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Micromet AG and Medlmmune Inc are developing MT-103, a single-chain bispecific recombinant antibody from Micromet's BiTE (bispecific T-cell engager) product platform that binds both the CD19 antigen and the T-cell receptor (CD3), for the potential treatment of B-cell lymphoma. The company is also investigating the compound for the potential treatment of chronic lymphocytic leukemia and acute lymphoblastic leukemia.

Adoptive immunotherapy with tumour-specific T cells is an emerging technology that may be applicable to a broad range of cancers. However, tumours can avoid T cell-mediated attack through multiple mechanisms including downregulation of major histocompatability complex (MHC). Consequently, engineering T cells to target intact protein antigen directly, thus bypassing the need for MHC presentation, can facilitate T cell targeting of tumour cells. Peripheral blood lymphocytes from nine of nine patients with non-Hodgkin lymphoma (NHL) were successfully gene-modified to express a receptor consisting of a CD19 single chain variable fragment (scFv) fused to the T cell CD3zeta signalling molecule. These T cells were functionally active against the CD19(+) Raji Burkitt's lymphoma cell line. Importantly, engineered T cells from seven of nine NHL patients efficiently lysed autologous lymph node tumour biopsy cells. There was a clear correlation between levels of CD19 expression on the tumour and effective killing by the engineered T cells. For two patients with a low or absent CD19(+) cells within the biopsy, no significant killing was observed. These results demonstrate that patients with CD19(+) NHL would be suitable candidates for this form of therapy in the setting of a phase I clinical trial.

Mycobacteria expressing recombinant antigens are already being developed as vaccines against both infections and tumors. Little is known about how dendritic cells might process such antigens. Two different mycobacterial species, the fast-growingMycobacterium smegmatisand the slow-growingM. bovis M. bovisBCG, were engineered to express a model tumor antigen, the Kb-restricted dominant cytotoxic T-lymphocyte epitope OVA257-264. RecombinantM. bovisBCG but not recombinantM. smegmatisconferred protection to mice challenged with the B16-OVA tumor cell line. We went on to investigate whether the contrast in antitumor efficacy could be due to differences in how dendritic cells process antigen from the two mycobacterial strains for class I presentation. Both strains of mycobacteria caused phenotypic maturation of dendritic cells, but recombinantM. smegmatisinfection led to a greater degree of dendritic cell maturation than recombinantM. bovisBCG infection. Antigen from recombinantM. smegmatiswas processed and presented as OVA257-264on Kbmolecules by the dendritic cell line DC2.4 but not by bone marrow-derived dendritic cells (BMDC) or splenic dendritic cells. In contrast, antigen from recombinantM. bovisBCG was presented by all three dendritic cell types as long as the mycobacteria were viable. Such presentation was dependent on proteasome function and nascent major histocompatibility complex (MHC) class I molecules in DC2.4 cells but independent of the proteasome and transporter associated with antigen processings (TAP) in BMDC and splenic dendritic cells. These data demonstrate for the first time that antigen vectored by the slow-growingM. bovisBCG but not that vectored by fast-growing, readily destroyedM. smegmatisis processed and presented on MHC class I by in vitro-generated dendritic cells, which has implications for recombinant microbial vaccine development.

Mycobacteria are potent adjuvants, can survive intracellularly and have been safely used for many years as vaccines against tuberculosis and leprosy. They are thus important potential vectors for recombinant vaccines. Many of their adjuvant properties are mediated following phagocytosis by dendritic cells (DC), which are in turn critical for priming naive T cells. Although the maturation of DC in response to mycobacteria, such as Mycobacterium bovis bacillus Calmette-Guerin (BCG), is well described the subsequent responses of autologous T cells to mycobacterium-infected DC remains uncharacterized. In our experiments DC infected with BCG expressed more co-stimulatory molecules than tumour-necrosis factor-alpha (TNF-alpha) -treated DC and stimulated more potent mixed leucocyte reactions. When autologous T cells were co-cultured with BCG-exposed DC they became highly activated, as determined by display of CD25, CD54 and CD71 on both CD4+ and CD8+ cells. In contrast, the response of T cells to TNF-alpha-matured DC was significantly less. Cytokine production from T cells cultured with BCG-exposed DC was enhanced with elevated secretion of interleukin-2 (IL-2), IL-10 and interferon-gamma (IFN-gamma) and was produced by both CD4+ and CD8+ lymphocytes as determined by intracellular staining. In particular, IFN-gamma secretion was increased from 50 pg/ml to 25 000 pg/ml and IL-10 secretion increased from 20 pg/ml to 300 pg/ml in BCG-exposed DC co-cultures. Blocking antibodies to B7.1 and B7.2 or IL-12 significantly reduced the secretion of IFN-gamma and reductions were also seen in the expression of CD25 and CD71 by CD4+ cells. These data demonstrate that mycobacterially infected DC are particularly potent activators of autologous T cells compared to TNF-alpha-exposed DC and that the resultant T cells are functionally superior.

Radiation therapy (RT) is administered to around 50% of all cancer patients making it one of the most important cancer treatments. In addition to the direct cytoreductive effect of RT there is increasing evidence that radiation-induces immunogenic tumor cell death. Despite the immunogenicity of RT-induced tumor cell death, RT delivered to tumours in the clinic rarely generates therapeutic systemic anti-cancer immune responses or ‘abscopal effects’. Here we show that tumor infiltrating CD8+ cytotoxic T lymphocytes (CTL) have increased expression of PD-1 following RT in vivo. Moreover, our data demonstrate that treatment of established syngeneic tumors with RT leads to upregulation of tumor cell expression of PD-L1 in vivo but not when cells are irradiated in vitro. Using depleting antibodies we determined that the depletion of CD8+ T cells but not CD4+ T cells or NK cells could abrogate this RT-induced increase in tumor cell expression of PD-L1 in vivo. Furthermore, silencing of IFNγR1 using ShRNA confirmed that this process was dependent on CD8+ T cell production of IFNγ suggesting an adaptive upregulation of PD-L1 following RT occurs in response to CTL activation. This novel finding suggests that the immunogenicity of RT may be limited via the PD-L1/PD-1 signalling axis and may contribute to treatment failure. We next sought to determine whether blockade of the PD-1/PD-L1 signalling axis could enhance the therapeutic response to RT. Our study demonstrates that administration of either an anti-PD-1 or anti-PD-L1 mAb in combination with RT leads to substantially improved survival when compared to either monotherapy alone with approximately 60% of treated mice undergoing a complete response. In addition, our data reveal that combination therapy generates long-term immunological memory in mice that have undergone complete response protecting against tumor rechallenge. This is the first report to our knowledge that demonstrates adaptive resistance through the upregulation of tumour cell PD-L1 expression in response to an RT-induced CTL response. This study demonstrates the potential for enhancing the efficacy of conventional RT through blockade of the PD-1/PD-L1 axis and warrants clinical evaluation. Citation Format: Simon J. Dovedi, Graznya Lipowska-Bhalla, Eleanor Cheadle, Edmund Poon, Michelle Morrow, Ross Stewart, Robert Wilkinson, Jamie Honeychurch, Timothy Illidge. The antitumor immune response generated by radiation therapy may be limited by tumor cell adaptive resistance and can be circumvented by PD-L1 blockade. [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 5034. doi:10.1158/1538-7445.AM2014-5034

The success of monoclonal antibodies in the treatment of certain cancers demonstrates that immune based therapies can work and are particularly effective in B cell malignancies. However, tumours can still avoid antibody mediate mechanisms of attack and there is currently no estalbished method of effectively recruiting T cells to harness their potential anti-tumour effects. We are exploring gene therapy approaches to endow T cells with antibody type specificity in order to more efficiently target and lyse tumours and thereby improve the overall immune therapy of cancer T cells grafted with a CD19 specific receptor consisting of a CD19 scFv linked to human CD3zeta (CD19z) were tested for their potency against B cell lymphoma lines in vivo. T cells were engineered using retroviral vestors to possess a CD19 specific receptor which endows the T cells with specificity for B cell lymphoma. The vector incorporates a truncated hCD34 gene as a marker to facilitate assessment of transduced cells using as clinically applicable, non-immunogenic marker gene. Mice bearing B cell lymphoma were treated with a systemic infusion of targeted T cells with or without non-myeloablative chemotherapy. Human T cells targeting CD19 cured 40% of SCID/beige mice with 6 day established metastatic tumour but only in conjunction with a single dose of cyclophosphamide. Murine T cells expressing the CD19z receptor were also effective with cure of 24hr established s.c human CD19+ tumour in SCID/beige and immunocompetent mice. Pretreatment with cyclophosphamide did not affect T cell engraftment or efficacy in immuno-compromised animals but was necessary for T cell engraftment in immuno-competent animals. These results which parallel the approach successfully used with tumour infiltrating lymhocytes in melanoma patients conclusively demonstrate that the combination of engineered T cells with “pre-conditioning” chemotherapy significantly impacts upon tumour growth in vivo and this evidence supports the development of phase I clinical trials targeting B cell lymphoma.

Abstract 1651 Background: Anti-CD20 monoclonal antibodies (mAbs), most notably rituximab, have revolutionized the treatment of B-cell malignancies with substantially improved clinical outcome for patients. However, a proportion of patients still relapse and become refractory to rituximab. Therefore, several next-generation mAbs are being developed to improve responses further and provide novel therapies for refractory patients. In addition to classical Fc-dependent mechanisms such as antibody-dependent cellular cytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC), certain mAbs can eliminate target cells by triggering intracellular signaling upon antigen ligation to directly induce programmed cell death (PCD). The role of direct PCD and its underlying mechanisms remain under-investigated and poorly understood. We recently demonstrated that certain mAbs (specifically type II anti-CD20 and anti-HLA DR mAbs) potently evoked PCD which was dependent on homotypic adhesion and the rearrangement of the actin cytoskeleton, which in turn triggered lysosome membrane permeabilization (LMP) and cathepsin-mediated cell death (Ivanov et al J Clin Invest 2009, Alduaij et al Blood 2011). Here, we further probe the mechanisms involved, specifically investigating the contribution of reactive oxygen species (ROS). Methods: The generation of ROS was detected using dihydroethidium (HE) and 5-(and-6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA) monitored using flow cytometry and fluorescence microscopy. Cell death was quantified using propidium iodide (PI) and annexin V staining, and Chromium-51 release assays. For primary B-cell chronic lymphocytic leukemia (B-CLL) cells, cell death was detected using Annexin V-Cy5.5 and 7-aminoactinomycin. Mitochondrial depolarization was monitored using the JC-1 dye. Results: The extent of ROS generated by a range of mAbs, including type II anti-CD20 mAbs (tositumomab and GA101) and anti-HLA DR mAbs (L243 and 1D10/apolizumab), was positively correlated with their ability to induce PCD, in human B-lymphoma cell lines and primary B-CLL cells. The ROS scavengers tiron and tempol abrogated mAb-induced PCD indicating that ROS are required for its execution. ROS was found to be generated downstream of the mAb-induced actin cytoskeletal reorganization and LMP. Although GA101 potently induced mitochondrial membrane permeabilization, the ability of mitochondrial respiration-deficient Raji subclones to efficiently produce ROS and undergo cell death upon mAb treatment was unaffected, strongly suggesting that mitochondria are not essential for mAb-induced ROS production and cell death. Instead, ROS generation and PCD were blocked by inhibitors of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases and reduced by siRNA-mediated knock down of the NADPH oxidase NOX2. Conclusion: These findings provide further novel insights into the non-apoptotic PCD pathway evoked by mAbs in B-cell malignancies, highlighting a previously unrecognized role for NADPH oxidase-derived ROS in the terminal effector phase of the death pathway. This newly characterized cell death pathway may be exploited to eliminate malignant lymphoid cells which are refractory to conventional chemotherapy and immunotherapy. Disclosures: No relevant conflicts of interest to declare.

Abstract 3245 Whilst modern treatment approaches cure a high number of patients with acute lymphoblastic leukemia (ALL), little progress has been made in the treatment of refractory and relapsed ALL and new treatment approaches are needed. We recently demonstrated that anti-HLA-DR class II monoclonal antibody (mAb) L243 induces a novel non-apoptotic mode of cell death in B-cell lymphoma lines, defined by homotypic adhesion (HA), actin reorganisation and lysosomal activity (Ivanov et al. J Clin Invest, 2009). Here, we extend these important observations and examine whether this novel form of mAb induced cell death occurs in pre-B ALL cell lines. Expression of HLA-DR was determined using flow cytometry in a panel of ALL cell lines (REH, SupB15 and SD1). HLA-DR was expressed at high levels on each of the cell lines. The ability of L243 to induce HA and cell death (Annexin V/PI positivity) was assessed using microscopy and flow cytometry respectively. L243 was able to evoke both strong HA and cell death in all of the ALL cell lines (e.g. in SupB15 cells 46±1.7% death versus 7±0.5% in controls, p These findings demonstrate that anti-HLA DR mAb L243 induces a novel model of cell death in ALL cell lines that is independent of caspase activation and dependent on actin reorganization. This data suggests that this novel mAb induced death pathway is independent of apoptosis and potentially exploitable in the clinic in leukemias resistant to chemotherapy apoptosis induction. Disclosures: No relevant conflicts of interest to declare.