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The progress in the isolation and characterization of tumor antigen (TA)-specific T lymphocytes and in the genetic modification of immune cells allowed the clinical development of adoptive cell therapy (ACT). Several clinical studies highlighted the striking clinical activity of T cells engineered to express either Chimeric Antigen (CAR) or T Cell (TCR) Receptors to target molecularly defined antigens expressed on tumor cells. The breakthrough of immunotherapy is represented by the approval of CAR-T cells specific for advanced or refractory CD19+ B cell malignancies by both the Food and Drug Administration (FDA) and the European Medicinal Agency (EMA). Moreover, advances in the manufacturing and gene editing of engineered immune cells contributed to the selection of drug products with desired phenotype, refined specificity and decreased toxicity. An important step toward the optimization of CAR-T cell therapy is the development of “off-the shelf” T cell products that allow to reduce the complexity and the costs of the manufacturing and to render these drugs available for a broad number of cancer patients. The Engineered Immune Cells in Cancer Immunotherapy (EICCI) workshop hosted in Doha, Qatar, renowned experts, from both academia and industry, to present and discuss the progress on both pre-clinical and clinical development of genetically modified immune cells, including advances in the “off-the-shelf” manufacturing. These experts have addressed also organizational needs and hurdles for the clinical grade production and application of these biological drugs., Qatar National Research Fund (QNRF)-Conference Workshop Sponsorship Program CWSP15-W-0911-18001, Associazione Italiana per la Ricerca sul Cancro (AIRC) Ig 18458, Associazione Italiana per la Ricerca sul Cancro (AIRC) 22737, Ministry of Education, Universities and Research (MIUR) PRIN 2017WC8499, Italian Ministry of Health (Research project on CAR T cells for hematological malignancies and solid tumors)

Naturally processed peptides, obtained by acid extraction of tumor cells, contain Ags able to activate specific CTL in vitro. We recently reported that the nonprofessional APC, RMA-S, expressing the B7.1 molecule (RMA-S/B7), pulsed with naturally processed peptides from the nonimmunogenic B16F1 melanoma (B16F1a.e.) primed syngenic CD8+ T cells against the tumor in vitro. Here, we show the rejection of B16F1 melanoma by C57BL/6 mice after immunization with RMA-S/B7 cells pulsed with B16F1a.e. This response is critically dependent on both CD4+ and CD8+ cells, but not on NK cells. However, only CD8+ T cells exert anti-B16F1 cytolitic activity in vitro. Moreover, RMA-S/B7 cells pulsed with B16F1a.e. can be used to prevent the growth of 24-h preestablished melanomas. These results may have important implications for the clinical use of natural peptide fractions of tumor cells as therapeutic cancer vaccines.

The clinical use of tumor necrosis factor alpha (TNF) as an anticancer drug is limited to local or locoregional administration because of dose-limiting systemic toxicity. We investigated in animal models whether the therapeutic index of systemically administered human or murine TNF can be increased by tumor pretargeting strategies based on the biotin-avidin system. Pretargeting of s.c. mouse WEHI-164 fibrosarcoma and RMA lymphoma genetically engineered to express the Thy 1.1 antigen on the cell membrane was achieved by i.p. injection of a biotinylated anti-Thy 1.1 antibody and avidin. This pretreatment increased the antitumor activity of systemically administered biotin-TNF conjugates by at least 5-fold. In contrast, pretargeting did not increase the toxicity of biotin-TNF, as judged by animal survival and weight loss after treatment. Ex vivo analysis of tumor cells 24 h after treatment showed that biotin-TNF persisted for several hours on the surface of pretargeted tumors, but not when avidin was omitted. The potentiation of the antitumor effects was related primarily to indirect mechanisms, involving a host-mediated response. The results indicate that tumor pretargeting improves the antitumor activity of TNF. Tumor pretargeting with avidin, which is currently used to increase the uptake of radioactive-labeled biotin in patients, could represent a new strategy for improving the therapeutic index of TNF.

Infection by 'high-risk' human papillomaviruses (HPV) is associated with the development of neoplastic lesions. HPV-18 is responsible for a very aggressive form of cancer and poor survival. As for other HPV types, immune surveillance has probably a role in the control of the infection. However, very little is known on HPV-18 immunogenicity. CD4(+) T cells from 16 healthy donors were tested ex vivo for reactivity to synthetic peptides corresponding to 3 sequences on the HPV-18 E6 transforming protein predicted by bioinformatics as promiscuous HLA-DR ligands, and to the recombinant E6 protein. We found 3 donors with CD4(+) T cells that specifically proliferated in the presence of HPV-18 E6 antigens and produced IFN-gamma in the presence of the E6 protein. We then propagated CD4(+) T cell lines and clones from the responsive subjects to better characterize the recognized sequences. We show that E6(52-66) and E6(97-111) are indeed promiscuous and, most importantly, they contain naturally processed epitopes. Collectively, our data indicate that healthy donors may develop spontaneous CD4 immunity against HPV-18 E6 epitopes, thus strongly suggesting the potential for this protein to elicit in the host a natural productive immune response.

We investigated the contribution of transfer of Ag-experienced donor T cells to the immune reconstitution of allogeneic bone marrow transplantation (BMT) recipients. To this purpose, we used a combination of cell culture methods to isolate tetanus toxoid (TT)-specific T cell clones, and a sensitive and specific heteroduplex analysis to monitor the presence of a particular clonotype using TCR N region sequences. We document that patients after BMT display a small response to TT, entirely accounted for by few donor-derived clones. These patients show a strong polyclonal response to TT vaccination; however, the T cell clones transferred with the transplant can still be detected within the polyclonal T cell lines for up to at least 5 yr after BMT. We also demonstrate that vaccination of donors with TT before BMT results in a more relevant transfer of Ag-experienced T cells, allowing the recipients to mount a strong polyclonal response without need of vaccination. These findings provide a rationale for vaccinating donors to optimize adoptive transfer of protective T cell immunity into recipients, and suggest the possibility of using preventive T cell adoptive therapy in conjunction with marrow infusion.

Among 60 retrospectively assessed patients with the lymphoproliferative disease of granular lymphocytes (LDGL), lymphocytes from only 2 patients had the CD3+, CD4-, CD8- phenotype, rarely observed in normal peripheral blood lymphocytes (about 3%). In this paper we report a detailed study of lymphocytes isolated from these two patients. The cells from patients 1 had the CD3+, CD4-, CD8-, WT31-, beta F1-, TCR delta 1+, Ti gamma A-, BB3+, CD7+, CD16-, CD57+ phenotype, while cells from patient 2 had a phenotype even more rarely observed on normal lymphocytes: CD3+, CD4-, CD8-, WT31+, beta F1+, TCR delta 1-, CD7+, CD16-, CD57+. Thus, in only the first case the cells expressed the gamma/delta T-cell receptor (TCR) on the membrane, while the cells from the second case had the alpha/beta TCR. Genetic studies showed that in case 1 the TCR gamma gene was rearranged and the beta chain gene configuration was germline; the TCR mRNA was of normal size for the gamma chain, while that of the beta chain was truncated. Case 2 had the beta and the gamma genes of the TCR rearranged, but only the alpha and beta mRNA were expressed. In agreement with these findings, the delta chain gene of the TCR was rearranged in case 1 and was deleted in case 2. Cytotoxic activity was absent in cells from case 1 and low in case 2; in the latter, the lytic activity could be up-regulated following incubation with IL-2 or an anti-CD3 monoclonal antibody. Our study indicates that CD3+, CD4-, CD8- lymphocytes are rarely expanded in patients with LDGL. The detection of a lymphoproliferative disease of a CD3+, CD4-, CD8-, alpha/beta + cell may contribute to a better characterization of this novel lymphocytic subpopulation.