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.

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. Here we report the initial findings from these correlative studies. Post infusion PBMCs were analyzed by flow cytometry evaluating memory and exhaustion markers (e.g. CD45RA, CCR7, PD-1) and polyfunctionality/cytotoxicity markers (e.g. IFN-γ and Granzyme B). NY-ESOc259-T cells were detected by pentamer staining and their phenotype was correlated with the clinical response. Once infused, NY-ESOc259-T rapidly expanded, peaking around day 21 and subsequently decreasing in frequency but remaining detectable up to one year after infusion. In MM, NY-ESOc259-T cells transitioned from a central memory phenotype to an effector phenotype, up-regulating markers of polyfunctionality and cytotoxicity. These markers decreased at later time points as the cells acquired an effector memory phenotype. In SS, NY-ESOc259-T also up-regulated these antitumor markers, but transitioned toward a phenotype closely related to T stem cell memory (see figure). In all clinical settings, no T regulatory NY-ESOc259-T cells were observed. Exhaustion markers analysis revealed no simultaneous expression of PD-1, LAG-3 and TIM-3 even at later time points, indicating that NY-ESOc259-T cells successfully clear the tumor without becoming exhausted. This correlated with up-regulation of anti-tumor molecules upon stimulation. These data show for the first time that affinity-enhanced, TCR transduced T cells exhibit robust expansion, durable persistence without exhaustion, and follow a natural immune expansion and contraction pattern consistent with an antigen-driven mechanism of action. Moreover the ability of the cells to respond to antigen even up to one year after tumor clearance indicates the onset of a functional memory that mimics a physiologic response to a pathogen.

Carl June and colleagues report the results of a phase I/II trial of adoptively transferred engineered T cells in patients with advanced multiple 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.

Meeting abstracts 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

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.

Abstract Although cancer patients frequently mount antigen specific T cell responses, the low affinity of T cell Receptors (TCRs) to self-antigens coupled with low levels of HLA-peptide on cancer cells contribute to the failure of the immune system to clear tumours. Peptide epitopes presented by Class I HLA represent the largest class of tumor associated antigens and are ideal targets for cancer immunotherapy, providing that the issues of low TCR affinity and immune-suppression in the tumor microenvironment can be overcome. The most attractive targets for minimising toxicity are expressed on a restricted number of normal tissue cells, for example cancer testis antigens. We have engineered ImmTAC reagents comprising soluble, high affinity TCRs fused to an anti-CD3 scFv domain that redirects a potent, polyclonal T cell response to kill tumour cells expressing as few as 10-20 epitopes. IMCgp100 targets the HLA-A2 presented epitope gp100 280-288 and kills malignant melanoma cells with pico-Molar potency, leaving other HLA-A2 positive cells untouched. This agent is undergoing clinical testing in a Phase I dose-escalation trial and is well tolerated. A second ImmTAC, IMCmage1, targets MAGE-A3 168-176 in the context of HLA-A1. It specifically kills the myeloma population within CD138+ cells extracted from the marrow of a stage III multiple myeloma patient and can re-direct T cells extracted from the tumor microenvironment. IMCmage1 will enter the clinic in 2012.

T cell receptor (TCR)-based immunotherapeutic approaches have so far had limited success because of a lack of specific immune recognition and activation by the TCR. Here Nathaniel Liddy and his colleagues describe the generation, optimization and characterization of a new set of reagents—immune-mobilizing monoclonal TCRs against cancer (or ImmTACs)—designed to overcome some of these limitations. The ImmTACs were used to redirect and activate T cells to lyse tumor cells both in vitro and in vivo , even those expressing very low epitope numbers on the cell surface.

Abstract 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 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

The human immune system can theoretically identify malignant cells by ‘scanning’ peptide antigens presented on the cell surface by Class I HLA molecules, and indeed many cancer patients generate CD8 cyto-toxic T cell responses to tumour-specific antigens. However, the majority of patients 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. We have engineered novel, bi-functional protein therapeutics termed ImmTACs which re-direct the immune system to target and destroy tumour cells with a high degree of potency and specificity. IMCgp100 comprises a soluble, high affinity T cell Receptor specific for the HLA-A2 presented melanoma-associated epitope gp100 280-288, fused to an anti-CD3 scFv domain. The anti-CD3 moiety activates a polyclonal T cell response when targeted to melanoma cells presenting as few as twenty gp100 epitopes, predominantly through activation of effector memory CD8 lymphocytes, leading to killing of the tumour cells. Extensive in vitro testing of IMCgp100 with a panel of tumour cells and normal human tissue cells demonstrates pico-Molar potency against cancer cells and specificity for HLA-A2 gp100 positive cells. The agent is able to inhibit tumour growth in mouse xenograft models. A phase I dose-escalation trial in late stage melanoma patients was initiated in October 2010 and interim results will be presented at this meeting.

Abstract In the last decade, major efforts in the fight against cancer have focused on galvanizing the adaptive immune system to kill tumors. Many of these endeavors 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. 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) recognize 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 recognized 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 tumor-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-mobilizing mTCRs Against Cancer). We present data showing the potential of two such ImmTACs, NY-ESO-ImmTAC and MAGE-A3-ImmTAC, to treat certain cancers. NY-ESO1 and MAGE-A3 are cancer testes antigens and therefore represent potentially very clean molecular targets. We demonstrate that both NY-ESO- and MAGE-A3-ImmTACs are capable of potently redirecting unstimulated CD8+ T cells against multiple myeloma, colorectal carcinoma and non-small cell lung cancer cell lines despite the presentation of extremely low antigen numbers (<100 epitopes/cell) on the cell surface. ImmTAC-redirected T cells respond with multiple effector functions including production of granzyme B, IFNγ and IL-2. Using the NY-ESO-ImmTAC we observed significant redirected degranulation of T cells against a primary lung tumor sample which was shown to express NY-ESO. We also present data from an established tumor model using the OV-79 cell line derived from an ovarian tumor which is shown to express MAGE. Administration of MAGE-A3-ImmTAC to animals with established tumors resulted in inhibition of tumor growth in all ImmTAC treated animals with regression or cure in some. Thus both MAGE-A3 and NY-ESO-ImmTACs possess the potential to be highly specific, potent cancer immunotherapies offering a targeting and therapeutic approach distinct from any other biologic in development. 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 4744. doi:10.1158/1538-7445.AM2011-4744

Abstract 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