Sara Colombetti

Efficient Induction of Tumor Antigen–Specific CD8+ Memory T Cells by Recombinant Lentivectors

The success of active cancer immunotherapy entails a robust induction of tumor-reactive effector and memory CD8+ T cells. We compared the in vivo immunogenicity of the melanoma-associated antigen Melan-A(26-35) encoded by third-generation recombinant lentivector (rec. lv) or as peptide admixed with a strong adjuvant. Ex vivo analyses of immunized HLA-A2/H-2K(b) mice showed that rec. lv triggered a stronger anti-Melan-A CD8+ T -cell response than peptide vaccine. Importantly, the majority of anti-Melan-A T cells elicited by rec. lv expressed the memory marker CD127 at the peak of the primary response. In those mice, memory T cells were detectable several months after priming and could be activated by recall peptide vaccination. These results show that immunization with rec. lv induces not only a strong antigen-specific CD8+ T -cell response but also a long-lasting T-cell memory against a bona fide tumor-associated antigen.

IL-7 adjuvant treatment enhances long-term tumor-antigen-specific CD8+ T-cell responses after immunization with recombinant lentivector.

Immunization with recombinant lentivector elicits higher frequencies of tumor antigen-specific memory CD8+ T cells than peptide-based vaccines. This finding correlates with our observation that, upon recombinant lentivector immunization, a higher fraction of antigen-specific effector CD8+ T cells does not down-regulate the expression of the survival/memory marker interleukin-7 receptor alpha chain (IL-7Ralpha). Here we show that, surprisingly, higher expression of IL-7Ralpha on recombinant lentivector-induced effector CD8+ T cells does not result in the up-regulation of survival molecules, such as Bcl-2. We thus hypothesized that physiologic levels of IL-7 might be limiting in vivo for delivering survival signals to the expanding population of effector cells. To test this hypothesis, we administered recombinant IL-7 during the effector phase of the response. We observed an up-regulation of Bcl-2 and a strong expansion of antigen-specific effector CD8+ T cells, and of naive CD8+ T cells. Strikingly, IL-7 treatment elicited also a significant increase in the number of antigen-specific memory CD8+ T cells in recombinant lentivector-immunized mice, but not in peptide-immunized mice. Altogether, these data show that IL-7 adjuvant treatment can enhance long-term antigen-specific CD8+ T-cell responses. However, its efficacy depends on the expression of IL-7Ralpha at the surface of effector CD8+ T cells.

Lentivector immunization induces tumor antigen‐specific B and T cell responses in vivo

Expression of the cancer/germ‐line antigen NY‐ESO‐1 by tumors elicits spontaneous humoral and cellular immune responses in some cancer patients. Development of vaccines capable of stimulating such comprehensive immune responses is desirable. We have produced recombinant lentivectors directing the intracellular synthesis of NY‐ESO‐1 (rLV/ESO) and have analyzed the in vivo immune response elicited by this vector. Single injection of rLV/ESO into HLA‐A2‐transgenic mice elicited long‐lasting Bu2004and T cell responses against NY‐ESO‐1. CD8+ T cells against the HLA‐A2‐restricted peptide NY‐ESO‐1157–165 were readily detectable ex vivo and showed restricted TCR Vβ usage. Moreover, rLV/ESO elicited a far greater anti‐NY‐ESO‐1157–165 CD8+ T cell response than peptide‐ or protein‐based vaccines. Anti‐NY‐ESO‐1 antibodies were rapidly induced after immunization and their detection preceded that of the antigen‐specific CD8+ T cells. The rLV/ESO also induced CD4+ T cells. These cells played an essential role as their depletion completely abrogated B cell and CD8+ T cell responses against NY‐ESO‐1. The induced CD4+ T cells were primarily directed against a single NY‐ESO‐1 epitope spanning amino acidsu200481–100. Altogether, our study shows that rLV/ESO induces potent and comprehensive immune responses in vivo.

In Vivo Fluorescence Imaging of the Activity of CEA TCB, a Novel T-Cell Bispecific Antibody, Reveals Highly Specific Tumor Targeting and Fast Induction of T-Cell-Mediated Tumor Killing.

Purpose: CEA TCB (RG7802, RO6958688) is a novel T-cell bispecific antibody, engaging CD3ϵ upon binding to carcinoembryonic antigen (CEA) on tumor cells. Containing an engineered Fc region, conferring an extended blood half-life while preventing side effects due to activation of innate effector cells, CEA TCB potently induces tumor lysis in mouse tumors. Here we aimed to characterize the pharmacokinetic profile, the biodistribution, and the mode of action of CEA TCB by combining in vitro and in vivo fluorescence imaging readouts. Experimental Design: CEA-expressing tumor cells (LS174T) and human peripheral blood mononuclear cells (PBMC) were cocultured in vitro or cografted into immunocompromised mice. Fluorescence reflectance imaging and intravital 2-photon (2P) microscopy were employed to analyze in vivo tumor targeting while in vitro confocal and intravital time-lapse imaging were used to assess the mode of action of CEA TCB. Results: Fluorescence reflectance imaging revealed increased ratios of extravascular to vascular fluorescence signals in tumors after treatment with CEA TCB compared with control antibody, suggesting specific targeting, which was confirmed by intravital microscopy. Confocal and intravital 2P microscopy showed CEA TCB to accelerate T-cell–dependent tumor cell lysis by inducing a local increase of effector to tumor cell ratios and stable crosslinking of multiple T cells to individual tumor cells. Conclusions: Using optical imaging, we demonstrate specific tumor targeting and characterize the mode of CEA TCB–mediated target cell lysis in a mouse tumor model, which supports further clinical evaluation of CEA TCB. Clin Cancer Res; 22(17); 4417–27. ©2016 AACR. See related commentary by Teijeira et al., p. 4277

Promises and Limitations of Murine Models in the Development of Anticancer T-Cell Vaccines

Murine models have been instrumental in defining the basic mechanisms of antitumor immunity. Most of these mechanisms have since been shown to operate in humans as well. Based on these similarities, active vaccination strategies aimed at eliciting antitumor T-cell responses have been elaborated and successfully implemented in various mouse models. However, the results of human antitumor vaccination trials have been rather disappointing thus far. This review summarizes the different experimental approaches used in mice to induce antitumor T-cell responses and identifies some critical parameters that should be considered when evaluating results from murine models.

Application of a MABEL Approach for a T-Cell-Bispecific Monoclonal Antibody: CEA TCB.

CEA TCB is a novel T-cell-bispecific (TCB) antibody targeting the carcinoembryonic antigen (CEA) expressed on tumor cells and the CD3 epsilon chain (CD3e) present on T cells, which is currently in Phase 1 clinical trials (NCT02324257) for the treatment of CEA-positive solid tumors. Because the human CEA (hCEA) binder of CEA TCB does not cross-react with cynomolgus monkey and CEA is absent in rodents, alternative nonclinical safety evaluation approaches were considered. These included the development of a cynomolgus monkey cross-reactive homologous (surrogate) antibody (cyCEA TCB) for its evaluation in cynomolgus monkey and the development of double-transgenic mice, expressing hCEA and human CD3e (hCEA/hCD3e Tg), as a potential alternative species for nonclinical safety studies. However, a battery of nonclinical in vitro/ex vivo experiments demonstrated that neither of the previous approaches provided a suitable and pharmacologically relevant model to assess the safety of CEA TCB. Therefore, an alternative approach, a minimum anticipated biological effect level (MABEL), based on an in vitro tumor lysis assay was used to determine the starting dose for the first-in-human study. Using the most conservative approach to the MABEL assessment, a dose of 52 &mgr;g was selected as a safe starting dose for clinical study.

Breast cancer suppressor candidate-1 (BCSC-1) is a melanoma tumor suppressor that down regulates MITF

Understanding the molecular aberrations involved in the development and progression of metastatic melanoma (MM) is essential for a better diagnosis and targeted therapy. We identified breast cancer suppressor candidate‐1 (BCSC‐1) as a novel tumor suppressor in melanoma. BCSC‐1 expression is decreased in human MM, and its ectopic expression in MM–derived cell lines blocks tumor formation in vivo and melanoma cell proliferation in vitro while increasing cell migration. We demonstrate that BCSC‐1 binds to Sox10, which down regulates MITF, and results in a switch of melanoma cells from a proliferative to a migratory phenotype. In conclusion, we have identified BCSC‐1 as a tumor suppressor in melanoma and as a novel regulator of the MITF pathway.