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1.
Sci Rep ; 6: 18851, 2016 Jan 13.
Article in English | MEDLINE | ID: mdl-26758806

ABSTRACT

Natural T-cell responses generally lack the potency to eradicate cancer. Enhanced affinity T-cell receptors (TCRs) provide an ideal approach to target cancer cells, with emerging clinical data showing significant promise. Nevertheless, the risk of off target reactivity remains a key concern, as exemplified in a recent clinical report describing fatal cardiac toxicity, following administration of MAGE-A3 specific TCR-engineered T-cells, mediated through cross-reactivity with an unrelated epitope from the Titin protein presented on cardiac tissue. Here, we investigated the structural mechanism enabling TCR cross-recognition of MAGE-A3 and Titin, and applied the resulting data to rationally design mutants with improved antigen discrimination, providing a proof-of-concept strategy for altering the fine specificity of a TCR towards an intended target antigen. This study represents the first example of direct molecular mimicry leading to clinically relevant fatal toxicity, mediated by a modified enhanced affinity TCR designed for cancer immunotherapy. Furthermore, these data demonstrate that self-antigens that are expressed at high levels on healthy tissue should be treated with extreme caution when designing immuno-therapeutics.


Subject(s)
Antigens, Neoplasm/immunology , Antigens, Neoplasm/metabolism , Cross Reactions/immunology , Immunotherapy/adverse effects , Immunotherapy/methods , Molecular Mimicry , Receptors, Antigen, T-Cell/metabolism , Antigen Presentation , Antigen-Presenting Cells/immunology , Antigen-Presenting Cells/metabolism , Antigens, Neoplasm/chemistry , Antigens, Neoplasm/genetics , Cardiotoxicity , Cell Line , Connectin/chemistry , Connectin/immunology , Connectin/metabolism , Epitopes, T-Lymphocyte/chemistry , Epitopes, T-Lymphocyte/immunology , Genetic Engineering , Humans , Models, Molecular , Mutation , Neoplasm Proteins/genetics , Neoplasm Proteins/immunology , Neoplasm Proteins/metabolism , Peptide Fragments/chemistry , Peptide Fragments/immunology , Protein Binding/immunology , Protein Conformation , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell, alpha-beta/genetics , Receptors, Antigen, T-Cell, alpha-beta/metabolism , T-Cell Antigen Receptor Specificity/immunology , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism
2.
Front Immunol ; 4: 168, 2013.
Article in English | MEDLINE | ID: mdl-23805144

ABSTRACT

Natural T cell receptors (TCRs) generally bind to their cognate pMHC molecules with weak affinity and fast kinetics, limiting their use as therapeutic agents. Using phage display, we have engineered a high affinity version of the A6 wild-type TCR (A6wt), specific for the human leukocyte antigen (HLA-A(∗)0201) complexed with human T cell lymphotropic virus type 111-19 peptide (A2-Tax). Mutations in just 4 residues in the CDR3ß loop region of the A6wt TCR were selected that improved binding to A2-Tax by nearly 1000-fold. Biophysical measurements of this mutant TCR (A6c134) demonstrated that the enhanced binding was derived through favorable enthalpy and a slower off-rate. The structure of the free A6c134 TCR and the A6c134/A2-Tax complex revealed a native binding mode, similar to the A6wt/A2-Tax complex. However, concordant with the more favorable binding enthalpy, the A6c134 TCR made increased contacts with the Tax peptide compared with the A6wt/A2-Tax complex, demonstrating a peptide-focused mechanism for the enhanced affinity that directly involved the mutated residues in the A6c134 TCR CDR3ß loop. This peptide-focused enhanced TCR binding may represent an important approach for developing antigen specific high affinity TCR reagents for use in T cell based therapies.

3.
Nat Med ; 18(6): 980-7, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22561687

ABSTRACT

T cell immunity can potentially eradicate malignant cells and lead to clinical remission in a minority of patients with cancer. In the majority of these individuals, however, there is a failure of the specific T cell receptor (TCR)­mediated immune recognition and activation process. Here we describe the engineering and characterization of new reagents termed immune-mobilizing monoclonal TCRs against cancer (ImmTACs). Four such ImmTACs, each comprising a distinct tumor-associated epitope-specific monoclonal TCR with picomolar affinity fused to a humanized cluster of differentiation 3 (CD3)-specific single-chain antibody fragment (scFv), effectively redirected T cells to kill cancer cells expressing extremely low surface epitope densities. Furthermore, these reagents potently suppressed tumor growth in vivo. Thus, ImmTACs overcome immune tolerance to cancer and represent a new approach to tumor immunotherapy.


Subject(s)
Cytotoxicity, Immunologic , Neoplasms, Experimental/therapy , Receptors, Antigen, T-Cell/physiology , Animals , CD8-Positive T-Lymphocytes/immunology , Humans , Immunologic Memory , Immunotherapy , Interferon-gamma/biosynthesis , Lymphocyte Activation , Mice , Mice, SCID , Neoplasms, Experimental/immunology
4.
Protein Cell ; 1(12): 1118-27, 2010 Dec.
Article in English | MEDLINE | ID: mdl-21213105

ABSTRACT

Using directed mutagenesis and phage display on a soluble fragment of the human immunoglobulin super-family receptor ILT2 (synonyms: LIR1, MIR7, CD85j), we have selected a range of mutants with binding affinities enhanced by up to 168,000-fold towards the conserved region of major histocompatibility complex (MHC) class I molecules. Produced in a dimeric form, either by chemical cross-linking with bivalent polyethylene glycol (PEG) derivatives or as a genetic fusion with human IgG Fc-fragment, the mutants exhibited a further increase in ligand-binding strength due to the avidity effect, with resident half-times (t(1/2)) on the surface of MHC I-positive cells of many hours. The novel compounds antagonized the interaction of CD8 co-receptor with MHC I in vitro without affecting the peptide-specific binding of T-cell receptors (TCRs). In both cytokine-release assays and cell-killing experiments the engineered receptors inhibited the activation of CD8(+) cytotoxic T lymphocytes (CTLs) in the presence of their target cells, with subnanomolar potency and in a dose-dependent manner. As a selective inhibitor of CD8(+) CTL responses, the engineered high affinity ILT2 receptor presents a new tool for studying the activation mechanism of different subsets of CTLs and could have potential for the development of novel autoimmunity therapies.


Subject(s)
Antigens, CD/genetics , Antigens, CD/pharmacology , Immunologic Factors/genetics , Immunologic Factors/pharmacology , Lymphocyte Activation/immunology , Receptors, Immunologic/genetics , Amino Acid Sequence , Antigens, CD/chemistry , Autoimmunity , Biological Assay , Cell Line , Cytotoxicity, Immunologic/genetics , Cytotoxicity, Immunologic/immunology , Dose-Response Relationship, Immunologic , Humans , Immunoglobulins/immunology , Immunoglobulins/metabolism , Immunologic Factors/chemistry , Kinetics , Leukocyte Immunoglobulin-like Receptor B1 , Lymphocyte Activation/genetics , Major Histocompatibility Complex/genetics , Major Histocompatibility Complex/immunology , Molecular Sequence Data , Molecular Targeted Therapy , Mutagenesis, Site-Directed , Peptide Library , Polyethylene Glycols , Protein Binding/genetics , Protein Binding/immunology , Receptors, Immunologic/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/metabolism
5.
Cancer Res ; 67(23): 11419-27, 2007 Dec 01.
Article in English | MEDLINE | ID: mdl-18056470

ABSTRACT

Proteolysis-inducing factor (PIF) is a sulfated glycoprotein produced by cachexia-inducing tumors, which induces atrophy of skeletal muscle. PIF has been shown to bind specifically with high affinity (K(d), in nanomolar) to sarcolemma membranes from skeletal muscle of both the mouse and the pig, as well as murine myoblasts and a human muscle cell line. Ligand binding was abolished after enzymatic deglycosylation, suggesting that binding was mediated through the oligosaccharide chains in PIF. Chondroitin sulfate, but not heparan or dermatan sulfate, showed competitive inhibition (K(d), 1.1 x 10(-7) mol/L) of binding of PIF to the receptor, suggesting an interaction with the sulfated oligosaccharide chains. Ligand blotting of [(35)S]PIF to triton solublized membranes from C(2)C(12) cells provided evidence for a binding protein of apparent M(r) of approximately 40,000. Amino acid sequence analysis showed the PIF receptor to be a DING protein. Antisera reactive to a 19mer from the N-terminal amino acid residues of the binding protein attenuated protein degradation and activation of the ubiquitin-proteasome pathway induced by PIF in murine myotubes. In addition, the antisera was highly effective in attenuating the decrease in body weight in mice bearing the MAC16 tumor, with a significant increase in muscle wet weight due to an increase in the rate of protein synthesis, together with a reduction in protein degradation through attenuation of the increased proteasome expression and activity. These results confirm that the PIF binding protein has a functional role in muscle protein atrophy in cachexia and that it represents a potential new therapeutic target.


Subject(s)
Muscle, Skeletal/metabolism , Proteoglycans/metabolism , Receptors, Neurotransmitter/metabolism , Ubiquitins/metabolism , Animals , Antibodies, Monoclonal/immunology , Binding, Competitive , Blotting, Western , Cells, Cultured/drug effects , Chondroitin Sulfates/pharmacology , DNA-Binding Proteins/metabolism , Dermatan Sulfate/pharmacology , Heparitin Sulfate/pharmacology , Mice , Mice, Inbred Strains , Muscle, Skeletal/drug effects , Myoblasts, Skeletal/drug effects , Myoblasts, Skeletal/metabolism , Polycomb Repressive Complex 1 , Proteasome Endopeptidase Complex/metabolism , Repressor Proteins , Sarcolemma/metabolism , Ubiquitin-Protein Ligases
6.
Protein Eng Des Sel ; 20(8): 397-403, 2007 Aug.
Article in English | MEDLINE | ID: mdl-17644531

ABSTRACT

Naturally selected T-cell receptors (TCRs) are characterised by low binding affinities, typically in the range 1-100 microM. Crystal structures of syngeneic TCRs bound to peptide major histocompatibility complex (pMHC) antigens exhibit a conserved mode of binding characterised by a distinct diagonal binding geometry, with poor shape complementarity (SC) between receptor and ligand. Here, we report the structures of three in vitro affinity enhanced TCRs that recognise the pMHC tumour epitope NY-ESO(157-165) (SLLMWITQC). These crystal structures reveal that the docking mode for the high affinity TCRs is identical to that reported for the parental wild-type TCR, with only subtle changes in the mutated complementarity determining regions (CDRs) that form contacts with pMHC; both CDR2 and CDR3 mutations act synergistically to improve the overall affinity. Comparison of free and bound TCR structures for both wild-type and a CDR3 mutant reveal an induced fit mechanism arising from restructuring of CDR3 loops which allows better peptide binding. Overall, an increased interface area, improved SC and additional H-bonding interactions are observed, accounting for the increase in affinity. Most notably, there is a marked increase in the SC for the central methionine and tryptophan peptide motif over the native TCR.


Subject(s)
Crystallography, X-Ray , Major Histocompatibility Complex/immunology , Peptides/immunology , Receptors, Antigen, T-Cell/chemistry , Receptors, Antigen, T-Cell/immunology , Complementarity Determining Regions/chemistry , Complementarity Determining Regions/genetics , Escherichia coli/genetics , Humans , Hydrogen Bonding , Kinetics , Ligands , Models, Molecular , Mutation , Peptides/chemistry , Protein Binding , Protein Conformation , Protein Structure, Secondary , Protein Structure, Tertiary , Receptors, Antigen, T-Cell/genetics , Surface Plasmon Resonance
7.
Protein Eng ; 16(9): 707-11, 2003 Sep.
Article in English | MEDLINE | ID: mdl-14560057

ABSTRACT

Antibody and T-cell receptors (TCRs) are the primary recognition molecules of the adaptive immune system. Antibodies have been extensively characterized and are being developed for a large number of therapeutic applications. This has been possible because of the ability to manufacture stable, soluble, monoclonal antibodies which retain the antigen specificity of B cells. Unlike antibodies, TCRs are not expressed in a soluble form, but are anchored to the T-cell surface by an insoluble trans-membrane domain. Characterization and development of TCRs has been hampered by the lack of suitable methods for producing them as soluble and stable proteins. Here we report the engineering of soluble human TCRs suitable for crystallization studies and potentially for in vivo therapeutic use.


Subject(s)
Receptors, Antigen, T-Cell/chemistry , T-Lymphocytes/chemistry , Crystallization , Crystallography, X-Ray , Humans , Models, Molecular , Protein Structure, Tertiary/physiology , T-Lymphocytes/immunology
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