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.

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.

In recent years significant advances in the treatment of metastatic melanoma have emerged. Small molecule drugs provide potent short-term responses for a significant proportion of the patient population; for a minority of patients, immunotherapy has elicited long-term responses with the promise of a cure. Despite these advances, long-term remission for the majority of patients remains elusive and much effort is focussed on combination therapies attempting to bring together the potency of small molecule drugs with the durability of immunotherapy. IMCgp100 is a novel bi-specific immunotherapy comprising a soluble, affinity- enhanced, T cell receptor (TCR) specific for the melanoma-associated antigen gp100, fused to an anti-CD3 specific antibody fragment (scfv). The engineered TCR portion of the drug targets the gp100 peptide 280-288 antigen, which is over- expressed and presented by HLA-A2 on the surface of melanoma cells, thereby effectively coating these cells with CD3-specific antibody fragments. The anti-CD3 scfv portion captures and redirects any T cells in physical contact with the melanoma cell to kill it. In vitro, IMCgp100 potently redirects T cells from the blood of late stage cancer patients to target melanoma cells exhibiting substantial HLA-down regulation, even in the presence of high numbers of regulatory T cells. Target cell killing is observed within hours, and is associated with the release of pro-inflammatory cytokines and dendritic cell cross-presentation of gp100 and other melanoma-specific antigens. Thus, IMCgp100 demonstrates the potential to elicit potent short-term responses and trigger longer-term anti melanoma durability in vivo (ref: paper to be published imminently in Nature Medicine). IMCgp100 is currently under investigation as part of a Phase 1 dose-finding study in patients with unresectable Stage III/Stage IV malignant melanoma. Safety, tolerability, pharmacokinetic, pharmacodynamic, and clinical activity are assessed after intravenous infusion of IMCgp100, and the maximum tolerated dose (MTD) will be established. We also have a Phase 0 trial open, in which IMCgp100 is injected directly into tumours to assess pharmacodynamic activity in human lesions. Both studies are actively enrolling and preliminary clinical data will be presented. 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 3525. doi:1538-7445.AM2012-3525

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