Daniel Williams

Identification of a Titin-Derived HLA-A1–Presented Peptide as a Cross-Reactive Target for Engineered MAGE A3–Directed T Cells

T cells engineered to express affinity-enhanced TCRs directed to a MAGE A3 peptide cross-react with a similar, but unrelated, self-peptide. Cross-Reactive Adoptive Therapy Engineering T cells with enhanced affinity to cancer targets is a promising therapy. However, one key bottleneck in this strategy is the identification of targets that are expressed on cancer cells but not on normal healthy tissue. One way to identify these antigens is by looking at the family of cancer-testis antigens, which have restricted expression in normal tissue but are frequently up-regulated in tumors. Cameron et al. now report that a T cell engineered to target one such antigen—MAGE A3—cross-reacts with a peptide from a muscle protein, Titin. The authors developed a T cell that targeted a MAGE A3 antigen for use in adoptive immunotherapy. Although extensive preclinical investigations demonstrated no off-target antigen recognition, patients who received these T cells had serious adverse events, including fatal cardiac toxicity. The authors then used amino acid scanning to search for potential cross-reactivity of these T cells with an off-target peptide and identified a peptide derived from the muscle protein Titin. Because affinity-enhanced T cells are highly potent, this cross-reactivity was likely the cause of the off-target toxicity. This study highlights methods that may be used to prevent cross-reactivity in future trials of adoptive immunotherapy. MAGE A3, which belongs to the family of cancer-testis antigens, is an attractive target for adoptive therapy given its reactivation in various tumors and limited expression in normal tissues. We developed an affinity-enhanced T cell receptor (TCR) directed to a human leukocyte antigen (HLA)–A*01–restricted MAGE A3 antigen (EVDPIGHLY) for use in adoptive therapy. Extensive preclinical investigations revealed no off-target antigen recognition concerns; nonetheless, administration to patients of T cells expressing the affinity-enhanced MAGE A3 TCR resulted in a serious adverse event (SAE) and fatal toxicity against cardiac tissue. We present a description of the preclinical in vitro functional analysis of the MAGE A3 TCR, which failed to reveal any evidence of off-target activity, and a full analysis of the post-SAE in vitro investigations, which reveal cross-recognition of an off-target peptide. Using an amino acid scanning approach, a peptide from the muscle protein Titin (ESDPIVAQY) was identified as an alternative target for the MAGE A3 TCR and the most likely cause of in vivo toxicity. These results demonstrate that affinity-enhanced TCRs have considerable effector functions in vivo and highlight the potential safety concerns for TCR-engineered T cells. Strategies such as peptide scanning and the use of more complex cell cultures are recommended in preclinical studies to mitigate the risk of off-target toxicity in future clinical investigations.

NY-ESO-1-specific TCR-engineered T cells mediate sustained antigen-specific antitumor effects in myeloma

Despite recent therapeutic advances, multiple myeloma (MM) remains largely incurable. Here we report results of a phase I/II trial to evaluate the safety and activity of autologous T cells engineered to express an affinity-enhanced T cell receptor (TCR) recognizing a naturally processed peptide shared by the cancer-testis antigens NY-ESO-1 and LAGE-1. Twenty patients with antigen-positive MM received an average 2.4 × 109 engineered T cells 2 d after autologous stem cell transplant. Infusions were well tolerated without clinically apparent cytokine-release syndrome, despite high IL-6 levels. Engineered T cells expanded, persisted, trafficked to marrow and exhibited a cytotoxic phenotype. Persistence of engineered T cells in blood was inversely associated with NY-ESO-1 levels in the marrow. Disease progression was associated with loss of T cell persistence or antigen escape, in accordance with the expected mechanism of action of the transferred T cells. Encouraging clinical responses were observed in 16 of 20 patients (80%) with advanced disease, with a median progression-free survival of 19.1 months. NY-ESO-1–LAGE-1 TCR–engineered T cells were safe, trafficked to marrow and showed extended persistence that correlated with clinical activity against antigen-positive myeloma.

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.

Abstract 4707: Genetically engineered NY-ESO-1-specific T cells in HLA-A2+ patients with synovial sarcoma

NY-ESO-1 is expressed in ∼70% of synovial sarcomas and not expressed in vital tissues. We report interim results of NCT01343043 evaluating safety and activity of autologous T cells engineered to express an HLA-A2+ restricted, affinity-enhanced T cell receptor (TCR) targeting NY-ESO-1. HLA-A2+ patients with unresectable, metastatic, or recurrent synovial sarcoma were eligible if tumors expressed NY-ESO-1 by IHC. Lymphocytes were activated using anti-CD3/28 microbeads, genetically modified with a lentivector, then cryopreserved. Subjects received fludarabine 30mg/m2/d (D-6 to -2) and cyclophosphamide 1800mg/m2/d (D-3,-2), and infusion of engineered T cells. Systemic IL-2 was not administered. Nine subjects have received NY-ESO-1 cell infusions. Median transduced T cell dose was 3.4 × 109 cells (range 0.4-14.4), 60×106 cells/kg (range 5.7-165.5), and median transduction efficiency was 45.8%. Toxicity likely attributable to the T cells included fever, and grade 1-2 cytokine release syndrome. No autoimmune toxicity has been observed. Circulating engineered NY-ESO-1 cells were detected in all patients, peaking 3-21 days post-infusion. Persistence has been evaluated beyond 3 months in 4 subjects, all of whom had detectable NY-ESO-1 cells at 4 mos, 6 mos+, 12 mos+ and 12 mos+. We identified persisting NY-ESO-1 T cells using dextamer and/or anti-vβ13.1 mAbs, and observed that a high fraction of CD4+ and CD8+ NY-ESO-1 TCR expressing T cells were CD45RA+CCR7+CD95+, consistent with a stem cell memory phenotype. Persisting cells also demonstrate a polyfunctional (IFN-γ and TNF-α) and cytotoxic (CD107a and granzyme B) signature without overexpression of exhaustion markers (PD-1, LAG-3, and TIM-3). Of 8 patients whose follow-up is sufficient to assess response, 4 experienced objective responses (1CR x 9 mos, 1PR x 9 mos, 2PR x 6 mos). Tumor shrinkage could not be attributed to chemotherapy alone as progressive decreases in tumor size were observed over several months following completion of the lymphodepleting regimen. All PR patients (2 upon signs of progression and 1 still responding to therapy) ultimately underwent resection for residual disease and two remain without evidence of disease. Adoptive immunotherapy with NY-ESO-1 engineered T cells shows promising results in synovial sarcoma with acceptable toxicity. High dose IL-2 is not required for therapeutic benefit with this regimen. Citation Format: Melinda S. Merchant, Sandra P. D9Angelo, Hua Zhang, Donna Bernstein, Gwen Binder-Scholl, Tom Holdich, Luca Melchiori, Dan Williams, Marylene Fortin, Yoav Peretz, Jason Howe, Michael Mehler, Bruce A. Hug, Matthew Wright, Stephen Grupp, Paul A. Meyers, William Tap, Bent Jakobsen, Crystal L. Mackall. Genetically engineered NY-ESO-1-specific T cells in HLA-A2+ patients with synovial sarcoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4707. doi:10.1158/1538-7445.AM2015-4707

192. Deep Phenotyping of Manufactured Enhanced-Affinity NY-ESO-1-Specific T Cells Reveals a Pattern of Effector and Memory Programming That Correlates with Clinical Outcome in Observed Cancer Indications

It is well established that human tumors express unique antigens; however, immunosuppressive mechanisms prevent natural immune responses. We hypothesized that infusion of T cells that have been genetically modified to express affinity-optimized tumor antigen-specific TCRs may overcome these barriers. We initiated several ongoing clinical studies to evaluate T cells engineered with an affinity-enhanced TCR specific for the NY-ESO-1 and LAGE-1 cancer testis antigens (NY-ESOc259-T), in synovial sarcoma (SS) and multiple myeloma (MM) patients with antigen-positive tumors. Here we report updated phenotyping results from two MM and SS cohorts of patients. Manufactured product (MP) and baseline samples were analyzed by flow cytometry evaluating memory and activation markers (e.g. CD45RA, CCR7, ICOS, OX40, etc) and polyfunctionality markers (e.g. IFN-γ and IL-2). NY-ESOc259-T was detected by pentamer staining and its phenotype was correlated with clinical response. Upon culture NY-ESOc259-T generated different memory phenotypes. The phenotype at baseline (day -50) did not predict the phenotype acquired at the end of culture. Interestingly, in MM a positive trend with clinical response was observed in MPs bearing a higher percentage of Central Memory (CM) cells secreting IL-2 and IFN-γ, while a negative trend was found in Effector Memory (EM) and Effectors (EMRA)-dominated MPs secreting high levels of TNF-α. This trend was confirmed by analysing the absolute number of CM cells infused in a small number of patients, hinting at the existence of an “effective CM cells dose” of around 3×108 CM cells necessary to achieve clinical response. Of note, this trend was observed also in the SS cohort. Upon stimulation T cells proliferate and differentiate in vitro, but at the same time cells need time in culture to reach an effective dose. To understand these dynamics we retrospectively analysed the evolution of CM cells in relation to the length of culture. We observed a negative correlation between percentage of CM cells and culture time, while EM and EMRA cells increased with the duration of culture. This suggests the importance of reconciling time of culture with phenotype and number of cells in order to achieve that “sweet spot” critical for clinical response. Activation/costimulation markers in the SS cohort were also analysed in relation to culture conditions. MPs consistently displayed up-regulation of ICOS, CD40L and OX40, suggesting that an activated phenotype is achieved even after several days of culture. Of note, markers of immunological memory like CD27 and CD28 were also maintained up to the end of culture highlighting the fitness of the product despite the duration of the culture. These data suggest that NY-ESOc259-T may acquire a CM phenotype that positively correlates with clinical response in the cancer indications observed. Additionally, we show how the manufacturing process successfully produces a population of activated cells that express important activation markers without losing markers of fitness and memory - properties that are key to driving the anti-tumour response observed in cancer patients.

511. Enhanced-Affinity NY-ESO-1-Specific T Cells Exhibit Extended Functionality without Exhaustion in a Pattern of Effector and Memory Programming in Multiple Cancer Indications

It is now well established that human tumors express unique antigens, however immunosuppressive mechanisms prevent natural immune responses. We hypothesized that infusion of T cells that have been genetically modified to express affinity-optimized tumor antigen-specific TCRs may overcome these barriers. We initiated several ongoing clinical studies to evaluate T cells engineered with an affinity-enhanced TCR specific for the NY-ESO-1 and LAGE-1 cancer testis antigens (NY-ESOc259-T), in synovial sarcoma (SS) and multiple myeloma (MM) patients with antigen-positive tumors.

Process improvement and comparability analysis for engineered T cell manufacture

Engineered T cell therapy (ECT) for oncology has met significant clinical proof of success for chemotherapy resistant B cell malignancies in multiple studies. These data have underscored the transformative potential of ECT in advanced oncology. Adaptimmune specializes in the generation and testing of affinity-enhanced T cell receptors (TCR) for engineered T cell therapy, and has several ongoing clinical studies to evaluate T cells engineered with an affinity enhanced TCR specific for the NY-ESO-1 and LAGE-1 cancer testis antigens (NY-ESO-T), in patients with antigen-positive tumors. A major challenge in successful commercialization of engineered cell therapy will be to maintain product safety and potency while streamlining the manufacturing process for cost reduction, process consistency, and manufacturing portability, in order to meet commercial-scale demands and reimbursement feasibility. A summary of NY-ESO-T manufacture is provided in Figure ​Figure1.1. Modifications to manipulation of incoming product (A), initial T cell enrichment (B), medium components (C), and final formulation (D) will be described. To evaluate safety and efficacy of the product after these process changes, comparability studies have been carried out, including 14-colour flow cytometry analysis of cell phenotype and functionality after expansion. Data will cover the phenotype of T cell sub-populations, cytokine expression profile, exhaustion and activation marker expression, to demonstrate product consistency and potency following process changes. Establishment of critical quality attributes will require iterative analysis of correlative and efficacy data from clinical trials. Figure 1 NY-ESO-T manufacturing process.

Abstract 3528: IMCmage1: A novel bi-specific biologic re-directing T cells to kill MAGE-A3/A6 presenting cancers

In a minority of cancer patients immunotherapy has shown the capacity to eradicate tumours leading to clinical remission and the promise of a cure. In the majority of patients however, a cure remains elusive due to active immune evasion by cancers; HLA-down-regulation and immunosuppression are two of the known mechanisms adopted by cancers to promote their survival and proliferation. To overcome these challenges we have developed bi-specific soluble biologics termed ImmTACs (Immune mobilising mTCR against cancer) to re-direct the immune system to recognise and kill cancers. Antigenic peptide fragments presented by HLA molecules on the surface of cancer cells constitute the largest class of cancer associated targets. T cells scan the HLA-peptide (pHLA) antigens being presented to them; sufficient recognition by the harboured T Cell Receptor (TCR) will result in T cell activation and killing of the antigen presenting cell. In cancer patients this process is inefficient partly due to the low affinity TCRs expressed by tumour specific T cells and the low abundance of pHLA on cancers. ImmTACs comprise a soluble TCR with an enhanced affinity for cancer associated pHLA (targeting end) fused to an anti-CD3 scfv, enabling potent T cell re-direction (effector end). Our pipeline constitutes a number of ImmTACs targeting various antigen pHLA complexes relevant to numerous cancer indications. IMCmage1 is a novel ImmTAC targeting MAGE-A3168-176 in the context of HLA-A1. MAGE-A3 is a well validated cancer testis antigen expressed in a variety of cancers including myeloma, NSCLC, prostate cancer, melanoma, bladder cancer, oesophageal cancer and others. IMCmage1 re-directs T cells from cancer patients or healthy donors to kill a range of MAGE positive cell-lines in vitro; this activity is observed against cells presenting as few as 40 epitopes per cell and is coupled with the release of pro-inflammatory cytokines including IFNα, TNFα, IL-2, MIP1α and others. We also demonstrate that IMCmage1 specifically targets and kills the myeloma associated population within CD138+ cells extracted from the marrow of a stage III myeloma patient. IMCmage1 specificity was confirmed by exposure to a panel of HLA-A1 MAGE negative primary cells derived from various organs such as the heart, skin, lung and others; no significant activity was detected. A phase I clinical trial in multiple myeloma to assess tolerability and establish a maximum tolerated dose is planned to commence in Q2 2012. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3528. doi:1538-7445.AM2012-3528

Abstract 1787: ImmTACs: Bi-functional reagents for redirected tumour cell killing

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL The human immune system can theoretically identify malignant cells by inspecting cell surface Class I HLA -peptide complexes for the presence of disease-associated epitopes. Indeed, many cancer patients generate CD8 cyto-toxic T cell responses to tumour-associated antigens; the majority of patients, however, fail to clear tumours since T cell avidity for self-antigens tends to be weak, and cancer cells employ escape mechanisms for avoiding destruction by T cells. To overcome these issues, we have engineered novel, bi-functional protein therapeutics termed ImmTACs (Immune Mobilising mTCR Against Cancer) which re-direct the immune system to target and destroy tumour cells with a high degree of potency and specificity. An ImmTAC comprises a high affinity ‘monoclonal’ T cell Receptor (mTCR) targeting a cancer-associated HLA-peptide complex, fused to an anti-CD3 scFv domain which activates an anti-tumour T cell response. We demonstrate that ImmTACs against a number of different cancer-associated antigens can target and kill tumour cells expressing as few as 10-20 epitopes per cell with pico-Molar potency. ImmTACs preferentially activate effector memory CD8 T cells, resulting in secretion of multiple cytokines and tumour cell killing; a single activated T cell can kill multiple antigen positive tumour cells. Furthermore, we demonstrate that the reagents are able to inhibit tumour growth in mouse xenograft models. In vitro ImmTAC potency translates to a dose of less than 1mg in humans, representing a significant advance over existing targeted anti-cancer therapies. Currently we are conducting a phase I dose-escalation trial and Phase 0 exploratory trial using ImmTAC-gp100 in late stage melanoma patients. In summary, ImmTACs offers a novel therapeutic approach for the treatment of various cancers with the potential to provide major benefits over current treatments including reduction in dose to sub-mg quantities and an improved safety profile. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1787. doi:10.1158/1538-7445.AM2011-1787

Abstract 5616: Soluble, high affinity TCRs fused to anti-CD3 redirect T cells to kill cancer cells presenting MAGE-A3 and NY-ESO antigens

In the last decade, major efforts in the fight against cancer have focussed on galvanising the adaptive immune system to kill tumours. Many of these endeavours are based on the development and clinical use of monoclonal antibodies (mAb) which are the most successful class of immune modulating agent identified to date. However, while mAbs show promise against certain cancers, their specificity is limited to integral membrane proteins; this hinders their extensive development for the purposes of targeting cancer cells. In contrast to mAbs, T cell receptors (TCRs) recognise peptides bound to major histocompatibility complex class I (MHC I) molecules. These peptides are derived from endogenously processed proteins, and therefore represent a different repertoire of targets to those recognised by mAbs. This alternate spectrum of antigens provides the potential to target cancers using an untapped source of well-validated epitopes. Naturally occurring TCRs, however, have relatively low affinities for their antigen compared to antibody binding. Advances in engineering techniques have allowed the generation of high affinity monoclonal TCRs (mTCRs) with picomolar affinities for their antigen. Using targeted mutagenesis and phage display, we have generated a number of soluble, high affinity mTCRs specific for several reported tumour-associated antigens. Through mTCR fusion to an anti-CD3 single chain variable fragment (scfv), we produced bifunctional proteins that redirect T cell immune specificity. These novel proteins are termed ImmTACs (Immune-mobilising mTCRs Against Cancer). We present data demonstrating the potential of two such ImmTAC molecules, NY-ESO-ImmTAC and MAGE-A3-ImmTAC, to treat certain cancers. NY-ESO1 and MAGE-A3 are both cancer testes antigens and therefore represent potentially very clean molecular targets. We demonstrate that both NY-ESO-ImmTAC and MAGE-A3-ImmTAC are capable of potently redirecting unstimulated peripheral blood mononuclear cells (PBMC) or CD8+ T cells against multiple myeloma, colorectal carcinoma and non-small cell lung cancer cell lines despite the presentation of extremely low antigen numbers ( Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5616.