Molecular engineering of high affinity T-cell receptors for bispecific therapeutics

Cytotoxic T lymphocytes are able to identify malignant cells by scanning for aberrant peptides presented on cell surface human leukocyte antigen (HLA) Class I molecules by virtue of an antigen binding receptor called the T-cell receptor (TCR). Peptides presented by HLA Class I complexes represent the largest array of tumour associated antigens (TAAs) and are therefore ideal targets for immunotherapeutic reagents. Cancer patients frequently mount T-cell responses to tumour-specific antigens, but these are in most cases ineffective at clearing the tumour. This is in part due to the low affinity of TCRs for self-antigens coupled with low-level expression of target peptides on the surface of cancer cells. To harness the exquisite antigen recognition property of TCRs for use as potential therapeutic proteins, the principal goal of this thesis was to generate ultra-high affinity TCRs against three clinically relevant HLA Class I melanoma-specific epitopes, including peptides derived from Melan-A/MART-1(26-35), gp100(280-288) and MAGE-A3(168-176). TCRs are membrane-bound disulphide (ds)-linked heterodimers consisting of an alpha and a beta chain. Each chain comprises three hypervariable or complementarity-determining region (CDR) loops, which assemble to form the antigen binding domains. As a general rule the CDR3 loops, and to a lesser extent the CDR1 loops, contact the peptide bound in the HLA groove and as such specificity is largely attributable to the CDR3 loops. The remaining CDR loops interact with the HLA surface and not the bound peptide. Each CDR loop was mutagenised using degenerative NNK oligonucleotides and expressed on the surface of bacteriophage as fusions to the phage coat protein pIII. Through a Darwinian process of in vitro evolution using pHLA ligand as the target molecule, mutated TCRs with improved affinity for pHLA were identified. TCRs engineered by phage display were produced as soluble ds-linked proteins and the contribution to affinity of each mutated CDR was measured by surface plasmon resonance (SPR). Using a combinatorial strategy, individual mutated CDRs were spliced into the same TCR molecule in a stepwise manner to further increase binding affinity. The final combination of mutated CDRs was shown to bind their cognate pHLA antigen with substantially improved KD values of 18 pM (Melan-A/MART-1(26-35)), 11 pM (gp100(280-288)) and 58 pM (MAGE-A3(168-176)), representing an increase over the wild-type TCR of approximately 1.8 million-fold, 1.7 million-fold and 3.7 million-fold respectively. In addition, having discovered an off-target binding profile for the high affinity MAGE-A3 TCR, the phage display methodologies were explored to 5 reestablish the specificity of this molecule. These results are significant because this has provided a platform on which, for the first time, to make TCR-based therapeutics. For example, the affinity enhanced gp100 TCR is currently undergoing clinical evaluation in a Phase I/II trial

Specificity of bispecific T cell receptors and antibodies targeting peptide-HLA

Tumor-associated peptide–human leukocyte antigen complexes (pHLAs) represent the largest pool of cell surface–expressed cancer-specific epitopes, making them attractive targets for cancer therapies. Soluble bispecific molecules that incorporate an anti-CD3 effector function are being developed to redirect T cells against these targets using 2 different approaches. The first achieves pHLA recognition via affinity-enhanced versions of natural TCRs (e.g., immune-mobilizing monoclonal T cell receptors against cancer [ImmTAC] molecules), whereas the second harnesses an antibody-based format (TCR-mimic antibodies). For both classes of reagent, target specificity is vital, considering the vast universe of potential pHLA molecules that can be presented on healthy cells. Here, we made use of structural, biochemical, and computational approaches to investigate the molecular rules underpinning the reactivity patterns of pHLA-targeting bispecifics. We demonstrate that affinity-enhanced TCRs engage pHLA using a comparatively broad and balanced energetic footprint, with interactions distributed over several HLA and peptide side chains. As ImmTAC molecules, these TCRs also retained a greater degree of pHLA selectivity, with less off-target activity in cellular assays. Conversely, TCR-mimic antibodies tended to exhibit binding modes focused more toward hot spots on the HLA surface and exhibited a greater degree of crossreactivity. Our findings extend our understanding of the basic principles that underpin pHLA selectivity and exemplify a number of molecular approaches that can be used to probe the specificity of pHLA-targeting molecules, aiding the development of future reagents

TCR‐induced alteration of primary MHC peptide anchor residue

The HLA‐A*02:01‐restricted decapeptide EAAGIGILTV, derived from melanoma antigen recognized by T‐cells‐1 (MART‐1) protein, represents one of the best‐studied tumor associated T‐cell epitopes, but clinical results targeting this peptide have been disappointing. This limitation may reflect the dominance of the nonapeptide, AAGIGILTV, at the melanoma cell surface. The decapeptide and nonapeptide are presented in distinct conformations by HLA‐A*02:01 and TCRs from clinically relevant T‐cell clones recognize the nonapeptide poorly. Here, we studied the MEL5 TCR that potently recognizes the nonapeptide. The structure of the MEL5‐HLA‐A*02:01‐AAGIGILTV complex revealed an induced fit mechanism of antigen recognition involving altered peptide–MHC anchoring. This “flexing” at the TCR–peptide–MHC interface to accommodate the peptide antigen explains previously observed incongruences in this well‐studied system and has important implications for future therapeutic approaches. Finally, this study expands upon the mechanisms by which molecular plasticity can influence antigen recognition by T cells

Abstract 1122‐000029: Pharmacological and Clinical Factors Associated with Gender Difference in Alzheimer Dementia Patients

Introduction: Alzheimer dementia (AD) has been reported in both men and women. However, factors contributing to gender differences are not fully understood. We tested the hypothesis that specific pharmacological, demographic, and risk factors contribute to gender difference in AD. Methods: A retrospective analytical approach was used to analyze data from 12,632 AD patients, comprising 4,584 men and 8,048 women. Univariate and multivariate analyses determined the factors contributing to the gender difference in AD patients. Results: About 36% of AD patients were men, and 64% were women. Citalopram (OR = 1.187, 95% CI, 1.044 – 1.350, P = 0.009) was associated with men, while escitalopram (OR = 1.213, 95% CI, 1.119 – 1.315, P<0.001) was associated with women. In both men and women, increasing age (OR = 1.075, 95% CI, 1.071 – 1.079, P<0.001/OR = 1.096, 95% CI, 1.093 – 1.100, P<0.001), tobacco use (OR = 1.150, 95% CI, 1.054 – 1.254, P = 0.002/OR = 1.150, 95% CI, 1.073 – 1.233, P<0.001), and black patients (OR = 2.380, 95% CI, 2.120 – 2.674, P<0.001/OR = 1.395, 95% CI, 1.268 – 1.535, P<0.001) were associated with AD. Conclusions: Our findings reveal similarities and differences in factors associated with both men and women AD patients, suggesting the development of management strategies for the care of AD

Specificity of bispecific T cell receptors and antibodies targeting peptide-HLA

Tumor-associated peptide–human leukocyte antigen complexes (pHLAs) represent the largest pool of cell surface–expressed cancer-specific epitopes, making them attractive targets for cancer therapies. Soluble bispecific molecules that incorporate an anti-CD3 effector function are being developed to redirect T cells against these targets using 2 different approaches. The first achieves pHLA recognition via affinity-enhanced versions of natural TCRs (e.g., immune-mobilizing monoclonal T cell receptors against cancer [ImmTAC] molecules), whereas the second harnesses an antibody-based format (TCR-mimic antibodies). For both classes of reagent, target specificity is vital, considering the vast universe of potential pHLA molecules that can be presented on healthy cells. Here, we made use of structural, biochemical, and computational approaches to investigate the molecular rules underpinning the reactivity patterns of pHLA-targeting bispecifics. We demonstrate that affinity-enhanced TCRs engage pHLA using a comparatively broad and balanced energetic footprint, with interactions distributed over several HLA and peptide side chains. As ImmTAC molecules, these TCRs also retained a greater degree of pHLA selectivity, with less off-target activity in cellular assays. Conversely, TCR-mimic antibodies tended to exhibit binding modes focused more toward hot spots on the HLA surface and exhibited a greater degree of crossreactivity. Our findings extend our understanding of the basic principles that underpin pHLA selectivity and exemplify a number of molecular approaches that can be used to probe the specificity of pHLA-targeting molecules, aiding the development of future reagents

Monoclonal TCR-redirected tumor cell killing

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