in SEQ ID NO:9 of US20120071420, which is incorporated herein by reference in its entirety; D27 is in bold and uppercase:









(SEQ ID NO: 1)



Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu 



1               5                   10                   



 



Ser Val Pro Glu Gly Ala Ile Ala Ser Leu Asn



            15                  20



 



Cys Thr Tyr Ser ASP Arg Gly Ser Gln Ser Phe



        25                  30 



 



Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro 



    35                  40                   



 



Glu Leu Ile Met Phe Ile Tyr Ser Asn Gly Asp



45                  50                  55



 



Lys Glu Asp Gly Arg Phe Thr Ala Gln Leu Asn



                60                  65



 



Lys Ala Ser Gln Tyr Val Ser Leu Leu Ile Arg 



            70                  75 



 



Asp Ser Gln Pro Ser Asp Ser Ala Thr Tyr Leu



        80                  85



 



Cys Ala Val Asn Phe Gly Gly Gly Lys Leu Ile



    90                  95



 



Phe Gly Gln Gly Thr Glu Leu Ser Val Lys Pro 



100                 105                 110



 



Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln



                115                 120



 



Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val



            125                 130



 



Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn 



        135                 140



 



Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile



    145                 150



 



Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met



155                 160                 165



 



Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser



                170                 175



 



Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe 



            180                 185 



 



Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe



        190                 195



 



Pro Ser Pro Glu Ser Ser Cys Asp Val Lys Leu



    200                 205



 



Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu 



210                 215                 220



 



Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg



                225                 230



 



Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu



            235                 240



 



Leu Met Thr Leu Arg Leu Trp Ser Ser



        245                 250






In some embodiments, the wild type beta chain sequence comprises a wild type sequence as set forth in SEQ ID NO:10 of US20120071420; Leu95 is in bold and uppercase:








SEQ ID NO: 2


Ile Ala Gly Ile Thr Gln Ala Pro Thr Ser Gln 


1               5                   10 


 


Ile Leu Ala Ala Gly Arg Arg Met Thr Leu Arg


            15                  20


 


Cys Thr Gln Asp Met Arg His Asn Ala Met Tyr


        25                  30


 


Trp Tyr Arg Gln Asp Leu Gly Leu Gly Leu Arg 


    35                  40                  


 


Leu Ile His Tyr Ser Asn Thr Ala Gly Thr Thr


45                  50                  55


 


Gly Lys Gly Glu Val Pro Asp Gly Tyr Ser Val


                60                  65


 


Ser Arg Ala Asn Thr Asp Asp Phe Pro Leu Thr 


            70                  75 


 


Leu Ala Ser Ala Val Pro Ser Gln Thr Ser Val


        80                  85


 


Tyr Phe Cys Ala Ser Ser LEU Ser Phe Gly Thr


    90                  95


 


Glu Ala Phe Phe Gly Gln Gly Thr Arg Leu Thr 


100                 105                 110


 


Val Val Glu Asp Leu Asn Lys Val Phe Pro Pro


                115                 120


 


Glu Val Ala Val Phe Glu Pro Ser Glu Ala Glu


            125                 130


 


Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys 


        135                 140


 


Leu Ala Thr Gly Phe Phe Pro Asp His Val Glu 


    145                 150 


 


Leu Ser Trp Trp Val Asn Gly Lys Glu Val His


155                 160                 165


 


Ser Gly Val Ser Thr Asp Pro Gln Pro Leu Lys


                170                 175


 


Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys 


            180                 185 


 


Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe


        190                 195


 


Trp Gln Asn Pro Arg Asn His Phe Arg Cys Gln


    200                 205


 


Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu 


210                 215                 220


 


Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln


                225                 230


 


Ile Val Ser Ala Glu Ala Trp Gly Arg Ala Cys


            235                 240


 


Gly Phe Thr Ser Ser Tyr Gln Gln Gly Val Leu 


        245                 250 


 


Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly


    255                 260


 


Lys Ala Thr Leu Tyr Ala Val Leu Val Ser Ala


265                 270                 275


 


Leu Val Leu Met Ala Met Val Lys Arg Lys Asp Phe


                280                 285






In some embodiments, the wild type alpha or beta chain sequence comprises a wild type sequence as set forth in Robbins et al., J. Immunol., 180: 6116-6131, 2008, which are SEQ ID NOs. 3 and 4, respectively:

DMF5 Valpha (D49 Corresponds to D27)










(SEQ ID NO: 3)



MMKSLRVLLV ILWLQLSWVW SQQKEVEQNS GPLSVPEGAI 



 



ASLNCTYSDR GSQSFFWYRQ YSGKSPELIM FIYSNGDKED



 



GRFTAQLNKA SQYVSLLIRD SQPSDSATYL CAVNFGGGKL



 



IFGQGTELSV KPN 






An exemplary nucleic acid sequence encoding wild type DMF5 Valpha follows:








(SEQ ID NO: 7)


ATGATGAAATCCTTGAGAGTTTTACTAGTGATCCTGTGGCTTCA


 


GTTGAGCTGGGTTTGGAGCCAACAGAAGGAGGTGGAGCAGAATT


 


CTGGACCCCTCAGTGTTCCAGAGGGAGCCATTGCCTCTCTCAAC


 


TGCACTTACAGTGACCGAGGTTCCCAGTCCTTCTTCTGGTACAG


 


ACAATATTCTGGGAAAAGCCCTGAGTTGATAATGTTCATATACT


 


CCAATGGTGACAAAGAAGATGGAAGGTTTACAGCACAGCTCAAT


 


AAAGCCAGCCAGTATGTTTCTCTGCTCATCAGAGACTCCCAGCC


 


CAGTGATTCAGCCACCTACCTCTGTGCCGTGAACTTCGGAGGAG


 


GAAAGCTTATCTTCGGACAGGGAACGGAGTTATCTGTGAAACCCAAT






DMF5 Vbeta (L114 Corresponds to L95)










(SEQ ID NO: 4)



MRIRLLCCVA FSLLWAGPVI AGITQAPTSQ ILAAGRRMTL



 



RCTQDMRHNA MYWYRQDLGL GLRLIHYSNT AGTTGKGEVP



 



DGYSVSRANT DDFPLTLASA VPSQTSVYFC ASSLSFGTEA



 



FFGQGTRLTV V 






An exemplary nucleic acid sequence encoding wild type DMF5 Vbeta follows:








(SEQ ID NO: 8)


ATGAGAATCAGGCTCCTGTGCTGTGTGGCCTTTTCTCTCCTGTGGGC


 


AGGTCCAGTGATTGCTGGGATCACCCAGGCACCAACATCTCAGATCC


 


TGGCAGCAGGACGGCGCATGACACTGAGATGTACCCAGGATATGAGA


 


CATAATGCCATGTACTGGTATAGACAAGATCTAGGACTGGGGCTAAG


 


GCTCATCCATTATTCAAATACTGCAGGTACCACTGGCAAAGGAGAAG


 


TCCCTGATGGTTATAGTGTCTCCAGAGCAAACACAGATGATTTCCCC


 


CTCACGTTGGCGTCTGCTGTACCCTCTCAGACATCTGTGTACTTCTG


 


TGCCAGCAGCCTAAGTTTCGGCACTGAAGCTTTCTTTGGACAAGGCA


 


CCAGACTCACAGTTGTA






Another exemplary sequence alpha chain sequence is shown below; D27 is in bold and upper case.











Alpha chain















LOCUS 3QEU_D 202 aa linear PRI 10-OCT-2012


DEFINITION Chain D, The Crystal Structure 


Of Tcr Dmf5.


ACCESSION 3QEU_D


VERSION 3QEU_D GI:339717586








  1 
akeveqnsgp lsvpegaias lnctysDrgs qsffwyrqys 



gkspelimfi ysngdkedgr


 


 61 
ftaqlnkasq yvsllirdsq psdsatylca vnfgggklif 



gqgtelsvkp niqnpdpavy


 


121 
qlrdskssdk svclftdfds qtnvsqskds dvyitdkcvl 



dmrsmdfksn savawsnksd


 


181 
facanafnns iipedtffps pe



(SEQ ID NO: 5)









Another exemplary sequence beta chain sequence is shown below; the amino acid corresponding to Leu95 (Leu96 in this sequence) is in bold and upper case.











Beta chain















LOCUS 3QEU_E 243 aa linear PRI 10-OCT-2012


DEFINITION Chain E, The Crystal Structure 


Of Tcr Dmf5.


ACCESSION 3QEU_E


VERSION 3QEU_E GI:339717587








  1
miagitqapt sqilaagrrm tlrctqdmrh namywyrqdl 



glglrlihys ntagttgkge


 


 61
vpdgysysra ntddfpltla savpsqtsvy fcassLsfgt 



eaffgqgtrl tvvedlnkvf


 


121
ppevavfeps eaeishtqka tlvclatgfy pdhvelswwv 



ngkevhsgvc tdpqplkeqp


 


181
alndsryals srlrvsatfw gdprnhfrcq vqfyglsend 



ewtqdrakpv tgivsaeawg


 


241
rad



(SEQ ID NO: 6)









Other sequences can include those used in Borbulevych et al., J Immunol 187:2453-2463 (2011) or Davis-Harrison et al., J Mol Biol 346: 533-550 (2005).
Provided herein are variants of the DMF5 TCR receptor that have mutations resulting in increased binding affinity. In some embodiments, a High-Affinity DMF5 Variant includes a D to Y or D to W mutation at amino acid 27 in the alpha chain (αD27Y or αD27W) and/or a L to W mutation at amino acid 95 in the beta chain (βL95W). In some embodiments, the Variant also or alternatively has one or more of an A to P mutation at amino acid 55 of the alpha chain (αA55P) and/or a T to F mutation at amino acid 102 in the beta chain (βT102F).
In some embodiments, the High-Affinity DMF5 Variant includes one or more additional mutations that do not decrease affinity. Thus, in some embodiments, the sequence can be at least 80%, 85%, 90%, 95%, or 99% identical to at least 60%, 70%, 80%, 90%, or 100% of a High-Affinity DMF5 Variant, e.g., SEQ ID NO:1 or 2; e.g., the sequence can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mutations. In some embodiments the variant includes one or more of the following mutations described in Robbins et al., J. Immunol., 180: 6116-6131, 2008 (e.g., (3G54A); US20120071420; or Borbulevych et al. (2011) J Immunol 187 p. 2453).
To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). The length of a reference sequence aligned for comparison purposes is typically at least 80% of the length of the reference sequence, and in some embodiments is at least 90% or 100%. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. In another embodiment, the percent identity of two amino acid sequences can be assessed as a function of the conservation of amino acid residues within the same family of amino acids (e.g., positive charge, negative charge, polar and uncharged, hydrophobic) at corresponding positions in both amino acid sequences (e.g., the presence of an alanine residue in place of a valine residue at a specific position in both sequences shows a high level of conservation, but the presence of an arginine residue in place of an aspartate residue at a specific position in both sequences shows a low level of conservation).
For purposes of the present invention, the comparison of sequences and determination of percent identity between two sequences can be accomplished using a BLOSUM 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
In some embodiments, the mutation is a conservative substitution. Such changes include substituting any of isoleucine (I), valine (V), and leucine (L) for any other of these hydrophobic amino acids; aspartic acid (D) for glutamic acid (E) and vice versa; glutamine (Q) for asparagine (N) and vice versa; and serine (S) for threonine (T) and vice versa. Other substitutions can also be considered conservative, depending on the environment of the particular amino acid and its role in the three-dimensional structure of the protein. For example, glycine (G) and alanine (A) can frequently be interchangeable, as can alanine (A) and valine (V). Methionine (M), which is relatively hydrophobic, can frequently be interchanged with leucine and isoleucine, and sometimes with valine. Lysine (K) and arginine (R) are frequently interchangeable in locations in which the significant feature of the amino acid residue is its charge and the differing pK's of these two amino acid residues are not significant. Still other changes can be considered “conservative” in particular environments (see, e.g. Table III of US2011/0201052; pages 13-15 “Biochemistry” 2nd ED. Stryer ed (Stanford University); Henikoff et al., PNAS 1992 Vol 89 10915-10919; Lei et al., J Biol Chem 1995 May 19; 270(20):11882-6).
Therapeutic Compositions and Imaging Reagents
Also described herein are compounds that include one or more non-TCR moieties in addition to the High-Affinity DMF5 Variants described herein, e.g., linked to the N or C terminus of the High-Affinity DMF5 Variant (preferably the C terminus), or internally, e.g., linked to an internal cysteine, so long as the binding activity of the variant is not affected. In some embodiments, the non-TCR moiety is a therapeutic agent or a detectable moiety. These compounds can be used to treat and/or detect cancers, i.e., MART-1 expressing cancers, including melanoma, e.g., malignant melanomas, including primary cutaneous malignant melanomas and mucosal melanomas; and other tumors of melanocytic origin or differentiation (i.e., melanosome producing), e.g., clear cell sarcoma, melanotic neurofibroma, melanotic schwannoma and other melanotic neural crest derived tumors, as well as perivascular epitheloid cell tumors, angiomyolipoma, lymphangioleiomyoma/tosis), and pulmonary sugar tumors. In preferred embodiments, the cancer is melanoma, e.g., melanoma with HLA-A201 tissue type. Methods for diagnosing a subject with a MART-1 expressing cancer are known in the art, and can include obtaining a sample comprising one or more cells of the cancer or tumor (e.g., cells that are known or suspected to be cancerous or from a tumor) and detecting expression of MART-1 in the cells, using methods know in the art (e.g., methods that detect the presence of MART-1 mRNA (GenBank Acc. No. NM_005511.1) or protein (GenBank Acc. No. NP_005502.1), e.g., PCR-based or antibody based methods).
Therapeutic Compositions
In some embodiments, the invention provides therapeutic compositions in which the High-Affinity DMF5 Variants described herein are linked to one or more anti-cancer therapeutic agents, i.e., agents that have a therapeutic effect against cancer, e.g., against melanoma. In some embodiments, the therapeutic effect is an anti-tumor effect, e.g., resulting in cancer cell death or stasis, tumor regression or shrinkage, reduction or delay in tumor growth, or a reduction or delay in tumor metastasis.
The High-Affinity DMF5 Variant as described herein can be used to deliver a variety of anti-cancer therapeutic agents, e.g., a radioisotope; an anticancer drug such as a genotoxin; or any other cytotoxic moiety, e.g., molecules of plant, fungal, or bacterial origin, or biological proteins (e.g., protein toxins) or particles (e.g., a recombinant viral particles, e.g., via a viral coat protein), or mixtures thereof. The therapeutic agent can be an intracellularly active drug or other agent, such as short-range radiation emitters, including, for example, short-range, high-energy α-emitters, as described herein. In some embodiments, the High-Affinity DMF5 Variant can be coupled to a molecule of plant or bacterial origin (or derivative thereof), e.g., a maytansinoid (e.g., maytansinol or the DM1 maytansinoid). DM1 is a sulfhydryl-containing derivative of maytansine that can be linked to the peptide, e.g., via a disulfide linker that releases DM1 when inside target cells. The disulfide linkers display greater stability in storage and in serum than other linkers. Maytansine is a cytotoxic agent that effects cell killing by preventing the formation of microtubules and depolymerization of extant microtubules. It is 100- to 1000-fold more cytotoxic than anticancer agents such as doxorubicin, methotrexate, and vinca alkyloid, which are currently in clinical use. Alternatively, the High-Affinity DMF5 Variant as described herein can be coupled to a taxane, a calicheamicin, a proteosome inhibitor, or a topoisomerase inhibitor. [(1R)-3-methyl-1-[[(2S)-1-oxo-3-phenyl-2-[(3-mercaptoacetyl)amino]propyl]amino]butyl]Boronic acid is a suitable proteosome inhibitor. N,N′-bis[2-(9-methylphenazine-1-carboxamido)ethyl]-1,2-ethanediamine is a suitable topoisomerase inhibitor.
Enzymatically active toxins and fragments thereof are exemplified by diphtheria toxin A fragment, nonbinding active fragments of diphtheria toxin, exotoxin A (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α-sacrin, certain Aleurites fordii proteins, certain Dianthin proteins, Phytolacca americana proteins (PAP, PAPII and PAP-S), Morodica charantia inhibitor, curcin, crotin, Saponaria officinalis inhibitor, gelonin, mitogillin, restrictocin, phenomycin, and enomycin. In some embodiments, the High-Affinity DMF5 Variant is conjugated to maytansinoids, e.g., maytansinol (see U.S. Pat. No. 5,208,020), CC-1065 (see U.S. Pat. Nos. 5,475,092, 5,585,499, 5,846,545). Procedures for preparing enzymatically active polypeptides of the immunotoxins are described in WO84/03508 and WO85/03508, which are hereby incorporated by reference. Examples of cytotoxic moieties that can be conjugated to the antibodies include adriamycin, chlorambucil, daunomycin, methotrexate, neocarzinostatin, and platinum.
To kill or ablate cancerous cells, a High-Affinity DMF5 Variant can be conjugated with a prodrug that is activated only when in close proximity with a prodrug activator. The prodrug activator is conjugated with a second High-Affinity DMF5 Variant, e.g., a second High-Affinity DMF5 Variant according to the present invention, preferably one that binds to a non-competing site on the same receptor (e.g., plectin-1) or cell. Whether two High-Affinity DMF5 Variant bind to competing or non-competing binding sites can be determined by conventional competitive binding assays. Drug-prodrug pairs suitable for use are known in the art, see, e.g., in Blakely et al., Cancer Research 56:3287-3292 (1996).
A drug attached to a variant as described herein can also include agents that are derived from, or that beneficially modulate host biological processes, such as interferons, tumor growth factors, tumor necrosis factors, growth factors such as GM-CSF and G-CSF and interleukins, for example, interleukin-2, interleukin-6, interleukin-7 and interleukin-12, and the like. An drug attached to a peptide of the present invention may comprise an agent which damages DNA and/or prevent cells from multiplying, such as genotoxins. A genotoxin includes but is not limited to alkylating agents, antimetabolites, DNA cutters, DNA binders, topoisomerase poisons and spindle poisons. Examples of alkylating agents are lomustine, carmustine, streptozocin, mechlorethamine, melphalan, uracil nitrogen mustard, chlorambucil, cyclosphamide, iphosphamide, cisplatin, carboplatin, mitomycin, thiotepa, dacarbazin, procarbazine, hexamethyl melamine, triethylene melamine, busulfan, pipobroman, mitotane and other platine derivatives.
Alternatively, the High-Affinity DMF5 Variant can be coupled to high energy radiation emitters, for example, a radioisotope, such as 131I, a γ-emitter, which, when localized at the tumor site, results in a killing of several cell diameters. See, e.g., Order, “Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody in Cancer Therapy”, in Monoclonal Antibodies for Cancer Detection and Therapy, R. W. Baldwin et al. (eds.), pp 303-316 (Academic Press 1985). Other suitable radioisotopes include α-emitters, such as 212Bi, 213Bi, and 211At, and β-emitters, such as 186Re and 90Y. Lu117 may also be used as both an imaging and cytotoxic agent.
Radioimmunotherapy (RIT) using High-Affinity DMF5 Variant labeled with 131I, 90Y, and 177Lu can also be used. There are significant differences in the physical characteristics of these three nuclides and as a result, the choice of radionuclide can be important in order to deliver maximum radiation dose to the tumor. The higher beta energy particles of 90Y may be good for bulky tumors, but it may not be necessary for small tumors and especially bone metastases, (e.g. those common to prostate cancer). The relatively low energy beta particles of 131I are ideal, but in vivo dehalogenation of radioiodinated molecules is a major disadvantage for internalizing High-Affinity DMF5 Variants. In contrast, 177Lu has low energy beta particle with only 0.2-0.3 mm range and delivers much lower radiation dose to bone marrow compared to 90Y. In addition, due to longer physical half-life (compared to 90Y), the tumor residence times are higher. As a result, higher activities (more mCi amounts) of 177Lu labeled agents can be administered with comparatively less radiation dose to marrow. There have been several clinical studies investigating the use of 177Lu labeled antibodies in the treatment of various cancers (see, e.g., Mulligan et al., Clin Cancer Res. 1: 1447-1454 (1995); Meredith et al., Nucl Med 37:1491-1496 (1996); Alvarez et al., Gynecologic Oncology 65: 94-101 (1997)).
The High-Affinity DMF5 Variant can also be conjugated or fused to viral surface proteins present on viral particles. For example, a High-Affinity DMF5 Variant could be fused (e.g., to form a fusion protein) to a viral surface protein. Alternatively, a High-Affinity DMF5 Variant could be chemically conjugated (e.g., via a chemical linker) to a viral surface protein. Preferably, the virus is one that fuses with endocytic membranes, e.g., an influenza virus, such that the virus is internalized along with the High-Affinity DMF5 Variant and thereby enters and kills cancer cells. The virus can be genetically engineered as a cellular toxin. For example, the virus could express or induce the expression of genes that are toxic to cells, e.g., cell death promoting genes. Preferably, such viruses would be incapable of viral replication.
Additional examples of cytotoxic peptides or proteins include Idarubicin; CRM9 (e.g., FN18-CRM9, Knechtle et al., Transplantation 1997; 63:1-6); or pokeweed antiviral protein. In some embodiments, the cytotoxic protein is a bacterial toxin, e.g., diphtheria toxin (DT) or portions or variants thereof, e.g., Met1-Thr387, e.g., as described in Aullo et al., EMBO J. 11(2):575-83 (1992); Abi-Habib et al., Blood. 104(7):2143-2148 (2004); Perentesis et al., Proc. Natl. Acad. Sci. USA 85:8386-8390 (1988); Zettlemeissl et al., Gene. 41(1):103-111 (1986); US 2009/0010966; US20090041797; U.S. Pat. No. 5,843,711; U.S. Pat. No. 7,585,942; U.S. Pat. No. 7,696,338; or US20080166375; or Pseudomonas exotoxin (PE), or portions or variants thereof, e.g., as described in U.S. Pat. Nos. 4,545,985; 4,892,827; 5,458,878; 7,314,632; Song et al., Protein Expression and Purification 44(1):52-57 (2005); Theuer et al., J. Biol. Chem. 267(24):16872-16877 (1992); Heimbrook et al., Proc Natl Acad Sci USA. 87(12):4697-4701 (1990); Debinski et al., Mol Cell Biol. 11(3):1751-1753 (1991); Chaudhary et al., Proc. Nadl. Acad. Sci. USA 87:308-312 (1990). In some embodiments, the cytotoxic protein is a plant toxin, e.g., a plant holotoxin (e.g., class II ribosome-inactivating proteins such as ricin (e.g., deglycosylated ricin A chain (dgA)), abrin, mistletoe lectin, or modeccin) or hemitoxin (class I ribosome-inactivating proteins, e.g., PAP, saporin, bryodin 1, bouganin, or gelonin), or fragments or variants thereof that retain cytotoxic activity. See, e.g., Neville et al., J Contr Rel 1993; 24:133-141; Vallera, Blood 1994; 83:309-317; Vitetta et al., Immunology Today 1993; 14:252-259; Kreitman et al., AAPS Journal. 2006; 8(3):E532-E551). Suitable sequences are known in the art.
Imaging Reagents
In some embodiments, the invention provides compounds that are useful as imaging reagents in which the non-TCR moiety is a detectable moiety, e.g., a radioactive moiety (e.g., 35S, 32P, 3H, or 14C), a chemiluminescent moiety (e.g., Streptavidin-Alkaline Phosphatase, Streptavidin-Horseradish Peroxidase, Streptavidin-Biotinylated Horseradish Peroxidase, e.g., for detection with ECLTM or a variant thereof (Amersham Biosciences, Piscataway, N.J.)), a fluorescent moiety (e.g., CyDyes (such as Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, Cy5Q, Cy7Q, Cy2-Streptavidin, Cy3-Streptavidin, Cy5-Streptavidin, Streptavidin-Fluorescein, Streptavidin-Texas Red (Amersham Biosciences, Piscataway, N.J.), fluorescein, rhodamine, Texas red, cyanine, Cascade Blue, or phycoerythrin), quantum dots (see, e.g., Watson et al., BioTechniques 2003 February; 34(2):296-300, 302-3; Goldman et al., J. Am. Chem. Soc. 2002 Jun. 5; 124(22):6378-82; Han et al., Nat. Biotechnol. 2001 July; 19(7):631-5; Chan et al., Science 1998 Sep. 25; 281(5385):2016-8), or other directly or indirectly detectable moiety (e.g., gold or other particles). Contrast agents, e.g., contrast agents for MRI or NMR, for X-ray CT, Raman imaging, optical coherence tomography, absorption imaging, ultrasound imaging, or thermal imaging can be used. Exemplary contrast agents include gold (e.g., gold nanoparticles), gadolinium (e.g., chelated Gd), iron oxides (e.g., superparamagnetic iron oxide (SPIO), monocrystalline iron oxide nanoparticles (MIONs), and ultrasmall superparamagnetic iron oxide (USPIO)), manganese chelates (e.g., Mn-DPDP), barium sulfate, iodinated contrast media (iohexyl), microbubbles, or perfluorocarbons. In a preferred embodiment, the variant is coupled to a paramagnetic agent, e.g., a paramagnetic nanoparticle, e.g., cross-linked iron oxide (CLIO) nanoparticles; see, e.g., US 20110046004; Josephson et al., Bioconjug. Chem., 10(2):186-91 (1999).
In some embodiments, the detectable moiety can be or include a fluorophore, e.g., a near infrared fluorophore (NIRF). A number of NIRFs useful in the methods and compositions described herein are known in the art, e.g., including Cy5.5, Cy5 and Cy7 (Amersham, Arlington Hts., IL; IRD41 and IRD700 (LI-COR, Lincoln, Nebr.); NIR-1, (Dejindo, Kumamoto, Japan); LaJolla Blue (Diatron, Miami, Fla.); indocyanine green (ICG) and its analogs (Licha et al., 1996, SPIE 2927:192-198; Ito et al., U.S. Pat. No. 5,968,479); indotricarbocyanine (ITC; WO 98/47538); and chelated lanthanide compounds. Fluorescent lanthanide metals include europium and terbium. Fluorescence properties of lanthanides are described in Lackowicz, 1999, Principles of Fluorescence Spectroscopy, 2nd Ed., Kluwar Academic, New York. The fluorophores can be covalently linked to the plectin-1 binding moiety, or to a nanoparticle, e.g., via a fluorochrome attachment moiety, backbone, or spacer using any suitable reactive group on the fluorochrome and a compatible functional group on the fluorochrome attachment moiety, backbone, or spacer. For example, a carboxyl group (or activated ester) on a fluorochrome can be used to form an amide linkage with a primary amine such as the epsilon-amino group of the lysyl side chain on polylysine. Alternatively or in addition, the fluorophores can be linked directly to the backbone or linked to the backbone through nonbiodegradable spacers. See, e.g., US P.G.Pub. 20060275775.
The High-Affinity DMF5 Variants can be linked to the detectable moieties directly, e.g., as a fusion protein with protein or peptide detectable moieties (with or without an optional linking sequence, e.g., a flexible linker sequence) or via a chemical coupling moiety. A number of such coupling moieties are known in the art, e.g., a peptide linker or a chemical linker, e.g., as described in International Patent Application Publication No. WO 2009/036092.
These moieties can be detected using methods known in the art. For example, a number of methods are known in the art for detection of fluorescent moieties, including, but not limited to, fluorescence polarization (FP), fluorescence resonance energy transfer (FRET), time-resolved fluorescence resonance energy transfer (TR-FRET), and fluorescence intensity (FI).
Nucleic Acids, Expression Vectors, and Host Cells
Also provided herein are polynucleotides (also referred to herein as nucleic acids) that encode a high-affinity variant as described herein, as well as vectors, preferably expression vectors, containing a nucleic acid encoding a polypeptide described herein. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked and can include a plasmid, cosmid or viral vector. The vector can be capable of autonomous replication or it can integrate into a host DNA. Viral vectors include, e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses.
A vector can include a nucleic acid that encodes a high-affinity variant as described herein in a form suitable for expression of the nucleic acid in a host cell. Preferably the recombinant expression vector includes one or more regulatory sequences operatively linked to the nucleic acid sequence to be expressed. The term “regulatory sequence” includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence, as well as tissue-specific regulatory and/or inducible sequences. The design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like. The expression vectors of the invention can be introduced into host cells to thereby produce a high-affinity variant as described herein, including fusion proteins as described herein.
The recombinant expression vectors of the invention can be designed for expression of a high-affinity variant as described herein in prokaryotic or eukaryotic cells. For example, polypeptides of the invention can be expressed in E. coli, insect cells (e.g., using baculovirus expression vectors), yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, (1990) Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. Alternatively, the recombinant expression vector can be transcribed and translated in vitro, e.g., using T7 promoter regulatory sequences and T7 polymerase.
Expression of proteins in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.), which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.
The expression vector can be, e.g., a yeast expression vector, a vector for expression in insect cells (e.g., a baculovirus expression vector), or a vector suitable for expression in mammalian cells.
When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.
In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Non-limiting examples of suitable tissue-specific promoters include the lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) and immunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748).
In some embodiments, the vector is pMSGV1, which is derived from pMSGV and includes a murine stem cell virus long terminal repeat containing an extended gag region and Kozak sequence (Hughes et al., Hum Gene Ther. 2005; 16:457-472). In some embodiments, the vector is pMSGV DMF5 furin 2-A, which was generated by introducing DMF5 TCR-α cDNA15 followed by a furin T2A cleavage sequence and DMF5 TCR-β, as described in Johnson et al., 2006, supra, and Johnson et al., Blood. 114: 535-546, 2009.
Another aspect the invention provides a host cell which includes a nucleic acid molecule described herein, e.g., a nucleic acid molecule encoding a high-affinity variant as described herein within a recombinant expression vector or a nucleic acid molecule containing sequences that allow it to homologously recombine into a specific site of the host cell's genome. The terms “host cell” and “recombinant host cell” are used interchangeably herein. Such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
A host cell can be any prokaryotic or eukaryotic cell. For example, a high-affinity variant as described herein can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (e.g., Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.
Vector DNA can be introduced into host cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation.
A host cell of the invention can be used to produce (i.e., express) a high-affinity variant as described herein. Accordingly, the invention further provides methods for producing a high-affinity variant as described herein using the host cells of the invention. In one embodiment, the method includes culturing the host cell of the invention (into which a recombinant expression vector encoding a high-affinity variant as described herein has been introduced) in a suitable medium such that a high-affinity variant as described herein is produced. In another embodiment, the method further includes isolating a high-affinity variant as described herein from the medium or the host cell.
Also provided herein are human cells, e.g., hematopoietic cells, e.g., human PBLs, transformed with a nucleic acid that encodes a high-affinity variant as described herein.
Gene Therapy
The nucleic acids encoding a high-affinity variant described herein can be incorporated into a nucleic acid construct to be used as a part of a nucleic acid therapy protocol. Thus described herein are targeted expression vectors for in vivo or in vitro transfection and expression of a polynucleotide that encodes a high-affinity variant as described herein, in particular cell types, especially autologous peripheral blood lymphocytes (PBLs), and methods of use thereof. Expression constructs of such components can be administered in any effective carrier, e.g., any formulation or composition capable of effectively delivering the polynucleotide that encodes a high-affinity variant as described herein to cells in vivo or in vitro. Approaches include insertion of the polynucleotide that encodes a high-affinity variant as described herein in viral vectors, including recombinant retroviruses, adenovirus, adeno-associated virus, lentivirus, and herpes simplex virus-1, or recombinant bacterial or eukaryotic plasmids. Viral vectors transfect cells directly; plasmid DNA can be delivered naked or with the help of, for example, cationic liposomes (lipofectamine) or derivatized (e.g., antibody conjugated), polylysine conjugates, gramacidin S, artificial viral envelopes or other such intracellular carriers, as well as direct injection of the nucleic acid construct or CaPO4 precipitation carried out in vivo. RNA (e.g., RNA encoding the variant) can also be delivered directly to the cell, e.g., using electroporation.
A preferred approach for in vivo introduction of nucleic acid into a cell is by use of a viral vector containing nucleic acid, e.g., a cDNA that encodes a high-affinity variant as described herein. Infection of cells with a viral vector has the advantage that a large proportion of the targeted cells can receive the nucleic acid. Additionally, molecules encoded within the viral vector, e.g., by a cDNA contained in the viral vector, are expressed efficiently in cells that have taken up viral vector nucleic acid.
Retrovirus vectors and adeno-associated virus vectors can be used as a recombinant nucleic acid delivery system for the transfer of exogenous nucleic acids in vivo or in vitro, particularly into humans. These vectors provide efficient delivery of nucleic acids into cells, and the transferred nucleic acids are stably integrated into the chromosomal DNA of the host. The development of specialized cell lines (termed “packaging cells”) which produce only replication-defective retroviruses has increased the utility of retroviruses for nucleic acid therapy, and defective retroviruses are characterized for use in nucleic acid transfer for gene therapy purposes (for a review see Miller, Blood 76:271 (1990)). A replication defective retrovirus can be packaged into virions, which can be used to infect a target cell through the use of a helper virus by standard techniques. Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in Ausubel, et al., eds., Current Protocols in Molecular Biology, Greene Publishing Associates, (1989), Sections 9.10-9.14, and other standard laboratory manuals. Examples of suitable retroviruses include pLJ, pZIP, pWE and pEM which are known to those skilled in the art. Examples of suitable packaging virus lines for preparing both ecotropic and amphotropic retroviral systems include ΨCrip, ΨCre, Ψ2 and ΨAm. Retroviruses have been used to introduce a variety of nucleic acids into many different cell types, including epithelial cells, in vitro and/or in vivo (see for example Eglitis, et al. (1985) Science 230:1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. USA 87:6141-6145; Huber et al. (1991) Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad. Sci. USA 88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; van Beusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay et al. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl. Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J. Immunol. 150:4104-4115; U.S. Pat. Nos. 4,868,116; 4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; and PCT Application WO 92/07573).
Another viral nucleic acid delivery system useful in the present methods utilizes adenovirus-derived vectors. The genome of an adenovirus can be manipulated, such that it encodes and expresses a protein or gene product of interest but is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. See, for example, Berkner et al., BioTechniques 6:616 (1988); Rosenfeld et al., Science 252:431-434 (1991); and Rosenfeld et al., Cell 68:143-155 (1992). Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 d1324 or other strains of adenovirus (e.g., Ad2, Ad3, or Ad7 etc.) are known to those skilled in the art. Recombinant adenoviruses can be advantageous in certain circumstances, in that they are not capable of infecting non-dividing cells and can be used to infect a wide variety of cell types, including epithelial cells (Rosenfeld et al., (1992) supra). Furthermore, the virus particle is relatively stable and amenable to purification and concentration, and as above, can be modified so as to affect the spectrum of infectivity. Additionally, introduced adenoviral DNA (and foreign DNA contained therein) is not integrated into the genome of a host cell but remains episomal, thereby avoiding potential problems that can occur as a result of insertional mutagenesis in situ, where introduced DNA becomes integrated into the host genome (e.g., retroviral DNA). Moreover, the carrying capacity of the adenoviral genome for foreign DNA is large (up to 8 kilobases) relative to other nucleic acid delivery vectors (Berkner et al., supra; Haj-Ahmand and Graham, J. Virol. 57:267 (1986).
Yet another viral vector system useful for delivery of nucleic acids is the adeno-associated virus (AAV). Adeno-associated virus is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle. (For a review see Muzyczka et al., Curr. Topics in Micro. and Immunol. 158:97-129 (1992). It is also one of the few viruses that may integrate its DNA into non-dividing cells, and exhibits a high frequency of stable integration (see for example Flotte et al., Am. J. Respir. Cell. Mol. Biol. 7:349-356 (1992); Samulski et al., J. Virol. 63:3822-3828 (1989); and McLaughlin et al., J. Virol. 62:1963-1973 (1989). Vectors containing as little as 300 base pairs of AAV can be packaged and can integrate. Space for exogenous DNA is limited to about 4.5 kb. An AAV vector such as that described in Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985) can be used to introduce DNA into cells. A variety of nucleic acids have been introduced into different cell types using AAV vectors (see for example Hermonat et al., Proc. Natl. Acad. Sci. USA 81:6466-6470 (1984); Tratschin et al., Mol. Cell. Biol. 4:2072-2081 (1985); Wondisford et al., Mol. Endocrinol. 2:32-39 (1988); Tratschin et al., J. Virol. 51:611-619 (1984); and Flotte et al., J. Biol. Chem. 268:3781-3790 (1993).
In addition to viral transfer methods, such as those illustrated above, non-viral methods can also be employed to cause expression of a nucleic acid compound described herein (e.g., a polynucleotide that encodes a high-affinity variant as described herein) in a cell or tissue of a subject. Typically non-viral methods of nucleic acid transfer rely on the normal mechanisms used by mammalian cells for the uptake and intracellular transport of macromolecules. In some embodiments, non-viral nucleic acid delivery systems can rely on endocytic pathways for the uptake of the subject nucleic acid by the targeted cell. Exemplary nucleic acid delivery systems of this type include liposomal derived systems, poly-lysine conjugates, and artificial viral envelopes. Other embodiments include plasmid injection systems such as are described in Meuli et al., J. Invest. Dermatol. 116(1):131-135 (2001); Cohen et al., Gene Ther. 7(22):1896-905 (2000); or Tam et al., Gene Ther. 7(21):1867-74 (2000).
In some embodiments, a polynucleotide that encodes a high-affinity variant as described herein is entrapped in liposomes bearing positive charges on their surface (e.g., lipofectins), which can be tagged with antibodies against cell surface antigens of the target tissue (Mizuno et al., No Shinkei Geka 20:547-551 (1992); PCT publication WO91/06309; Japanese patent application 1047381; and European patent publication EP-A-43075).
In clinical settings, the nucleic acid delivery systems for the polynucleotide that encodes a high-affinity variant as described herein can be introduced into a cell or subject by any of a number of methods, each of which is familiar in the art. For instance, a pharmaceutical preparation of the nucleic acid delivery system can be introduced systemically, e.g., by intravenous injection, and specific transduction of the protein in the target cells will occur predominantly from specificity of transfection, provided by the nucleic acid delivery vehicle, cell-type or tissue-type expression due to the transcriptional regulatory sequences controlling expression of the receptor nucleic acid, or a combination thereof. In other embodiments, initial delivery of the recombinant nucleic acidis more limited, with introduction into the subject being quite localized. For example, the nucleic acid delivery vehicle can be introduced by catheter (see U.S. Pat. No. 5,328,470) or by stereotactic injection (e.g., Chen et al., PNAS USA 91: 3054-3057 (1994)).
A pharmaceutical preparation of can consist essentially of a nucleic acid delivery system in an acceptable diluent, or can comprise a slow release matrix in which the nucleic acid delivery vehicle is embedded. Alternatively, where the complete nucleic acid delivery system can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can comprise one or more cells, which produce the nucleic acid delivery system.
Pharmaceutical Compositions and Methods of Administration
The methods described herein include the manufacture and use of pharmaceutical compositions, which include the High Affinity Variants described herein as active agents. Also included are the pharmaceutical compositions themselves.
Pharmaceutical compositions typically include a pharmaceutically acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
Pharmaceutical compositions are typically formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.
Methods of formulating suitable pharmaceutical compositions are known in the art, see, e.g., Remington: The Science and Practice of Pharmacy, 21st ed., 2005; and the books in the series Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY). For example, solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
Pharmaceutical compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying, which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
The compositions described herein can also be administered orally, by inhalation, or topically.
Therapeutic compounds that are or include nucleic acids can be administered by any method suitable for administration of nucleic acid agents, such as a DNA vaccine.
These methods include gene guns, bio injectors, and skin patches as well as needle-free methods such as the micro-particle DNA vaccine technology disclosed in U.S. Pat. No. 6,194,389, and the mammalian transdermal needle-free vaccination with powder-form vaccine as disclosed in U.S. Pat. No. 6,168,587. Additionally, intranasal delivery is possible, as described in, inter alia, Hamajima et al., Clin. Immunol. Immunopathol., 88(2), 205-10 (1998). Liposomes (e.g., as described in U.S. Pat. No. 6,472,375) and microencapsulation can also be used. Biodegradable targetable microparticle delivery systems can also be used (e.g., as described in U.S. Pat. No. 6,471,996).
In one embodiment, the therapeutic compounds are prepared with carriers that will protect the therapeutic compounds against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid.
Such formulations can be prepared using standard techniques, or obtained commercially, e.g., from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to selected cells with monoclonal antibodies to cellular antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.
The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
Methods of Use
The High-Affinity DMF5 Variants described herein can be used, e.g., to treat or detect cancer, e.g., melanoma, in a subject. As used in this context, to “treat” means to ameliorate at least one symptom of the cancer. Administration of a therapeutically effective amount of a high-affinity variant as described herein for the treatment of melanoma will result in one or more of cancer cell death or stasis, tumor regression or shrinkage, reduction or delay in tumor growth, or a reduction or delay in cancer metastasis. The methods of treatment can include administering a composition comprising a High-Affinity DMF5 Variant as described herein, e.g., with or without a therapeutic agent or a detectable moiety, to a subject who is in need of, or who has been determined to be in need of, such treatment.
In some embodiments, the methods of treatment include administering to the subject (i.e., a subject who has cancer, e.g., melanoma) T cells (preferably autologous T cells) that have been genetically engineered to express a high-affinity variant as described herein. Methods for doing so are known in the art, see, e.g., Johnson et al. 2009. Blood. 114: 535-546; Morgan et al., 2006. Science. 314: 126-129; Borbulevych et al., J. Immunol. 2011 Sep. 1; 187(5): 2453-2463. In some embodiments, the methods include obtaining cells, preferably PBLs, from a human, preferably from the subject who is to be treated (i.e., autologous cells). The cells are transfected with a vector comprising a nucleic acid encoding a high-affinity variant as described herein, and maintained in culture, optionally for a time sufficient for expansion to occur. Expression of the high-affinity variant as described herein can be optionally confirmed using methods known in the art, e.g., tetramer staining and flow cytometric analysis, and cell function can also be evaluated, e.g., by overnight coculture with cognate antigen-bearing target cells (1×105:1×105) and enzyme-linked immunosorbent assay (ELISA) measurement (Pierce Endogen) of interferon-γ (IFN-γ) produced in the culture supernatant. The cells are then administered to the subject, e.g., by infusion into the subject intravenously.
The methods of use can also include imaging methods. In these methods, an imaging reagent comprising a high-affinity variant as described herein linked to a detectable moiety is administered to the subject, and an appropriate method is used to detect localization of the moiety in the subject's body. For example, MRI or NMR, for X-ray CT, Raman imaging, optical coherence tomography, absorption imaging, ultrasound imaging, or thermal imaging can be used when a high-affinity variant linked to an appropriate detectable moiety, i.e., an appropriate contrast agent, is used.
Imaging methods can also be carried out in vitro. For example, one other potential use is imaging to detect antigen-expressing cells in a T cell population that might be cultured from a patient. For example, the T cells are removed, grown up, and the high affinity variant, coupled to a detectable moiety, e.g., a fluorescent tag, is used to identify T cells expressing target antigen, e.g., using flow cytometry/FACS.
EXAMPLES
The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.
Example 1
Evidence for a Dynamically Driven T Cell Signaling Mechanism
Oligomerization, stronger associations with neighboring subunits, and conformational changes can all be facilitated by changes in molecular flexibility. Global reductions in flexibility can help overcome the entropy barrier associated with binding and enhance complementarity at interfaces (6). More defined conformational changes that occur upon binding often take place at regions with heightened intrinsic flexibility (7). Recent work in a number of systems has demonstrated how ligand binding can alter protein flexibility across time and length scales, impacting binding behavior at distant sites in the absence of crystallographically-observed structural changes (8-10). This process, termed dynamically-driven allostery, has attracted attention as a unifying mechanism for the general phenomenon of allostery and is of particular interest in cases where signaling occurs in the absence of clear structural changes (reviewed in ref 11).
As large conformational changes that could communicate pMHC binding have not been observed in the TCR, we wished to explore the potential for alterations in protein flexibility as a mechanism to communicate receptor engagement.
Materials and Methods
The following materials and methods were used in Example 1.
Protein Expression and Purification
Recombinant A6, DMF5, and HLA-A2 were refolded from inclusion bodies and purified as described previously (12). Constructs were stabilized either with an engineered disulfide bond (13) or a C-terminal leucine zipper (14). Synthetic peptide was purchased from Genscript.
Hydrogen/Deuterium Exchange
Exchange was initiated by diluting samples in at 25° C. in 25 mM HEPES, 50 mM NaCl, pH 7.4 10-fold with the same buffer made with 99.9% 2H2O. For analysis of free A6, DMF5, and Tax/HLA-A2, protein concentration after dilution was 10 μM. For analysis of bound A6, concentrations after dilution were 10 μM TCR, 40 μM pMHC, resulting in 94% occupancy. These concentrations were reversed for analysis of bound Tax/HLA-A2. For examining DMF5, concentrations after dilution were 20 μM TCR and 80 μM pMHC, resulting in 100% occupancy. For A6 with non-cognate MART-126-35/HLA-A2, concentrations after dilution were 10 μM TCR and 45 μM pMHC. After dilution into 2H2O, 5 μL aliquots were removed at time points ranging from five seconds to 10 hours. For each aliquot, exchange was quenched at 0° C. with 100 μL of 0.1% trifluoracetic acid at pH 2.4, conditions under which the rate of hydrogen/deuterium exchange is minimal (15). Pepsin (8 μL at 1 mg/mL) was added and digestion performed for 5 minutes before freezing in liquid nitrogen. Samples were stored at −80° C. until analysis.
Identification of Peptic Fragments of TCR and HLA-A2
Peptic fragments were identified by LC/MS/MS. 500 ng of the pepsin digest was loaded onto a 75 μm×15 cm C12 column. Peptides were eluted using an Eksigent nano UltraLC into the nanospray source of an AB Sciex 5500 QTrap spectrometer. An individual survey scan was performed and up to six dependent MS/MS acquisitions were collected every cycle. Data were searched using the Paragon algorithm in thorough search mode with no enzyme specificity (16). The sequences of the TCR, HLA-A2, β2m, and potential contaminants (keratins, pepsin, and the E. coli genome) were searched. False detection rates were determined using a decoy-database strategy (17). In all cases, the relevant proteins were the top hit and all peptides passed on for further analysis identified at a 99% CI.
MALDI Mass Spectrometry
A matrix solution of 2,5-dihydroxybenzoic acid in 50:50 0.1% trifluoroacetic acid (pH 2.4) and acetonitrile was prepared and mixed 1:1 with the acidified protein on a chilled MALDI target. The target was dried under vacuum to minimize back exchange; experiments in which fully deuterated fragments were processed the same way indicated back exchange never exceeded 5%. Spectra were acquired on a Bruker Autoflex III Smartbeam MALDI-TOF mass spectrometer. The instrument was calibrated with a standard peptide mixture prior to collection of each dataset.
MALDI Data Processing
The centroid (mass weighted average) of the isotopic envelope for each peptide was calculated via a spreadsheet after data export. The level of backbone deuterium incorporation for each peptide was calculated as:




  
    %
    ⁢
    
      
    
    ⁢
    deuteration
  
  =
  
    100
    ×
    
      
        
          (
          m
          )
        
        -
        
          m
          
            0
            ⁢
            %
          
        
      
      
        
          m
          
            100
            ⁢
            %
          
        
        -
        
          m
          
            0
            ⁢
            %
          
        
      
    
  




where <m> is the centroid mass, m0% is the mass of the undeuterated sample, and m100% is the mass of the peptide in which the backbone is fully deuterated. In all cases examined, exchange was complete by 10 minutes as shown in FIG. 1B; thus percent deuteration at 10 minutes was used for comparisons.

Surface Plasmon Resonance

Surface plasmon resonance experiments with the wild-type and high affinity variant of DMF5 were performed using a steady state assay with a Biacore 3000 instrument as previously described (12). Solution conditions were 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% surfactant P-20, pH 7.4, 25° C.
Anisotropic Network Model Calculations
Anisotropic Network Model calculations were performed using the Anisotropic Network Model server with a cutoff for interactions between α carbon atoms of 15 Å and a distance weight for interactions between α carbon atoms of 2.5 (18). Calculations were performed on the separately solved structures of A6 and its complex with pMHC (14, 19).
Structure-based Computational Design of a High Affinity DMF5 Variant
Design of the high affinity DMF5 variant was performed using the ZAFFI algorithm (20) and the structure of wild-type DMF5 in complex with the decameric MART-1 epitope (21). Briefly, mutations were simulated using the interface protocol of Rosetta 2.3 (22), using gradient-based minimization to refine backbone and torsion angles and rigid-body position before and after mutation (this was superior for reproducing binding affinities compared to the fixed backbone protocol used originally). From this analysis, the threshold of the ZAFFI filter function was raised to 0.3. Computational mutagenesis of 31 DMF5 residues resulted in 589 mutants. Ten of the highest scoring mutants were evaluated experimentally, and two that led to enhanced binding (αD27Y, βL95W) combined to generate the high affinity variant.
Results and Discussion
Ligation by pMHC Globally Rigidifies the A6 TCR
Although NMR remains the most powerful method for investigating protein motions, complications resulting from the size, complexity, and stability of TCRs and their complexes have limited NMR studies of TCR-pMHC interactions, requiring the use of simplified systems (23). To circumvent these challenges, we utilized hydrogen/deuterium exchange monitored by proteolysis and mass spectrometry (HDX-MS) as a tool to probe flexibility (reviewed in ref. 24). In HDX-MS, protein prepared in aqueous solution is incubated in 2H2O, digested into fragments, and backbone deuteron incorporation for each fragment determined via mass spectrometry. Increased levels of deuteron incorporation indicate that backbone amides become more accessible to exchange though greater sampling of more open, solvent-exposed states, while lower levels of incorporation indicate that amides are protected from exchange through less frequent excursions into solvent exposed states. HDX-MS has been utilized in a number of cases to explore protein motions and their changes, and has been particularly useful in characterizing low-frequency motions in samples not easily adaptable to NMR (24).
We began by performing HDX-MS on the ectodomain of the well-characterized A6 TCR in the absence and presence of its cognate pMHC ligand, the Tax peptide presented by the class I MHC HLA-A*0201 (HLA-A2). The A6 TCR is particularly amenable to this analysis, as its high affinity towards Tax/HLA-A2 allows full receptor occupancy to be achieved without requiring concentrations of pMHC that would hinder detection of TCR signals in the analysis of the exchange reactions (12). As shown in FIG. 1A, the A6 TCR showed decreased levels of deuteration upon ligation. Data for each peptide fragment were collected as a function of time out to several hours, but in all cases examined, exchange was essentially complete within 10 minutes as shown in FIG. 1B. The average deuteration of the free TCR after 10 minutes of exchange was 35%, whereas in the complex the average deuteration after 10 minutes of exchange was 18% (FIG. 1C). These values were reproducible, with a repeat experiment yielding free and bound levels of 34% and 16%, respectively. As shown in FIG. 1C, the reduced deuteration was distributed across the TCR, indicating that TCR ligation results in a global reduction in structural fluctuations, extending from the variable to the constant domains, although the extent varies throughout the molecule. Similar global reductions in flexibility upon ligand binding have been seen in other protein systems, such as the DNA binding protein BirA (25).
As expected, peptide fragments covering the A6 CDR loops showed reduced deuteration. Beyond the CDR loops, reduced deuteration was also seen for other key regions of the TCR, including a more than three-fold reduction in deuteration for a fragment spanning the β chain FG loop, a prominent structural feature that forms part of a cavity that has been proposed as a docking site for CD3εγ (26). A molecular dynamics simulation has shown that the CD3εδ subunit also rigidifies upon binding (in this case a monoclonal antibody) (27). It is not known if CD3εγ likewise rigidifies upon ligand binding; however, stronger binding of one or both of the CD3ε subunits to the TCR resulting from pMHC-induced rigidification could play a role in T cell triggering.
Although the FG loop is the most prominent feature impacted in the TCR constant domains, the reduction in flexibility includes other elements also implicated in either TCR oligomerization or interactions with CD3 subunits. This includes the α chain AB loop, which has been suggested to play a role in mediating interactions between adjacent TCRs on T cell membranes (3). Reduced AB loop flexibility could underlie a prior observation of a conformational change occurring upon TCR ligation (4), which is observed to a small extent when comparing the bound and free structures of A6 (19).
The ectodomain of the A6 TCR utilized for the data in FIG. 1 included an engineered disulfide bond near the base of the TCR constant domains to ensure heterodimer stability and facilitate proper chain pairing during protein refolding (13). An experiment with an alternate construct stabilized instead by a C-terminal heterodimeric coiled coil (14) also showed reduced deuteration upon binding at regions distant from the antigen binding site, indicating that the results in FIG. 1 are not a consequence of the method used to stabilize the receptor. A control experiment in which HDX-MS was performed with A6 before and after mixing with non-cognate ligand did not show reduced deuteration.
Ligation by pMHC Globally Rigidifies the DMF5 TCR
To ask if the reduction in exchange occurring upon binding was general or specific to A6, we repeated the HDX-MS experiments using the DMF5 TCR, which recognizes the MART-126-35 peptide presented by HLA-A2 (21). However, DMF5 binds more weakly than A6, and achieving high receptor occupancy required excess ligand concentrations that negatively impacted detection of TCR signals by MALDI mass spectrometry. We thus utilized a high affinity DMF5 variant, produced using the recently developed structure-guided TCR design algorithm ZAFFI (20). Surface plasmon resonance analysis of 10 DMF5 point mutants predicted to improve affinity identified two that when combined yielded a high-affinity DMF5 variant that included a D to Y mutation at amino acid 27 in the alpha chain (αD27Y) and a L to W mutation at amino acid 98 in the beta chain (βL95W), which bound with significantly improved affinity as compared to the wild-type (Table 1 and FIGS. 2A-B).
HDX-MS experiments with the high affinity DMF5 variant showed a reduction in hydrogen/deuterium exchange upon ligation that closely resembled that observed with A6 (FIG. 2C). The average deuteration of DMF5 before and after binding was 37% and 19%, respectively, with a repeat experiment yielding values of 35% and 18%. As with A6, the reduction was distributed across the TCR, and included the FG loop and the AB loop, as well as other regions implicated in TCR oligomerization or association with CD3 subunits (3).
Additional Mutations that Increase Affinity
Three additional mutations were made using the methods described above, including a D to W mutation at amino acid 27 in the alpha chain (αD27W), an A to P mutation at amino acid 55 of the alpha chain (αA55P), and a T to F mutation at amino acid 102 in the beta chain (βT102F).
The affinities of the mutants were tested and the results are shown in Table 1.









TABLE 1









AAGIGILTV/HLA-A2











ELAGIGILTV/HLA-A2

ΔΔG,











DMF5 Mutant
KD (μM)
ΔΔG, kcal/mol
KD (μM)
kcal/mol














wild-type
9.5
—
43
—


αD27W
0.68
−1.6
1.1
−2.2


αD27Y
0.46
−1.8
4.5
−1.4


βL95W
2.9
−0.7
11
−0.8


αD27W/βL95W
0.033
−3.3
0.60
−2.6


αD27Y/βL95W
0.024
−3.5
1.7
−1.9









Mutations at other positions were also tested, and demonstrated lower affinity; for example, αR28W had a KD of 26 μM; βF97W had a KD of 46 μM; and βT99F had an slightly improved KD of 8.9 μM.

Summary

In conclusion, the present data demonstrate that formation of a TCR-pMHC complex results in global TCR rigidification. Via entropic and packing effects, rigidification will promote associations with other neighboring proteins and strengthen the stability of existing complexes. New or strengthened assemblages could impact the density, positions, and accessibility of cytoplasmic domains and their associated kinases, as well as prime a complex to communicate a mechanical stress. Overall, these data raise the possibility that the TCR and its pMHC ligand are components of a dynamically-modulated, allosteric T cell signaling system.
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OTHER EMBODIMENTS
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

## Claims

1. A high-affinity DMF5 T cell receptor variant α chain, wherein the aspartic amino acid at position 27 or 49 is a Y (αD27Y or αD49Y) or W (αD27W or αD49W).

2. A high-affinity DMF5 T cell receptor variant β chain, wherein the leucine amino acid at position 95, 96 or 114 is a W (βL95W, βL96W βL114W).

3. A high-affinity DMF5 T cell receptor variant comprising:
a high-affinity DMF5 T cell receptor variant α chain, wherein the aspartic amino acid at
position 27 or 49 is a Y (αD27Y or αD49Y) or W (αD27W or αD49W), and
a high-affinity DMF5 T cell receptor variant β chain, wherein the leucine amino acid at position 95, 96 or 114 is a W (βL95W, βL96W βL114W).

4. A fusion protein comprising the high-affinity DMF5 T cell receptor variant of claim 3 linked to a non-TCR sequence.

5. A therapeutic composition comprising:
a therapeutic agent linked to
the high-affinity DMF5 T cell receptor variant of claim 3, optionally linked to a non-TCR sequence.

6. The therapeutic composition of claim 5, wherein the therapeutic agent is a radioisotope; an anticancer drug; or a cytotoxic moiety.

7. A composition comprising:
a detectable moiety linked to
the high-affinity DMF5 T cell receptor variant of claim 3, optionally linked to a non-TCR sequence.

8. The composition of claim 7, wherein the detectable moiety is a contrast agent.

9. The composition of claim 8, wherein the contrast agent is selected from the group consisting of gold, gadolinium, iron oxides, manganese chelates, barium sulfate, iodinated contrast media, microbubbles, and perfluorocarbons.

10. A nucleic acid encoding:
the high-affinity DMF5 T cell receptor variant of claim 3, optionally linked to a non-TCR sequence.

11. A vector comprising the nucleic acid of claim 10.

12. The vector of claim 11, which is a retroviral vector.

13. A host cell expressing:
the high-affinity DMF5 T cell receptor variant of claim 3, optionally linked to a non-TCR sequence.

14. A host cell comprising the nucleic acid of claim 10.

15. The host cell of claim 14, wherein the cell is a peripheral blood lymphocyte (PBL).

16. A method of treating a subject who has melanoma, the method comprising administering to the subject a therapeutically effective amount of the therapeutic composition of claim 5.

17. A method of treating a subject who has melanoma, the method comprising administering to the subject a therapeutically effective amount of the host cells of claim 14.

18. The method of claim 17, wherein the host cells are autologous to the subject.

19. A method of treating a subject who has melanoma, the method comprising:
obtaining cells from the subject;
transducing the cells with a vector expressing:
the high-affinity DMF5 T cell receptor variant of claim 3, optionally linked to a non-TCR sequence;
optionally maintaining the cells in culture for a time sufficient for the cells to express the variant or fusion protein and/or to proliferate; and
administering a therapeutically effective amount of the cells to the subject thereafter.

20. The method of claim 19, wherein the cells are PBLs.