Peptides derived from almost all proteins, including disease-associated proteins, can be presented on the cell surface as peptide-human leukocyte antigen (pHLA) complexes. T cells specifically recognize pHLA with their clonally rearranged T-cell receptors (TCRs), whose natural affinities are limited to approximately 1-100 muM. Here we describe the display of ten different human TCRs on the surface of bacteriophage, stabilized by a nonnative interchain disulfide bond. We report the directed evolution of high-affinity TCRs specific for two different pHLAs: the human T-cell lymphotropic virus type 1 (HTLV-1) tax(11-19) peptide-HLA-A(*)0201 complex and the NY-ESO-1(157-165) tumor-associated peptide antigen-HLA-A(*)0201 complex, with affinities of up to 2.5 nM and 26 pM, respectively, and we demonstrate their high specificity and sensitivity for targeting of cell-surface pHLAs.

Single and dual amino acid substitution variants were generated in the TCR CDRs of three TCRs that recognize tumor-associated Ags. Substitutions that enhance the reactivity of TCR gene-modified T cells to the cognate Ag complex were identified using a rapid RNA-based transfection system. The screening of a panel of variants of the 1G4 TCR, that recognizes a peptide corresponding to amino acid residues 157–165 of the human cancer testis Ag NY-ESO-1 (SLLMWITQC) in the context of the HLA-A*02 class I allele, resulted in the identification of single and dual CDR3α and CDR2β amino acid substitutions that dramatically enhanced the specific recognition of NY-ESO-1+/HLA-A*02+ tumor cell lines by TCR gene-modified CD4+ T cells. Within this group of improved TCRs, a dual substitution in the 1G4 TCR CDR3α chain was identified that enhanced Ag-specific reactivity in gene-modified CD4+ and CD8+ T cells. Separate experiments on two distinct TCRs that recognize the MART-1 27–35 (AAGIGILTV) peptide/HLA-A*02 Ag complex characterized single amino acid substitutions in both TCRs that enhanced CD4+ T cell Ag-specific reactivity. These results indicate that simple TCR substitution variants that enhance T cell function can be identified by rapid transfection and assay techniques, providing the means for generating potent Ag complex-specific TCR genes for use in the study of T cell interactions and in T cell adoptive immunotherapy.

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.

HIV’s phenomenal capacity to vary its HLA-I-restricted peptide antigens allows it to escape from host cytotoxic T-lymphocytes (CTLs). Nevertheless, therapeutics able to target HLA-I-associated antigens, with specificity for the spectrum of preferred CTL escape mutants, could prove effective. Here we use phage display to isolate and enhance a T-cell receptor (TCR) originating from a patient CTL line and specific for the immunodominant HLA-A*02-restricted, HIVgag-specific peptide SLYNTVATL (SL9). High affinity (KD <400pM) TCRs were produced that bound with a half-life in excess of three hours, retained specificity, targeted HIV-infected cells and recognized all common escape variants of this epitope. CD8 T-cells transduced with supraphysiologic TCR produced a greater range of soluble factors and more IL2, than those transduced with natural SL9-specific TCR, and effectively controlled wildtype and mutant strains of HIV at effector-to-target ratios that could be achieved by T-cell therapy.

We examined the activity of human T cells engineered to express variants of a single TCR (1G4) specific for the cancer/testis Ag NY-ESO-1, generated by bacteriophage display with a wide range of affinities (from 4 μM to 26 pM). CD8+ T cells expressing intermediate- and high-affinity 1G4 TCR variants bound NY-ESO-1/HLA-A2 tetramers with high avidity and Ag specificity, but increased affinity was associated with a loss of target cell specificity of the TCR gene-modified cells. T cells expressing the highest affinity TCR (KD value of 26 pM) completely lost Ag specificity. The TCRs with affinities in the midrange, KD 5 and 85 nM, showed specificity only when CD8 was absent or blocked, while the variant TCRs with affinities in the intermediate range—with KD values of 450 nM and 4 μM—demonstrated Ag-specific recognition. Although the biological activity of these two relatively low-affinity TCRs was comparable to wild-type reactivity in CD8+ T cells, introduction of these TCR dramatically increased the reactivity of CD4+ T cells to tumor cell lines.

The mammalian a/b T cell receptor (TCR) repertoire plays a pivotal role in adaptive immunity by recognizing short, processed, peptide antigens bound in the context of a highly diverse family of cellsurface major histocompatibility complexes (pMHCs). Despite the extensive TCR-MHC interaction surface, peptide-independent cross-reactivity of native TCRs is generally avoided through cellmediated selection of molecules with low inherent affinity for MHC. Here we show that, contrary to expectations, the germ line-encoded complementarity determining regions (CDRs) of human TCRs, namely the CDR2s, which appear to contact only the MHC surface and not the bound peptide, can be engineered to yield soluble low nanomolar affinity ligands that retain a surprisingly high degree of specificity for the cognate pMHC target. Structural investigation of one such CDR2 mutant implicates shape complementarity of the mutant CDR2 contact interfaces as being a key determinant of the increased affinity. Our results suggest that manipulation of germ line CDR2 loops may provide a useful route to the production of high-affinity TCRs with therapeutic and diagnostic potential.

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.

Background Measurement of anti‐GM1 IgM antibodies in multifocal motor neuropathy (MMN) sera is confounded by relatively low sensitivity that limits clinical usefulness. Combinatorial assay methods, in which antibodies react to heteromeric complexes of two or more glycolipids, are being increasingly applied to this area of diagnostic testing. Methods A newly developed combinatorial glycoarray able to identify antibodies to 45 different heteromeric glycolipid complexes and their 10 individual glycolipid components was applied to a randomly selected population of 33 MMN cases and 57 normal or disease controls. Comparison with an enzyme‐linked immunosorbent assay (ELISA) was conducted for selected single glycolipids and their complexes. Results By ELISA, 22/33 MMN cases had detectable anti‐GM1 IgM antibodies, whereas 19/33 MMN samples were positive for anti‐GM1 antibodies by glycoarray. Analysis of variance (anova) revealed that of the 55 possible single glycolipids and their 1:1 complexes, antibodies to the GM1:galactocerebroside (GM1:GalC) complex were most significantly associated with MMN, returning 33/33 MMN samples as positive by glycoarray and 29/33 positive by ELISA. Regression analysis revealed a high correlation in absolute values between ELISA and glycoarray. Receiver operator characteristic analysis revealed insignificantly different diagnostic performance between the two methods. However, the glycoarray appeared to offer slightly improved sensitivity by identifying antibodies in four ELISA‐negative samples. Conclusions The use of combinatorial glycoarray or ELISA increased the diagnostic sensitivity of anti‐glycolipid antibody testing in this cohort of MMN cases, without significantly affecting specificity, and may be a useful assay modification for routine clinical screening.