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EBV infection is associated with a number of malignancies of clinical unmet need, including Hodgkin lymphoma, nasopharyngeal carcinoma, gastric cancer, and posttransplant lymphoproliferative disease (PTLD), all of which express the EBV protein latent membrane protein 2A (LMP2A), an antigen that is difficult to target by conventional antibody approaches. To overcome this, we utilized phage display technology and a structure-guided selection strategy to generate human T cell receptor–like (TCR-like) monoclonal antibodies with exquisite specificity for the LMP2A-derived nonamer peptide, C426LGGLLTMV434 (CLG), as presented on HLA-A*02:01. Our lead construct, clone 38, closely mimics the native binding mode of a TCR, recognizing residues at position P3–P8 of the CLG peptide. To enhance antitumor potency, we constructed dimeric T cell engaging bispecific antibodies (DiBsAb) of clone 38 and an affinity-matured version clone 38-2. Both DiBsAb showed potent antitumor properties in vitro and in immunodeficient mice implanted with EBV transformed B lymphoblastoid cell lines and human T cell effectors. Clone 38 DiBsAb showed a stronger safety profile compared with its affinity-matured variant, with no activity against EBV– tumor cell lines and a panel of normal tissues, and was less cross-reactive against HLA-A*02:01 cells pulsed with a panel of CLG-like peptides predicted from a proteomic analysis. Clone 38 was also shown to recognize the CLG peptide on other HLA-A*02 suballeles, including HLA-A*02:02, HLA-A*02:04, and HLA-A*02:06, allowing for its potential use in additional populations. Clone 38 DiBsAb is a lead candidate to treat EBV malignancies with one of the strongest safety profiles documented for TCR-like mAbs.

Ability to cross-present exogenous antigens in the human leukocyte antigen class I pathway is key to the antigen presenting function of mature tumor cell-loaded dendritic cells (DC). Conditions of DC maturation have been shown to be important for DCs ability to produce proinflammatory cytokines and induce T cell effector functions. However, it remains unknown if the different pathways of maturation are associated with modulation of the ability of mature DCs to cross-present tumor antigens (TA). Here, we compare DC matured with 3 clinically relevant cytokine combinations including interleukin (IL)-1 beta, tumor necrosis factor-alpha, IL-6 (termed DC-0), DC-0 cells incubated with prostaglandin-2 (termed DC-0+prostaglandin-2), or DC treated with interferon-gamma, interferon-alpha, tumor necrosis factor-alpha, Poly I:C, and IL1-beta (termed DC-1). We found that these DC vary in their ability to cross-present TA to cytotoxic T lymphocytes (CTL), with the DC-1 cytokine combination being significantly more effective than the other 2. TA cross presentation and CTL priming were strongly correlated with level of expression of the antigen processing machinery components, TAP1 and TAP2, indicating that these components could be used as biomarkers to standardize DC preparations for optimal function. However, the up-regulation of TAP1/TAP2 was not sufficient to explain the enhanced cross-presentation ability of DC-1 cells, as the use of IFN-gamma alone to up-regulate TAP1/TAP2 did not generate DC as effective at cross-presentation as the full DC-1 maturation cytokine combination. These data indicate for the first time that the pathways of DC maturation modulate antigen processing machinery component expression to different extents and that differently matured DC vary in the ability to cross-present TA to human leukocyte antigen class I-restricted CTL.

T-cell-based therapies have emerged as one of the most clinically effective ways to target solid and non-solid tumors. HER2 is responsible for the oncogenesis and treatment resistance of several human solid tumors. As a member of the HER family of tyrosine kinase receptors, its over-activity confers unfavorable clinical outcome. Targeted therapies directed at this receptor have achieved responses, although development of resistance is common. We explored a novel HER2/CD3 bispecific antibody (HER2-BsAb) platform that while preserving the anti-proliferative effects of trastuzumab, it recruits and activates non-specific circulating T-cells, promoting T cell tumor infiltration and ablating HER2(+) tumors, even when these are resistant to standard HER2-targeted therapies. Its in vitro tumor cytotoxicity, when expressed as EC50, correlated with the surface HER2 expression in a large panel of human tumor cell lines, irrespective of lineage or tumor type. HER2-BsAb-mediated cytotoxicity was relatively insensitive to PD-1/PD-L1 immune checkpoint inhibition. In four separate humanized mouse models of human breast cancer and ovarian cancer cell line xenografts, as well as human breast cancer and gastric cancer patient-derived xenografts (PDXs), HER2-BsAb was highly effective in promoting T cell infiltration and suppressing tumor growth when used in the presence of human peripheral blood mononuclear cells (PBMC) or activated T cells (ATC). The in vivo and in vitro antitumor properties of this BsAb support its further clinical development as a cancer immunotherapeutic.

Problem: Development of adjuvant cancer immunotherapy in head and neck squamous cell carcinoma (HNSCC). Methods: The MAGE gene family, collectively referred to as cancer-testis antigens to describe their restricted expression pattern in tumor tissues, have been successfully targeted through immunotherapy of melanoma. Expression of MAGE-3 and -6 have been documented in up to 60% of HNSCC tumors, but a quantitative evaluation of expression and T cell immunogenicity of these proteins in HNSCC have not been performed. Further, the determinants of MAGE expression in primary and metastatic HNSCC cells has not been specifically addressed. Results: Frozen HNSCC lesions (25 primary tumors and 17 cervical metastases) were microdissected from cryostat tissue sections. Using a novel quantitative RT-PCR (qRT-PCR) assay, we evaluated the expression of MAGE-3 and -6, as well as HLA A2.1, in these tissues and in 10 HNSCC cell lines. T cell recognition of the HLA A2.1-restricted MAGE-3271-279 peptide was studied in HNSCC cell lines using the Interferon-g ELISPOT. Conclusion: MAGE-3 or -6 is expressed frequently and at variable levels in HNSCC tumors and was detected in over 50% of HNSCC cell lines and tissues in our study. Importantly, expression was retained at high levels in metastatic tumor-positive nodes. CD8+ T cells specific for a cross-reactive MAGE-3271-279 peptide have detectable T cell reactivity against MAGE-positive HNSCC cell lines but not against MAGE-negative cells. MAGE-3271-279 loaded tetrameric HLA A2.1 molecules can be used to quantify these T cells in the peripheral blood of HNSCC patients. MAGE3 also appears to be a potentially useful marker of metastatic tumor deposits in cervical nodes. Significance: MAGE-3 and -6 appear to be promising targets for T cell immunotherapy of cancer patients, including those with HNSCC. Our study relates quantitative expression levels of these tumor antigens with their potential as immunotherapeutic targets, using functional assays and strategies for enhancing expression of MAGE gene expression in tumor cells. Support: FAMRI Clinical Investigator Award (RLF)