Zhinan Xia

Vaccination of Cancer Patients Against Telomerase Induces Functional Antitumor CD8+ T Lymphocytes

Purpose: High-level expression of the telomerase reverse transcriptase (hTERT) in >85% of human cancers, in contrast with its restricted expression in normal adult tissues, points to hTERT as a broadly applicable molecular target for anticancer immunotherapy. CTLs recognize peptides derived from hTERT and kill hTERT+ tumor cells of multiple histologies in vitro. Moreover, because survival of hTERT+ tumor cells requires functionally active telomerase, hTERT mutation or loss as a means of escape may be incompatible with sustained tumor growth. Experimental Design: A Phase I clinical trial was performed to evaluate the clinical and immunological impact of vaccinating advanced cancer patients with the HLA-A2-restricted hTERT I540 peptide presented with keyhole limpet hemocyanin by ex vivo generated autologous dendritic cells. Results: As measured by peptide/MHC tetramer, enzyme-linked immunospot, and cytotoxicity assays, hTERT-specific T lymphocytes were induced in 4 of 7 patients with advanced breast or prostate carcinoma after vaccination with dendritic cells pulsed with hTERT peptide. Tetramer-guided high-speed sorting and polyclonal expansion achieved highly enriched populations of hTERT-specific cells that killed tumor cells in an MHC- restricted fashion. Despite concerns of telomerase activity in rare normal cells, no significant toxicity was observed. Partial tumor regression in 1 patient was associated with the induction of CD8+ tumor infiltrating lymphocytes. Conclusions: These results demonstrate the immunological feasibility of vaccinating patients against telomerase and provide rationale for targeting self-antigens with critical roles in oncogenesis.

Phase I/II combined chemoimmunotherapy with carcinoembryonic antigen-derived HLA-A2-restricted CAP-1 peptide and irinotecan, 5-fluorouracil, and leucovorin in patients with primary metastatic colorectal cancer

Purpose: We conducted a phase I/II randomized trial to evaluate the clinical and immunologic effect of chemotherapy combined with vaccination in primary metastatic colorectal cancer patients with a carcinoembryonic antigen–derived peptide in the setting of adjuvants granulocyte macrophage colony-stimulating factor, CpG-containing DNA molecules (dSLIM), and dendritic cells. Experimental Design: HLA-A2–positive patients with confirmed newly diagnosed metastatic colorectal cancer and elevated serum carcinoembryonic antigen (CEA) were randomized to receive three cycles of standard chemotherapy (irinotecan/high-dose 5-fluorouracil/leucovorin) and vaccinations with CEA-derived CAP-1 peptide admixed with different adjuvants [CAP-1/granulocyte macrophage colony-stimulating factor/interleukin-2 (IL-2), CAP-1/dSLIM/IL-2, and CAP-1/IL-2]. After completion of chemotherapy, patients received weekly vaccinations until progression of disease. Immune assessment was done at baseline and after three cycles of combined chemoimmunotherapy. HLA-A2 tetramers complexed with the peptides CAP-1, human T-cell lymphotrophic virus type I TAX, cytomegalovirus (CMV) pp65, and EBV BMLF-1 were used for phenotypic immune assessment. IFN-γ intracellular cytokine assays were done to evaluate CTL reactivity. Results: Seventeen metastatic patients were recruited, of whom 12 completed three cycles. Therapy resulted in five complete response, one partial response, five stable disease, and six progressive disease. Six grade 1 local skin reactions and one mild systemic reaction to vaccination treatment were observed. Overall survival after a median observation time of 29 months was 17 months with a survival rate of 35% (6 of 17) at that time. Eight patients (47%) showed elevation of CAP-1–specific CTLs. Neither of the adjuvants provided superiority in eliciting CAP-1–specific immune responses. During three cycles of chemotherapy, EBV/CMV recall antigen–specific CD8+ cells decreased by an average 14%. Conclusions: The presented chemoimmunotherapy is a feasible and safe combination therapy with clinical and immunologic efficacy. Despite concurrent chemotherapy, increases in CAP-1–specific T cells were observed in 47% of patients after vaccination.

Viral antigen-specific CD8+ T-cell responses are impaired in multiple myeloma

Summary. Multiple myeloma (MM) is associated with defects of humoral and cellular immunity, however, little is known about the frequency and function of antigen‐specific CD8+ T cells. Such information might be critical for the development of immunotherapy for MM patients. As a model, we assessed the frequency and proliferation of CD8+ T cells specific for HLA‐A*0201‐restricted immunodominant epitopes from influenza A (Inf A) and Epstein–Barr virus (EBV). Experiments in identical twins demonstrated reduced numbers of antigen‐specific T cells after ex‐vivo antigenic challenge in the MM twin when compared with the healthy twin. Similarly, the proliferation and frequency of EBV‐ and Inf A‐specific T cells was also significantly reduced in a cohort of 24 previously untreated or conventionally treated MM patients when compared with 19 healthy individuals. In contrast, MM patients studied after receiving an autologous stem cell transplantation showed strikingly higher frequencies of EBV‐specific T cells with potential to proliferate ex vivo, suggesting that EBV‐specific T cells are readily expandable under these circumstances. These data identify an impaired response of CD8+ T cells in MM patients, which might in part explain the relatively limited success of anti‐MM immunisations. Prospective studies will determine whether such immune assessment strategies may identify patients more likely to benefit from cancer immunotherapy.

Efficient Presentation of Naturally Processed HLA Class I Peptides by Artificial Antigen-Presenting Cells for the Generation of Effective Antitumor Responses

Appropriate presentation of tumor-associated antigens (TAA) by antigen-presenting cells (APC) is required for the development of clinically relevant antitumor T-cell responses. One common approach, which uses APC pulsed with synthetic peptides, can sometimes generate ineffective immune responses. This failure may, in part, be attributed to the formation of HLA/synthetic pulsed peptide complexes that possess different conformations compared with those of endogenously presented peptides. In addition, endogenous peptides may undergo post-translational modifications, which do not occur with synthetic peptides. Because our goal is to induce immunity that can recognize TAA that are endogenously presented by tumors, we designed an APC that would not only express the required immunoaccessory molecules but also naturally process and present target antigenic peptides. In this study, we generated an artificial APC (aAPC) that can endogenously present any chosen HLA-A*0201 (A2)–restricted peptide by processing a fusion protein that contains a unique “LTK” sequence linked to the antigenic peptide. Proteasome-dependent processing is so effective that the presented peptide can be directly eluted from the cell surface and identified by biochemical methods. Furthermore, we found that aAPC, engineered to endogenously present peptide derived from the melanoma antigen MART1, can be used to prime and expand antitumor CTL that target MART1-expressing tumor cells in a HLA-A2-restricted manner. Our engineered aAPC could serve as an “off-the-shelf” APC designed to constitutively express class I–restricted TAA peptides and could be used to generate effective T-cell responses to treat human disease.

Gene Expression Profiling Identifies BAX-δ as a Novel Tumor Antigen in Acute Lymphoblastic Leukemia

The identification of new tumor-associated antigens (TAA) is critical for the development of effective immunotherapeutic strategies, particularly in diseases like B-cell acute lymphoblastic leukemia (B-ALL), where few target epitopes are known. To accelerate the identification of novel TAA in B-ALL, we used a combination of expression profiling and reverse immunology. We compared gene expression profiles of primary B-ALL cells with their normal counterparts, B-cell precursors. Genes differentially expressed by B-ALL cells included many previously identified as TAA in other malignancies. Within this set of overexpressed genes, we focused on those that may be functionally important to the cancer cell. The apoptosis-related molecule, BAX, was highly correlated with the ALL class distinction. Therefore, we evaluated BAX and its isoforms as potential TAA. Peptides from the isoform BAX-δ bound with high affinity to HLA-A*0201 and HLA-DR1. CD8+ CTLs specific for BAX-δ epitopes or their heteroclitic peptides could be expanded from normal donors. BAX-δ–specific T cells lysed peptide-pulsed targets and BAX-δ–expressing leukemia cells in a MHC-restricted fashion. Moreover, primary B-ALL cells were recognized by BAX-δ–specific CTL, indicating that this antigen is naturally processed and presented by tumor cells. This study suggests that ( a ) BAX-δ may serve as a widely expressed TAA in B-ALL and ( b ) gene expression profiling can be a generalizable tool to identify immunologic targets for cancer immunotherapy.