Affinity-enhanced T-cell Receptor Research Articles

Identification and affinity enhancement of T-cell receptor targeting a KRASG12V cancer neoantigen

Neoantigens derived from somatic mutations in Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS), the most frequently mutated oncogene, represent promising targets for cancer immunotherapy. Recent research highlights the potential role of human leukocyte antigen (HLA) allele A*11:01 in presenting these altered KRAS variants to the immune system. In this study, we successfully generate and identify murine T-cell receptors (TCRs) that specifically recognize KRAS8–16G12V from three predicted high affinity peptides. By determining the structure of the tumor-specific 4TCR2 bound to KRASG12V-HLA-A*11:01, we conduct structure-based design to create and evaluate TCR variants with markedly enhanced affinity, up to 15.8-fold. This high-affinity TCR mutant, which involved only two amino acid substitutions, display minimal conformational alterations while maintaining a high degree of specificity for the KRASG12V peptide. Our research unveils the molecular mechanisms governing TCR recognition towards KRASG12V neoantigen and yields a range of affinity-enhanced TCR mutants with significant potential for immunotherapy strategies targeting tumors harboring the KRASG12V mutation.

NY-ESO-1-specific T cell receptor-engineered T cells and Tranilast, a TRPV2 antagonist bivalent treatment enhances the killing of esophageal cancer: a dual-targeted cancer therapeutic route

BackgroundEsophageal cancer (EC) is a global canker notorious for causing high mortality due to its relentless incidence rate, convoluted with unyielding recurrence and metastasis. However, these intricacies of EC are associated with an immoderate expression of NY-ESO-1 antigen, presenting a lifeline for adoptive T cell therapy. We hypothesized that naturally isolated higher-affinity T cell receptors (TCRs) that bind to NY-ESO-1 would allow T lymphocytes to target EC with a pronounced antitumor response efficacy. Also, targeting TRPV2, which is associated with tumorigenesis in EC, creates an avenue for dual-targeted therapy. We exploited the dual-targeting antitumor efficacy against EC.MethodsWe isolated antigen-specific TCRs (asTCRs) from a naive library constructed with TCRs obtained from enriched cytotoxic T lymphocytes. The robustness of our asTCRs and their TCR-T cell derivatives, Tranilast (TRPV2 inhibitor), and their bivalent treatment were evaluated with prospective cross-reactive human-peptide variants and tumor cells.ResultsOur study demonstrated that our naive unenhanced asTCRs and their TCR-Ts perpetuated their cognate HLA-A*02:01/NY-ESO-1(157–165) specificity, killing varying EC cells with higher cytotoxicity compared to the known affinity-enhanced TCR (TCRe) and its wild-type (TCR0) which targets the same NY-ESO-1 antigen. Furthermore, the TCR-Ts and Tranilast bivalent treatment showed superior EC killing compared to any of their monovalent treatments of either TCR-T or Tranilast.ConclusionOur findings suggest that dual-targeted immunotherapy may have a superior antitumor effect. Our study presents a technique to evolve novel, robust, timely therapeutic strategies and interventions for EC and other malignancies.

Enhancing Efficacy of TCR-engineered CD4 + T Cells Via Coexpression of CD8α.

Adoptive cell therapy with T cells expressing affinity-enhanced T-cell receptors (TCRs) is a promising treatment for solid tumors. Efforts are ongoing to further engineer these T cells to increase the depth and durability of clinical responses and broaden efficacy toward additional indications. In the present study, we investigated one such approach: T cells were transduced with a lentiviral vector to coexpress an affinity-enhanced HLA class I-restricted TCR directed against MAGE-A4 alongside a CD8α coreceptor. We hypothesized that this approach would enhance CD4 + T-cell helper and effector functions, possibly leading to a more potent antitumor response. Activation of transduced CD4 + T cells was measured by detecting CD40 ligand expression on the surface and cytokine and chemokine secretion from CD4 + T cells and dendritic cells cultured with melanoma-associated antigen A4 + tumor cells. In addition, T-cell cytotoxic activity against 3-dimensional tumor spheroids was measured. Our data demonstrated that CD4 + T cells coexpressing the TCR and CD8α coreceptor displayed enhanced responses, including CD40 ligand expression, interferon-gamma secretion, and cytotoxic activity, along with improved dendritic cell activation. Therefore, our study supports the addition of the CD8α coreceptor to HLA class I-restricted TCR-engineered T cells to enhance CD4 + T-cell functions, which may potentially improve the depth and durability of antitumor responses in patients.

Novel ImmTAVTM and ImmTABTM molecules for the treatment of infectious diseases

Immunotherapeutic strategies harness the body’s own immune system to eradicate cancer cells and pathogens, such as bacteria or viruses. T cells play a critical role in these strategies, directing potent and antigen-specific immune responses. T cell receptors (TCRs) recognise foreign antigenic peptides presented by human leukocyte antigens (HLAs) on the cell surface and upon recognition, cytotoxic T cell responses are activated to eliminate the target cells. However, natural antigen-specific TCRs exhibit moderate binding affinities (µM to nM range) and may not effectively bind to target-specific antigens. At Immunocore, we have overcome this limitation by developing a novel class of soluble bispecific molecules that combines an affinity-enhanced TCR (pM affinity) with an anti-CD3 effector function called ImmTAC (Immune mobilising monoclonal TCRs Against Cancer). Our most advanced ImmTAC, IMCgp100, is currently in pivotal trials and has demonstrated encouraging preliminary anti-tumour activity against metastatic uveal melanoma. We have expanded our platform to address the unmet need in infectious diseases, such as hepatitis B virus (HBV) and tuberculosis (TB). These diseases constitute a global burden of 240-350 million people chronically infected with HBV and 10 million people affected by TB. Furthermore, chronic HBV patients exhibit defective T cell responses, producing liver cirrhosis and/or hepatocellular carcinoma in 10-30% of the cases. Our pipeline involves isolating antigenic-specific TCRs from either from PBMCs or naïve libraries. Isolated TCRs are expressed in E. coli and their binding affinities assessed by surface plasmon resonance (SPR). The affinity of antigen-specific TCRs is enhanced through directed evolution by phage display and specificity and potency tested by assessing in vitro killing capacity towards antigen-positive target cell lines. Here, we describe the process of developing ImmTAV/B molecules (Immune mobilising monoclonal TCRs Against Viruses/Bacteria) targeting HBV and TB.

Combination strategies to durably suppress HIV-1: Soluble T cell receptors

Immunotherapeutic interventions to enhance natural HIV-specific CD8+ T cell responses, such as vaccination or adoptive T cell transfer, have been a major focus of HIV cure efforts. However, these approaches have not been effective in overcoming viral immune evasion mechanisms. Soluble T cell receptor (TCR) bispecifics are a new class of ‘off-the-shelf’ therapeutic designed to address these limitations. These biologics are built on the Immune mobilising monoclonal TCRs against X disease (ImmTAX) platform, which was pioneered in oncology and recently validated by the FDA's approval of tebentafusp for treatment of metastatic uveal melanoma. ImmTAV® are an application of this technology undergoing clinical development for the elimination of chronic viral infections. ImmTAV molecules comprise an affinity-enhanced virus-specific TCR fused to an anti-CD3 effector domain. Engineering of the TCR confers extraordinary specificity and affinity for cognate viral antigen and the anti-CD3 enables retargeting of non-exhausted cytolytic T cells, irrespective of their specificity. These features enable ImmTAV molecules to detect and kill infected cells, even when expressing very low levels of antigen, bypassing ineffective host immune responses. Furthermore, the modularity of the platform allows for engineering of TCRs that effectively target viral variants. In this review, we discuss the progress made in the development of ImmTAV molecules as therapeutics for functional cure of chronic hepatitis B and HIV, from concept to the clinic.

ZENYTH-ESO: Master protocol to assess the safety and recommended phase II dose of next generation NY-ESO-1-specific TCR T-cells in HLA-A*02 patients with synovial sarcoma and myxoid/round cell liposarcoma [Substudy 3, GSK4427296

TPS2681 Background: Letetresgene autoleucel (lete-cel; GSK3377794) is an autologous T-cell therapy expressing an affinity-enhanced T-cell receptor (TCR) to improve recognition of cancer cells expressing NY-ESO-1 and/or LAGE-1a. Next generation NY-ESO-1 TCR T-cell therapy, GSK4427296, utilizes the same TCR as lete-cel, as well as an epigenetic reprogramming process (Epi-R) developed by Lyell Immunopharma to alter the phenotypic T-cell profile of the manufactured product, and is intended to increase the proportion of cells with properties of durable stemness. T cells with properties of durable stemness are able to proliferate, persist, and self-renew with anti-tumor functionality. A first-time-in-human master protocol (NCT04526509) is underway to evaluate the safety, tolerability, and recommended phase II dose (RP2D) of next generation NY-ESO-1 TCR T-cell therapies. Substudy 3 is added to this master protocol to assess GSK4427296 in patients with advanced synovial sarcoma (SS) or myxoid/round cell liposarcoma (MRCLS). Methods: This substudy includes a dose confirmation stage to assess RP2D and a dose expansion stage, aiming to dose 10 participants at the RP2D. Key inclusion criteria: age ≥18 years; measurable disease per RECIST v1.1; HLA-A*02:01, A*02:05, or A*02:06 positivity; NY-ESO-1/LAGE-1a tumor expression; advanced (metastatic/unresectable) SS with t(X;18) translocation or MRCLS with a translocation involving DDIT3 and/or FUS and/or EWSR1 genes; and anthracycline-based therapy receipt/completion/intolerance. Key exclusion criteria: prior malignancy that is not in complete remission or clinically significant systemic illness; prior receipt of gene or allogenic stem cell/solid organ transplant; and central nervous system metastases. Primary endpoints: safety (adverse events) and tolerability (dose-limiting toxicities). Secondary endpoints: investigator-assessed overall response rate, duration of response, maximum transgene expansion (Cmax), Tmax, and AUC(0-t). Analyses will be descriptive. The master protocol is open for recruitment. Clinical trial information: NCT04526509.

Quantitative contributions of TNF receptor superfamily members to CD8+ T-cell responses.

T‐cell responses to infections and cancers are regulated by co‐signalling receptors grouped into the binary categories of co‐stimulation or co‐inhibition. The co‐stimulation TNF receptor superfamily (TNFRSF) members 4‐1BB, CD27, GITR and OX40 have similar signalling mechanisms raising the question of whether they have similar impacts on T‐cell responses. Here, we screened for the quantitative impact of these TNFRSFs on primary human CD8+ T‐cell cytokine production. Although both 4‐1BB and CD27 increased production, only 4‐1BB was able to prolong the duration over which cytokine was produced, and both had only modest effects on antigen sensitivity. An operational model explained these different phenotypes using shared signalling based on the surface expression of 4‐1BB being regulated through signalling feedback. The model predicted and experiments confirmed that CD27 co‐stimulation increases 4‐1BB expression and subsequent 4‐1BB co‐stimulation. GITR and OX40 displayed only minor effects on their own but, like 4‐1BB, CD27 could enhance GITR expression and subsequent GITR co‐stimulation. Thus, different co‐stimulation receptors can have different quantitative effects allowing for synergy and fine‐tuning of T‐cell responses.

Overall Survival Benefit with Tebentafusp in Metastatic Uveal Melanoma

BackgroundUveal melanoma is a disease that is distinct from cutaneous melanoma, with a low tumor mutational burden and a 1-year overall survival of approximately 50% in patients with metastatic uveal melanoma. Data showing a proven overall survival benefit with a systemic treatment are lacking. Tebentafusp is a bispecific protein consisting of an affinity-enhanced T-cell receptor fused to an anti-CD3 effector that can redirect T cells to target glycoprotein 100–positive cells.MethodsIn this open-label, phase 3 trial, we randomly assigned previously untreated HLA-A*02:01–positive patients with metastatic uveal melanoma in a 2:1 ratio to receive tebentafusp (tebentafusp group) or the investigator’s choice of therapy with single-agent pembrolizumab, ipilimumab, or dacarbazine (control group), stratified according to the lactate dehydrogenase level. The primary end point was overall survival.ResultsA total of 378 patients were randomly assigned to either the tebentafusp group (252 patients) or the control group (126 patients). Overall survival at 1 year was 73% in the tebentafusp group and 59% in the control group (hazard ratio for death, 0.51; 95% confidence interval [CI], 0.37 to 0.71; P<0.001) in the intention-to-treat population. Progression-free survival was also significantly higher in the tebentafusp group than in the control group (31% vs. 19% at 6 months; hazard ratio for disease progression or death, 0.73; 95% CI, 0.58 to 0.94; P=0.01). The most common treatment-related adverse events in the tebentafusp group were cytokine-mediated events (due to T-cell activation) and skin-related events (due to glycoprotein 100–positive melanocytes), including rash (83%), pyrexia (76%), and pruritus (69%). These adverse events decreased in incidence and severity after the first three or four doses and infrequently led to discontinuation of the trial treatment (2%). No treatment-related deaths were reported.ConclusionsTreatment with tebentafusp resulted in longer overall survival than the control therapy among previously untreated patients with metastatic uveal melanoma. (Funded by Immunocore; ClinicalTrials.gov number, NCT03070392; EudraCT number, 2015-003153-18.)

1013P Similar overall survival in tebentafusp-treated 2L+ metastatic uveal melanoma regardless of prior immunotherapy

Tebentafusp is a bispecific consisting of an affinity-enhanced T-cell receptor fused to an anti-CD3 effector that can redirect T-cells to target gp100+ cells. In a randomized phase III study of tebentafusp in 1L metastatic uveal melanoma (mUM) [NCT03070392], tebentafusp demonstrated OS benefit vs. investigator’s choice including checkpoint immunotherapy (IO). As checkpoint inhibitors (CPI) may alter the tumor immune profile, we explored the impact of prior IO treatment on clinical outcomes (efficacy and on-target adverse events) and immune phenotype in a phase II study of 2L+ patients with mUM treated with tebentafusp [NCT02570308].

1014P Characterization of cytokine release syndrome (CRS) following treatment with tebentafusp in previously untreated patients with metastatic uveal melanoma

Tebentafusp (tebe), a bispecific fusion protein consisting of an affinity-enhanced T-cell receptor fused to an anti-CD3 effector that can redirect T-cells to target gp100+ cells, has shown an overall survival benefit for patients (pts) with untreated metastatic uveal melanoma (mUM) in a Ph3 trial (NCT03070392). In a post-hoc analysis of a Ph2 trial of 2L+ pts with mUM, mild to moderate episodes of CRS, a cytokine-mediated adverse event (AE), were common following each of the first 3 doses of tebe (Carvajal RD et al. ASCO 2021). Here, we reviewed the incidence, kinetics, and outcome of CRS in tebe-treated pts on the IMCgp100-202 trial of 1L pts with mUM. 245 HLA-A*02:01+ 1L mUM pts were treated weekly with tebentafusp following intra-patient dose escalation: 20mcg dose 1, 30mcg dose 2 and 68mcg dose 3+. Pts were monitored overnight during escalation to allow management of hypotension and other cytokine-related AEs; prophylactic corticosteroids, antihistamines or acetaminophen were not mandated. CRS was evaluated post-hoc according to ASTCT Consensus Grading criteria (Lee, DW et al. 2019). Analysis was conducted on primary analysis snapshot (Jan 2021). The most frequent treatment-related AEs, likely cytokine-mediated, included fever (76%), chills (47%), nausea (43%), hypotension (38%). Using ASTCT criteria, 89% of tebe treated pts (n=217) had any grade CRS. Most pts had either grade (G) 1 (12%; n=29) or G2 (76%; n=186), with 2 G3 (0.8%), no G4 and no deaths. Most CRS events occurred within the first 3 doses, reducing in frequency and severity thereafter; 3 episodes of G3 CRS in 2 patients occurred following dose 1, 3 and 4. Only 2 pts discontinued tebe due to CRS (1 G2; 1 G3). Treatment of CRS included antipyretics (n=192), IV fluids (n=93), IV steroids (n=57), oxygen (n=20), and vasopressor use (n=2). Two pts received tocilizumab. CRS, a common AE observed with T-cell engaging therapies, was frequently observed within 24 hours of initial tebe treatment. Most CRS events were mild or moderate in severity, reversible with standard management strategies, decreased in frequency and severity with subsequent doses, and rarely led to treatment discontinuation.

Probing the Conformational Dynamics of Affinity-Enhanced T Cell Receptor Variants upon Binding the Peptide-Bound Major Histocompatibility Complex by Hydrogen/Deuterium Exchange Mass Spectrometry.

Binding of the T cell receptor (TCR) to its cognate, peptide antigen-loaded major histocompatibility complex (pMHC) is a key interaction for triggering T cell activation and ultimately elimination of the target cell. Despite the importance of this interaction for cellular immunity, a comprehensive molecular understanding of TCR specificity and affinity is lacking. We conducted hydrogen/deuterium exchange mass spectrometry (HDX-MS) analyses of individual affinity-enhanced TCR variants and clinically relevant pMHC class I molecules (HLA-A*0201/NY-ESO-1157-165) to investigate the causality between increased binding affinity and conformational dynamics in TCR-pMHC complexes. Differential HDX-MS analyses of TCR variants revealed that mutations for affinity enhancement in TCR CDRs altered the conformational response of TCR to pMHC ligation. Improved pMHC binding affinity was in general observed to correlate with greater differences in HDX upon pMHC binding in modified TCR CDR loops, thereby providing new insights into the TCR-pMHC interaction. Furthermore, a specific point mutation in the β-CDR3 loop of the NY-ESO-1 TCR associated with a substantial increase in binding affinity resulted in a substantial change in pMHC binding kinetics (i.e., very slow kon, revealed by the detection of EX1 HDX kinetics), thus providing experimental evidence for a slow induced-fit binding mode. We also examined the conformational impact of pMHC binding on an unrelated TRAV12-2 gene-encoded TCR directed against the immunodominant MART-126-35 cancer antigen restricted by HLA-A*0201. Our findings provide a molecular basis for the observed TRAV12-2 gene bias in natural CD8+ T cell-based immune responses against the MART-1 antigen, with potential implications for general ligand discrimination and TCR cross-reactivity processes.

Molecular Rules Underpinning Enhanced Affinity Binding of Human T Cell Receptors Engineered for Immunotherapy

Immuno-oncology approaches that utilize T cell receptors (TCRs) are becoming highly attractive because of their potential to target virtually all cellular proteins, including cancer-specific epitopes, via the recognition of peptide-human leukocyte antigen (pHLA) complexes presented at the cell surface. However, because natural TCRs generally recognize cancer-derived pHLAs with very weak affinities, efforts have been made to enhance their binding strength, in some cases by several million-fold. In this study, we investigated the mechanisms underpinning human TCR affinity enhancement by comparing the crystal structures of engineered enhanced affinity TCRs with those of their wild-type progenitors. Additionally, we performed molecular dynamics simulations to better understand the energetic mechanisms driving the affinity enhancements. These data demonstrate that supra-physiological binding affinities can be achieved without altering native TCR-pHLA binding modes via relatively subtle modifications to the interface contacts, often driven through the addition of buried hydrophobic residues. Individual energetic components of the TCR-pHLA interaction governing affinity enhancements were distinct and highly variable for each TCR, often resulting from additive, or knock-on, effects beyond the mutated residues. This comprehensive analysis of affinity-enhanced TCRs has important implications for the future rational design of engineered TCRs as efficacious and safe drugs for cancer treatment.

Preclinical evaluation of an affinity-enhanced MAGE-A4-specific T-cell receptor for adoptive T-cell therapy

ABSTRACT A substantial obstacle to the success of adoptive T cell-based cancer immunotherapy is the sub-optimal affinity of T-cell receptors (TCRs) for most tumor antigens. Genetically engineered TCRs that have enhanced affinity for specific tumor peptide-MHC complexes may overcome this barrier. However, this enhancement risks increasing weak TCR cross-reactivity to other antigens expressed by normal tissues, potentially leading to clinical toxicities. To reduce the risk of such adverse clinical outcomes, we have developed an extensive preclinical testing strategy, involving potency testing using 2D and 3D human cell cultures and primary tumor material, and safety testing using human primary cell and cell-line cross-reactivity screening and molecular analysis to predict peptides recognized by the affinity-enhanced TCR. Here, we describe this strategy using a developmental T-cell therapy, ADP-A2M4, which recognizes the HLA-A2-restricted MAGE-A4 peptide GVYDGREHTV. ADP-A2M4 demonstrated potent anti-tumor activity in the absence of major off-target cross-reactivity against a range of human primary cells and cell lines. Identification and characterization of peptides recognized by the affinity-enhanced TCR also revealed no cross-reactivity. These studies demonstrated that this TCR is highly potent and without major safety concerns, and as a result, this TCR is now being investigated in two clinical trials (NCT03132922, NCT04044768).

Systemic and local immunity following adoptive transfer of NY-ESO-1 SPEAR T cells in synovial sarcoma

BackgroundGene-modified autologous T cells expressing NY-ESO-1c259, an affinity-enhanced T-cell receptor (TCR) reactive against the NY-ESO-1-specific HLA-A*02-restricted peptide SLLMWITQC (NY-ESO-1 SPEAR T-cells; GSK 794), have demonstrated clinical activity in patients with advanced synovial sarcoma (SS). The factors contributing to gene-modified T-cell expansion and the changes within the tumor microenvironment (TME) following T-cell infusion remain unclear. These studies address the immunological mechanisms of response and resistance in patients with SS treated with NY-ESO-1 SPEAR T-cells.MethodsFour cohorts were included to evaluate antigen expression and preconditioning on efficacy. Clinical responses were assessed by RECIST v1.1. Engineered T-cell persistence was determined by qPCR. Serum cytokines were evaluated by immunoassay. Transcriptomic analyses and immunohistochemistry were performed on tumor biopsies from patients before and after T-cell infusion. Gene-modified T-cells were detected within the TME via an RNAish assay.ResultsResponses across cohorts were affected by preconditioning and intra-tumoral NY-ESO-1 expression. Of the 42 patients reported (data cut-off 4June2018), 1 patient had a complete response, 14 patients had partial responses, 24 patients had stable disease, and 3 patients had progressive disease. The magnitude of gene-modified T-cell expansion shortly after infusion was associated with response in patients with high intra-tumoral NY-ESO-1 expression. Patients receiving a fludarabine-containing conditioning regimen experienced increases in serum IL-7 and IL-15. Prior to infusion, the TME exhibited minimal leukocyte infiltration; CD163+ tumor-associated macrophages (TAMs) were the dominant population. Modest increases in intra-tumoral leukocytes (≤5%) were observed in a subset of subjects at approximately 8 weeks. Beyond 8 weeks post infusion, the TME was minimally infiltrated with a TAM-dominant leukocyte infiltrate. Tumor-associated antigens and antigen presentation did not significantly change within the tumor post-T-cell infusion. Finally, NY-ESO-1 SPEAR T cells trafficked to the TME and maintained cytotoxicity in a subset of patients.ConclusionsOur studies elucidate some factors that underpin response and resistance to NY-ESO-1 SPEAR T-cell therapy. From these data, we conclude that a lymphodepletion regimen containing high doses of fludarabine and cyclophosphamide is necessary for SPEAR T-cell persistence and efficacy. Furthermore, these data demonstrate that non-T-cell inflamed tumors, which are resistant to PD-1/PD-L1 inhibitors, can be treated with adoptive T-cell based immunotherapy.Trial registrationClinicalTrials.gov, NCT01343043, Registered 27 April 2011.

Long‐term safety and activity of NY-ESO‐1 SPEAR T cells after autologous stem cell transplant for myeloma

AbstractThis study in patients with relapsed, refractory, or high-risk multiple myeloma (MM) evaluated the safety and activity of autologous T cells engineered to express an affinity-enhanced T-cell receptor (TCR) that recognizes a peptide shared by cancer antigens New York esophageal squamous cell carcinoma-1 (NY-ESO-1) and L-antigen family member 1 (LAGE-1) and presented by HLA-A*02:01. T cells collected from 25 HLA-A*02:01-positive patients with MM expressing NY-ESO-1 and/or LAGE-1 were activated, transduced with self-inactivating lentiviral vector encoding the NY-ESO-1c259TCR, and expanded in culture. After myeloablation and autologous stem cell transplant (ASCT), all 25 patients received an infusion of up to 1 × 1010 NY-ESO-1 specific peptide enhanced affinity receptor (SPEAR) T cells. Objective response rate (International Myeloma Working Group consensus criteria) was 80% at day 42 (95% confidence interval [CI], 0.59-0.93), 76% at day 100 (95% CI, 0.55-0.91), and 44% at 1 year (95% CI, 0.24-0.65). At year 1, 13/25 patients were disease progression-free (52%); 11 were responders (1 stringent complete response, 1 complete response, 8 very good partial response, 1 partial response). Three patients remained disease progression-free at 38.6, 59.2, and 60.6 months post-NY-ESO-1 SPEAR T-cell infusion. Median progression-free survival was 13.5 months (range, 3.2-60.6 months); median overall survival was 35.1 months (range, 6.4-66.7 months). Infusions were well tolerated; cytokine release syndrome was not reported. No fatal serious adverse events occurred during study conduct. NY-ESO-1 SPEAR T cells expanded in vivo, trafficked to bone marrow, demonstrated persistence, and exhibited tumor antigen-directed functionality. In this MM patient population, NY-ESO-1 SPEAR T-cell therapy in the context of ASCT was associated with antitumor activity. This trial was registered at www.clinicaltrials.gov as #NCT01352286.

Abstract B024: Using single-cell paired sequencing to isolate cancer-specific T-cell receptors for cancer immunotherapy

Abstract Immunocore’s ImmTAC™ (Immune Mobilising Monoclonal TCR Against Cancer) platform combines affinity-enhanced T-cell receptor (TCR)-based targeting with an anti-CD3 scFv effector function to activate a cytotoxic T-cell response against cancer cells. A key part to this process is the identification of tumour epitope specific TCRs from tumor antigen-reactive T-cells. Here, we describe an integrated in-house process leading to the isolation of TCRs specific for validated cancer epitopes, coupled with rapid identification of TCR chains from individual clones using single cell sequencing. The process involves first strand cDNA generation and universal amplification using SmartSeq2 chemistry, followed by targeted sequencing of the TCR alpha and beta chains using next-generation sequencing (NGS). We have also leveraged the 10x Genomics VDJ/5’ counting platform to label and pool multiple experimental clones for repertoire sequencing within a single run. Together with Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-Seq), we can reliably assign each T-cell clone to its sample of origin paired with transcriptomic information of epitope specific T-cell populations, linking TCR sequences to their functional phenotype. Citation Format: Karolina Lech, Lucia Correia, Max Beckmann, Maria Busz, Sean Collison, Sterenn Davis, Paraskevi Mallini, Sarah Scaife, Joseph Dukes, Bent K. Jakobsen, Luke Williams, Michelle Teng. Using single-cell paired sequencing to isolate cancer-specific T-cell receptors for cancer immunotherapy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B024.

Abstract A214: An in vitro preclinical package to assess the safety and efficacy of ImmTAC™ molecules

Abstract Immune Mobilising Monoclonal TCRs Against Cancer (ImmTAC) molecules are a new class of bi-specific biologic specifically designed to recognize and eradicate cancer cells. ImmTAC molecules are composed of an affinity-enhanced T-cell receptor (TCR) fused to an anti-CD3 effector function engineered to recognize peptides presented by human leukocyte antigen (HLA) and induce T-cell activation and T-cell mediated killing of tumor cells. TCR-based immunotherapeutic strategies offer distinct advantages over traditional antibody-based therapies, most notably providing access to up to nine-fold more cancer targets by targeting intracellularly processed peptides presented by HLA as opposed to cell surface proteins. Preclinical testing of ImmTAC molecules can prove particularly challenging as these molecules are exquisitely human-specific, requiring a human peptidome and intact human immune system; thus the use of conventional animal models is inappropriate. Given that these molecules harness the full force of the immune system, robust preclinical testing is essential to predict safety and efficacy and inform on clinical dosing for first-in-human studies. Here we present, to our knowledge, the first entirely in vitro preclinical package that comprises a range of cellular and molecular assays to assess the efficacy, safety and specificity of ImmTAC molecules. To exemplify our approach, we draw upon the preclinical testing of multiple ImmTAC molecules, including IMCgp100, the first ImmTAC molecule to reach the clinic. We propose that this preclinical package can be applied to the testing of other TCR-based therapies to deliver a more physiologically relevant model. Citation Format: Ana Ribeiro, Tomasz Dobrzycki, Jane Harper, Giovanna Bossi, Debbie Wright, Andrea Stacey, Nicole Bedke, Ruth Martinez-Hague, Dan Blat, Laure Humbert, Zoe Donellan, Samantha Paston, Luise Weigand, Martina Canestraro, Sophie Botta Gordon-Smith, Bent K. Jakobsen, Joseph Dukes. An in vitro preclinical package to assess the safety and efficacy of ImmTAC™ molecules [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A214.

Affinity-enhanced T-cell receptors for adoptive T-cell therapy targeting MAGE-A10: strategy for selection of an optimal candidate

ABSTRACTCirculating T-cells that have passed thymic selection generally bear T-cell receptors (TCRs) with sub-optimal affinity for cancer-associated antigens, resulting in a limited ability to detect and eliminate tumor cells. Engineering TCRs to increase their affinity for cancer targets is a promising strategy for generating T-cells with enhanced potency for adoptive immunotherapy in cancer patients. However, this manipulation also risks generating cross-reactivity to antigens expressed by normal tissue, with potentially serious consequences. Testing in animal models might not detect such cross-reactivity due to species differences in the antigenic repertoire. To mitigate the risk of off-target toxicities in future clinical trials, we therefore developed an extensive in vitro testing strategy. This approach involved systematic substitution at each position of the antigenic peptide sequence using all natural amino acids to generate a profile of peptide specificity (“X-scan”). The likelihood of off-target reactivity was investigated by searching the human proteome for sequences matching this profile, and testing against a panel of primary cell lines. Starting from a diverse panel of parental TCRs, we engineered several affinity-enhanced TCRs specific for the cancer-testis antigen MAGE-A10. Two of these TCRs had affinities and specificities which appeared to be equally optimal when tested in conventional biochemical and cellular assays. The X-scan method, however, permitted us to select the most specific and potent candidate for further pre-clinical and clinical testing.

Orphan designation: autologous CD4+ and CD8+ T cells transduced with lentiviral vector containing an affinity-enhanced T-cell receptor targeting the New York esophageal antigen-1, Treatment of soft tissue sarcoma

Orphan designation: autologous CD4+ and CD8+ T cells transduced with lentiviral vector containing an affinity-enhanced T-cell receptor targeting the New York esophageal antigen-1, Treatment of soft tissue sarcoma

An approved in vitro approach to preclinical safety and efficacy evaluation of engineered T cell receptor anti-CD3 bispecific (ImmTAC) molecules.

Robust preclinical testing is essential to predict clinical safety and efficacy and provide data to determine safe dose for first-in-man studies. There are a growing number of examples where the preclinical development of drugs failed to adequately predict clinical adverse events in part due to their assessment with inappropriate preclinical models. Preclinical investigations of T cell receptor (TCR)-based immunotherapies prove particularly challenging as these biologics are human-specific and thus the conventional testing in animal models is inadequate. As these molecules harness the full force of the immune system, and demonstrate tremendous potency, we set out to design a preclinical package that would ensure adequate evaluation of these therapeutics. Immune Mobilising Monoclonal TCR Against Cancer (ImmTAC) molecules are bi-specific biologics formed of an affinity-enhanced TCR fused to an anti-CD3 effector function. ImmTAC molecules are designed to activate human T lymphocytes and target peptides within the context of a human leukocyte antigen (HLA), thus require an intact human immune system and peptidome for suitable preclinical screening. Here we draw upon the preclinical testing of four ImmTAC molecules, including IMCgp100, the first ImmTAC molecule to reach the clinic, to present our comprehensive, informative and robust approach to in vitro preclinical efficacy and safety screening. This package comprises a broad range of cellular and molecular assays using human tissues and cultured cells to test efficacy, safety and specificity, and hence predict human responses in clinical trials. We propose that this entirely in vitro package offers a potential model to be applied to screening other TCR-based biologics.