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IMCgp100, a first in class immunotherapy, is a T cell redirecting bispecific biologic comprising an affinity enhanced T-cell receptor specific for gp100 and an anti-CD3 scFV. Phase I/IIa data has provided evidence of a favourable safety profile, and durable responses in both cutaneous and uveal melanoma have been observed. To complement the clinical studies we have developed a comprehensive biomarker strategy to aid our understanding of pharmacodynamics, patient response and potential mechanisms of resistance which includes analysis of markers in both the tumour and periphery. The data obtained to date provide evidence of the pharmacodynamic effects of the molecule including chemokine/cytokine release, in both the tumor and periphery, and lymphocyte infiltration into tumors. In addition, changes in the levels of certain chemokines following the first dose of IMCgp100 were associated with tumor shrinkage. The biomarker strategy we have developed forms the basis for the support of the on-going Ph II development of IMCgp100 in both cutaneous and uveal melanoma and for other ImmTAC molecules, as single agents and in combination, for the treatment of solid tumours. Citation Format: Cheryl McAlpine, David Krige, Sandra Herrero-Gonzalez, Sarah Franklin, Jacob Hurst, William Shingler, Sanjay Patel, Andy Johnson, Debbie Parker, Christina M. Coughlin, Namir J. Hassan, Bent K. Jakobsen. Biomarker strategy to guide the clinical development of ImmTACTM molecules, a novel class of bispecific T cell engaging biologic drugs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3655. doi:10.1158/1538-7445.AM2017-3655

Immunotherapeutic strategies that drive activation of cytotoxic T cells possess significant potential to eradicate tumours. Whereas monoclonal antibodies are restricted to targeting secreted or cell surface proteins, T cell receptors (TCRs) are able to recognise a wider range of targets. This is achieved through binding to short peptide fragments derived from proteins that are degraded intracellularly and presented at the cell surface by human leukocyte antigens (HLAs). Natural cancer specific TCRs however, have weak affinities and cancer cells often develop escape mechanisms to avoid destruction by T cells. To overcome this, we have developed Immune mobilising monoclonal TCRs Against Cancer (ImmTACs); a new class of soluble bi-specific molecules comprising affinity-enhanced, monoclonal T cell receptors (mTCRs) fused to an anti-CD3 scFv. ImmTACs target peptides presented by HLA, and through the anti-CD3 effector, re-direct cytotoxic T cells to achieve highly specific and potent tumour cell killing. At Immunocore, we have developed an integrated in-house process for the generation of ImmTACs and here describe the critical engineering steps involved. T-cell clones that specifically recognise validated cancer antigens are isolated from peripheral blood lymphocytes and the TCR-encoding sequences are identified by RACE. To confirm antigen binding, TCR á and â chains are expressed as inclusion bodies in bacteria, co-refolded in vitro, and their binding to the target peptide:HLA tested by Surface Plasmon Resonance (SPR). The affinity of the TCR is then enhanced up to a million-fold through directed evolution, utilising phage display. Individual mutants are screened by SPR and combined to generate ImmTACs with pM affinities (KD) and binding half-lives of many hours. A range of biochemical and cellular assays are then performed to assess the potency and specificity of each ImmTAC generated. This process has been successfully applied to produce ImmTACs for a wide range of targets, demonstrating the robustness of the platform. Our lead candidate, IMCgp100, is undergoing Phase IIa clinical trials in patients with advanced malignant melanoma. This reagent, which specifically targets the gp100 (280-288) peptide presented by HLA-A2 on melanoma cells, is well tolerated and shows very promising therapeutic potential. Citation Format: Andrew Knox, Fiona Chester, Frayne Bianchi, Sarah Bailey, Lucie Bouard, Nathaniel Liddy, Giovanna Bossi, Jane Harper, Joseph Dukes, Samantha Paston, Tara Mahon, Jessie Gavarret, Peter Molloy, Malkit Sami, Emma Baston, Brian Cameron, Alex Powlesland, Penio Todorov, Andrew Johnson, Martin Ebner, Yvonne McGrath, Namir Hassan, Annelise Vuidepot, Bent Jakobsen. Developing high affinity, soluble T cell receptors for the treatment of cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4872.

Immunotherapy strategies that are able to induce T cell infiltration into tumors and activate a cytotoxic T cell response have the potential to destroy the tumor. Although T cells can mediate clearance of a tumor, thymic selection and the suppressive microenvironment limit their effectiveness. To overcome poor tumor immunogenicity, we have developed a unique platform that enables the generation of ImmTACs (Immune-mobilising monoclonal TCRs Against Cancer); these are comprised of an affinity enhanced TCR specific for a cancer antigen fused to an anti-CD3 scFv. The TCR end targets and binds MHC class I/peptide complex displayed on cancerous cells while the anti-CD3 scFv end engages polyclonal T cells to mediate a potent anti-tumor response. The most advanced ImmTAC, IMCgp100, targets the HLA-A2/gp100280-288 epitope presented by melanoma cells. IMCgp100 is currently in a Phase I/IIa clinical trial for advance malignant melanoma and is showing promising clinical efficacy in some patients. Here we report a series of in vitro experiments evaluating IMCgp100 mechanism of action. IMCgp100 is able to redirect T cells from healthy donors or from melanoma patients to destroy cancer cells and secrete a range of inflammatory cytokines and chemokines associated with T cell trafficking into tumors; some of these cytokines also induce upregulation of inhibitory pathway molecules. We show that effector memory cells in the CD8+ and CD4+ T cell compartments are very efficient in eliminating melanoma cells and in expanding upon IMCgp100 engagement. The effects of combining IMCgp100 with agents that relieve the suppression imposed by immune check-point molecules have also been investigated. Citation Format: Giovanna Bossi, Rupert Kenefeck, Caroline Caroline Boudousquie, Jane Harper, Joseph Dukes, Nathaniel Liddy, Samantha Paston, Tara Mahon, Peter Molloy, Malkit Sami, Emma Baston, Brian Cameron, Annelise Vuidepot, Namir Hassan, Bent K. Jakobsen. ImmTACs re-direct the immune system efficiently to eradicate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4873.

Background: IMCgp100 comprises an affinity-enhanced T cell receptor (TCR) specific for the HLA-A2 restricted melanoma gp100 peptide (YLEPGPVTA) fused to an anti-CD3 antibody fragment. Binding of IMCgp100 to melanoma cells redirects T cell cytotoxicity even in the presence of significant levels of PD-1 and PD-L1. Importantly, IMCgp100 enables T cells to kill HLA down-regulated melanoma cells otherwise invisible to cancer specific T cells. A Phase I study was conducted to determine the Maximum Tolerated Dose (MTD) and toxicity of IMCgp100 in patients with metastatic melanoma. Methods: HLA A2 positive patients with Stage IV or unresectable Stage III melanoma, with an ECOG Performance Status (PS) ≤1 and a lymphocyte count of ≥0.5x109/L were enrolled. Those with symptomatic, unstable brain metastases requiring steroids and those who might benefit from immediate vemurafenib treatment were excluded. IMCgp100 was administered on day 1 and patients followed to day 30. Those who tolerated the first dose received repeated cycles of six weekly doses. The dose of IMCgp100 was escalated in cohorts of 3 (+3) patients until criteria for MTD were met. MTD was defined as the highest dose level with an observed incidence of Dose Limiting Toxicity (DLT) in fewer than 33% of patients enrolled at that level. Transient Grade 3 lymphopenia and Grade 3 skin toxicity were excluded from the definition of a DLT. Results: 31 patients were enrolled in 8 cohorts (doses from 5 to 900ng/Kg). 58% were male, median age was 61, ECOG PS 0 in 52% and 1 in 48%. All but 1 patient had stage IV disease. 60% of patients had received systemic treatment with DTIC (29%), ipilimumab (6%), vemurafenib (3%) or one or more experimental therapies (22%) prior to enrolment. At dose 900ng/Kg, 2/4 patients developed Grade 3 hypotension, therefore the MTD was defined at 600ng/Kg. Common toxicities included transient grade 3 pruritic rash and grade 2 pyrexia. Patients experienced profound lymphocyte trafficking to skin as evidenced by immunohistochemical analysis of skin biopsies, accompanied by chemokine/cytokine release. Rash was initially observed at dose level 45ng/Kg. Tumor flare was observed in patients with cutaneous or subcutaneous disease. Four partial responses (PR) and multiple lesser responses (not meeting RECIST PR) have been documented to date. One PR was achieved after a single dose. Two PRs continue after >9 months of continued treatment. One patient continues with stable disease for >10 months. Sites of response included the lung, the liver, the lymphatic system and various soft tissues. Conclusions: Toxicities of IMCgp100 were consistent with the mode of action of the drug i.e. IMCgp100 mediated T cell mobilisation, activation and tumor killing. Objective durable responses were observed. The study has been expanded to accrue additional patients at 600ng/Kg and to explore potentially superior dosing regimens. Citation Format: Mark Middleton, Jeff Evans, Neil Steven, Pippa Corrie, Clive Mulatero, Mario Sznol, William H. Shingler, Dominic Smethurst, Namir Hassan, Yvonne McGrath, Bent Jakobsen. A Phase I study of IMCgp100: durable responses with a novel first-in-class immunotherapy for advanced melanoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr CT329. doi:10.1158/1538-7445.AM2014-CT329

Despite significant advances in the treatment of metastatic melanoma, long-term remission for the majority of patients remains elusive. Kinase inhibitors provide potent but short-term responses for a significant proportion of patients and immunotherapy elicits long-term responses with the prospect of cure, but only in a minority. 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 and binds the gp100 peptide 280-288 antigen, which is over-expressed and presented by HLA-A2 on the surface of melanoma cells. The anti-CD3 scFv portion captures and redirects T cells to kill the melanoma cells, while normal antigen negative tissues are unaffected. Here, we present data which provides the foundation for the clinical observations. In vitro, IMCgp100 is demonstrated to potently redirect T cells from late stage cancer patients to target melanoma tumors exhibiting HLA-down regulation, even in the presence of high numbers of regulatory T cells. Target cell killing is observed within hours and is specific for gp100. In addition killing is associated with the release of various pro-inflammatory cytokines and chemokines as well as cross-presentation of gp100 and other melanoma-associated antigens by dendritic cells. Thus, IMCgp100 demonstrates the potential to elicit potent short-term responses and trigger longer-term anti melanoma activity in vivo. IMCgp100 is undergoing Phase I clinical testing in patients with advanced malignant melanoma; with the maximum tolerated dose having been established. The drug is well tolerated with evidence of tumor shrinkage. Analyses of serum samples obtained from patients on the trial demonstrate T cell mobilisation and transient drug mediated increases in various cytokines and chemokines, some of which are reported to play a key role in anti-melanoma responses. These data support the potential of IMCgp100 as an effective treatment for malignant melanoma. Citation Format: Namir J. Hassan, Giovanna Bossi, Debbie Baker, Katherine Adams, Jane Harper, Joseph Dukes, Nathaniel Liddy, Samantha Paston, Yvonne McGrath, Tara Mahon, Peter Molloy, Malkit Sami, Emma Baston, Brian Cameron, Andrew Johnson, Annelise Vuidepot, Gerry Linette, Michael Kalos, Carl June, Bent Jakobsen. IMCgp100: A novel bi-specific biologic for the treatment of malignant melanoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2900. doi:10.1158/1538-7445.AM2014-2900

Immunotherapeutic strategies that are able to drive activation of T cells, and in particular CD8+ cells, possess significant potential to eradicate tumors. Although it is well-established that CD8+ T cells can mediate potent destruction of tumor tissue, the immunosuppressive tumor microenvironment and thymic selection serve to limit the availability and effectiveness of tumor-specific T cells. These factors typically prevent an effective immune response in cancer patients. Despite some successful attempts to counter immune-suppression using bi-specific, immune-activating antibodies, the paucity of tumor-specific antibody targets limits this approach. ImmTACs (immune mobilising monoclonal TCRs against cancer) are a new class of soluble bi-specific molecules comprising, pico-molar affinity T cell receptors (TCRs) fused to an anti-CD3 specific scFv. Unlike antibodies, TCRs target peptide epitopes, derived from intracellular antigens, and presented on the cell surface by Class I MHC, thus providing access to a large pool of tumor-associated antigens. The high affinity recognition mediated by the engineered TCR portion of ImmTACs overcomes the problems posed by thymic selection, as well as HLA down-regulation on tumor cells, while the anti-CD3 moiety re-directs T cells to mediate a potent anti-tumor response. The most advanced ImmTAC reagent to-date, IMCgp100, targets the HLA-A2 presented gp100280-288 epitope. This reagent represents a first-in-class treatment for malignant melanoma, and is currently in Phase I clinical testing, with maximum tolerated dose having been established. Here we examine the processes which underpin the mechanism of action of IMCgp100, as well as the broader ImmTAC platform. These studies demonstrate that IMCgp100 is able to redirect T cells from healthy donors and from melanoma patients to destroy cancer cells with low cell-surface epitope densities (25 to 70 epitopes per cell), and to secrete a range of inflammatory cytokines. Effector and central memory cells in the CD8+ and CD4+ repertoire rapidly respond to ImmTAC engagement on melanoma cells, while the naïve cells provide a slower but nonetheless potent response. ImmTACs can induce an anti-tumor T cell response despite the presence of immunosuppressive regulatory T cells and expression of inhibitory molecules such as PD-L1. In addition, ImmTAC activated cytotoxic T cells are capable of serially killing a number of target cells. Finally, we have been able to demonstrate that IMCgp100 can trigger DC X presentation of melanoma associated antigens providing a potential mechanism for inducing a long-term self-sustained anti-cancer response. Taken together these data demonstrate the potential of the ImmTAC platform for targeted cancer therapy. Citation Format: Giovanna Bossi, Debbie Baker, Katherine Adams, Jane Harper, Joseph Dukes, Nathaniel Liddy, Samantha Paston, Yvonne McGrath, Tara Mahon, Pater Molloy, Malkit Sami, Emma Baston, Brian Cameron, Andrew Johnson, Annelise Vuidepot, Namir Hassan, Bent Jakobsen. ImmTACs: Bi-specific TCR-anti-CD3 fusions for potent re-directed killing of cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 670. doi:10.1158/1538-7445.AM2014-670

Malignant cells may be recognized by T cells binding cell surface Class I HLA-peptide complexes presenting disease-associated epitopes. Many cancer patients generate CD8 cytotoxic T cell responses to tumor-associated antigens, however due to the low avidity of these T cells and cancer cells developing escape mechanisms for avoiding destruction by T cells, the immune system fails to clear tumors. To overcome these issues, we have engineered novel, bi-functional protein therapeutics termed ImmTACs (Immune Mobilising monoclonal TCR Against Cancer) which re-direct the immune system to target and destroy tumor 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-tumor T cell response. In order to produce ImmTACs, we have developed an integrated in-house process leading to the isolation of TCRs specific for validated cancer epitopes. The critical steps in this process are: antigen selection, epitope identification, T cell cloning, TCR isolation and binding to soluble peptide:MHC on the BIAcore. We describe each of these steps in more detail and present data to illustrate the successful isolation of a number of TCRs specific for various tumor associated antigens such as prostate and cancer testis antigens leading from this procedure. Citation Format: Luise U. Weigand, Samantha Paston, Linda Hibbert, Ruth K. Ryan, Debbie E. Baker, Ruth A. Simmons, Jane V. Harper, Joseph D. Dukes, Giovanna Bossi, Francis Grand, Emma Hickman, Alex Powlesland, Annelise Vuidepot, Namir J. Hassan, Bent K. Jakobsen. Cancer-specific T cell receptor isolation for cancer immunotherapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2805. doi:10.1158/1538-7445.AM2014-2805

Despite significant advances in the treatment of metastatic melanoma long-term remission for the majority of patients remains elusive. Kinase inhibitors provide potent but short-term responses for a significant proportion of patients and immunotherapy elicits long-term responses but only in a minority. 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 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 activity. As part of a Phase 1 dose-finding study in HLA-A2 positive patients with advanced melanoma, 18 patients have received single IV infusions of 5, 15, 45, 135, 270 or 405ng/Kg of IMCgp100. Seven patients have subsequently received weekly IMCgp100 infusions for 6 weeks, with a brief clinical response in 1 patient at 270ng/Kg. To date, the most frequent drug related toxicities observed are rash (12 patients), pruritus (7), hypotension (5) and oedema (7), starting at the 45ng/Kg dose level. The frequency, severity and duration of adverse events demonstrate a clear dose response relationship. Pharmacodynamic data show a transient reduction in peripheral lymphocyte count 24h after dosing, consistent with the proposed mechanism of action of IMCgp100. Pharmacokinetic analysis indicates a linear dose relationship, with Cmax at 4h and T1/2 of approximately 9h. IMCgp100 is well-tolerated, with evidence of T-cell trafficking after dosing. Toxicity to date has been consistent with on target effects. Citation Format: Mark Middleton, Jeff Evans, Neil Steven, Pippa G. Corrie, Clive Mulatero, Debbie Baker, Giovanna Bossi, Katherine Adams, Jane Harper, Andy Johnson, William Shingler, Dominic Smethurst, Namir Hassan, Yvonne McGrath, Bent Jakobsen. IMCgp100: a novel bispecific biologic for the treatment of malignant melanoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1238. doi:10.1158/1538-7445.AM2013-1238

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

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

We have utilized highly purified hematopoietic progenitor and stem cells (HPCs, HSCs) from normal peripheral blood to develop methodology for: (a) efficient transfer into HPCs of a non-hematopoietic membrane reporter, i.e., the nerve growth factor receptor complementary DNA; and (b) effective gene transduction of putative HSCs, i.e., cells initiating Dexter-type long-term culture (LTC-ICs). Purified HPCs induced into cycling by growth factors (interleukin 3, interleukin 6, c-kit ligand) were transduced with the N2 retroviral vector containing the neomycin resistance (neor) gene. More than 80% of transduced HPCs were resistant to the toxic G418 level. Thereafter, the HPCs were effectively transduced with the LNSN retroviral vector containing a nerve growth factor receptor complementary DNA; the nerve growth factor receptor was detected onor = 18% of the transduced HPCs. These experiments provide a new tool from which (a) to monitor expression of a transduced membrane report on hematopoietic cells, particularly at the level of HPCs/HSCs, and (b) to characterize the transduced cells by double- and triple-labeling membrane antigen analysis. Purified HPCs/HSCs grown in Dexter-type LTC were transduced at 1 week by exposure to supernatant N2 retroviral particles in the absence of exogenous hematopoietic growth factors. The procedure, devoid of toxic effects, allowed an efficient neor transduction into LTC-ICs. Thus, we consistently detected neomycin-resistant mRNA in the clonal progeny of HPCs produced in LTC at 5-8 weeks in both the nonadherent and adherent fractions; this timing of expression coincides with that of HPC production by LTC-ICs, thereby indicating the effective transduction of the LTC-ICs. These experiments represent a first step toward development of preclinical models for gene transfer into human peripheral blood HSCs by complex retroviral vectors.

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

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 ( 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