Walter C. Olson

A Multipeptide Vaccine is Safe and Elicits T-cell Responses in Participants With Advanced Stage Ovarian Cancer

Nine participants with epithelial ovarian, fallopian tube, or primary peritoneal carcinoma, who were human leukocyte antigen (HLA)-A1+, HLA-A2+, or HLA-A3+, were eligible to enroll in a phase 1 study designed to assess the safety and immunogenicity of a peptide-based vaccine. Participants received 5 class I major histocompatibility complex-restricted synthetic peptides derived from multiple ovarian cancer-associated proteins plus a class II major histocompatibility complex-restricted synthetic helper peptide derived from tetanus toxoid protein. The vaccines were administered with granulocyte macrophage-colony stimulating factor in Montanide ISA-51 adjuvant over a 7-week period. All vaccine-related toxicities were grade 1 to 2, the most common being injection site reaction (grade 2, 100%), fatigue (grade 1, 78%), and headache (grade 1, 67%). Lymphocytes from the peripheral blood and a node draining a secondary vaccine site (sentinel immunized node) were harvested during the course of vaccination and T-cell responses to the peptides were evaluated using an enzyme-linked immunosorbent spot assay. CD8+ T-cell responses were detected in 1 participant ex vivo and in 8 of 9 participants (89%) after in vitro stimulation. All 4 HLA-A2 and HLA-A3–restricted peptides were immunogenic. This includes 2 peptides, folate binding protein (FBP191−199) and Her-2/neu754−762, which had not previously been evaluated in vaccines in humans. Responding T cells required over 200 nM for half-maximal reactivity. These data support continued investigation of these peptides as immunogens for patients with ovarian cancer but, owing to low potency, also suggest a need for additional immunomodulation in combination with vaccines to increase the magnitude and to improve the quality of the T-cell responses.

Disease-Stage Variance in Functional CD4 T-Cell Responses Against Novel Pan-Human Leukocyte Antigen-D Region Presented Human Papillomavirus-16 E7 Epitopes

Given the anticipated clinical importance of helper and regulatory CD4+ T cells reactive against human papillomavirus-16 E7 in the cervical carcinoma setting, we performed this study to identify novel E7-derived T helper (Th) epitopes and to characterize functional anti-E7 Th responses in normal donors and patients with cervical intraepithelial neoplasia I-III or cervical cancer. Candidate pan-HLA-DR (D region) binding peptides were identified and synthesized based on results obtained using a predictive computer algorithm, then applied in short-term in vitro T-cell sensitization assays. Using IFN-γ/IL-5 (interleukin 5) enzyme-linked immunospot assays as readouts for Th1-type and Th2-type CD4+ T-cell responses, respectively, we identified three E7-derived T helper epitopes (E71–12, E748–62, and E762–75), two of which are novel. Normal donor CD4+ T cells failed to react against these E7 peptides, whereas patients with premalignant cervical intraepithelial neoplasia I-III lesions displayed preferential Th1-type responses against all three E7 epitopes. Th1-type responses were still observed to the E748–62 but not to the E71–12 and E762–75 peptides in cancer patients, where these latter two epitopes evoked Th2-type responses. Notably all responders to the E71–12 and E762–75 peptides expressed the HLA-DR4 or -DR15 alleles, whereas all responders to the E748–62 peptide failed to express the HLA-DR4 allele. Our results are consistent with a model in which cervical cancer progression is linked to an undesirable Th1- to Th2-type shift in functional CD4+ T cell responses to two novel E7-derived epitopes. These peptides may prove important in vaccines to promote and maintain protective Th1-type antihuman papillomavirus immunity and in the immune monitoring of treated patients harboring HPV-16+ malignancies.

Differentiation of immunostimulatory stem-cell- and monocyte-derived dendritic cells involves maturation of intracellular compartments responsible for antigen presentation and secretion

Dendritic cells (DCs) are important for the induction of primary T‐cell responses and may serve as “biologic adjuvants” in therapeutic protocols. However, given the “plasticity” of this antigen‐presenting cell, it remains unclear which DC type (source, subtype, and stage of differentiation) should be applied clinically. To provide additional insight in this selection process, we have, for the first time, analyzed the in vitro differentiation of CD34+ precursor‐derived and monocyte‐derived DCs for ultrastructure, phenotype, and function. The ultrastructural intracytoplasmic differentiation of DCs correlated with increasing T‐cell stimulatory activity of these cells. “Early‐stage”‐DCs proliferate, exhibit high levels of soluble antigen uptake, and moderate T‐cell stimulatory capacity, and are characterized by centrally located nuclei and numerous enlarged mitochondria. “Intermediate‐stage”‐DCs are enlarged cells with enhanced T‐cell stimulatory activity and pronounced cytoplasmic protein synthesis machinery. “Late‐stage” (LS)‐DCs exhibit a mature secretory cell phenotype and low proliferative index. They express high levels of the HLA‐DR, CD40L, B7‐1, and B7‐2 molecules and CD83, a specific marker of mature DCs, and appear maximally stimulatory to T cells. Ultrastructurally, LS‐DCs feature an accentric nucleus, an enlarged cytoplasm, containing numerous secretory storage vesicles, along with a fully developed Golgi complex. LS‐DCs exhibited numerous multivesicular and multilaminar structures containing major histocompatibility complex class II molecules, consistent with the MIIC (peptide‐loading) compartment. In extended studies, cultured CD14+ monocyte‐derived DCs displayed a similar, but accelerated, temporal differentiation staging pattern.

A Mycoplasma Peptide Elicits Heteroclitic CD4+ T Cell Responses against Tumor Antigen MAGE-A6

Purpose: Although T-helper (Th) epitopes have been previously reported for many tumor antigens, including MAGE-A6, the relevant HLA-DR alleles that present these peptides are expressed by only a minority of patients. The identification of tumor antigenic epitopes presented promiscuously by many HLA-DR alleles would extend the clinical utility of these peptides in vaccines and for the immunomonitoring of cancer patients. Experimental Design: A neural network algorithm and in vitro sensitization assays were employed to screen candidate peptides for their immunogenicity. Results: The MAGE-A6140-170, MAGE-A6172-187, and MAGE-A6280-302 epitopes were recognized by CD4+ T cells isolated from the majority of normal donors and melanoma patients evaluated. Peptide-specific CD4+ T cells also recognized autologous antigen-presenting cell pulsed with recombinant MAGE-A6 (rMAGE) protein, supporting the natural processing and MHC presentation of these epitopes. Given the strong primary in vitro sensitization of normal donor CD4+ T cells by the MAGEA6172-187 epitope, suggestive of potential cross-reactivity against an environmental stimulus, we identified a highly homologous peptide within the Mycoplasma penetrans HF-2 permease (MPHF2) protein. MPHF2 peptide–primed CD4+ T cells cross-reacted against autologous APC pulsed with the MAGE-A6172-187 peptide or rMAGE protein and recognized HLA-matched MAGE-A6+ melanoma cell lines. These responses seemed heteroclitic in nature because the functional avidity of MPHF2 peptide-primed CD4+ T cells for the MAGE-A6172-187 peptide was ∼1,000 times greater than that of CD4+ T cells primed with the corresponding MAGE-A6 peptide. Conclusions: We believe that these novel “promiscuous” MAGE-A6/MPHF2 Th epitopes may prove clinically useful in the treatment and/or monitoring of a high proportion of cancer patients.

IL-12 production by human monocyte-derived dendritic cells : Looking at the single cell

Dendritic cells (DCs) are under investigation as immunotherapeutic agents in the treatment of cancer and infectious diseases. One of the important factors in skewing the immune response toward clinically beneficial TH1-type immunity is interleukin-12p70. IL-12p70 is synthesized and secreted in response to inflammatory cytokines, bacterial/viral components, and CD40 ligation. This study investigated the production of IL-12 by DCs at the single-cell level using a sensitive intracellular cytokine flow cytometry-based assay system. The authors observed that immature DCs could be stimulated with several compounds to produce IL-12, but that IL-12 production was a feature of a minority of activated DCs. IL-12+ DCs were characterized as being partially matured (ie, absent or low CD83 expression, with variable expression of CD1a and CD64). Interestingly, activated DCs lacked expression of the CD16 and CD64 FcγR, which may have important implications for exogenous antigen-loading strategies.

Progenipoietin‐generated dendritic cells exhibit anti‐tumor efficacy in a therapeutic murine tumor model

Progenipoietin (ProGP‐4) is a chimeric molecule, exhibiting both Flt‐3 and granulocyte‐colony stimulating factor (G‐CSF) receptor agonist activities. Subcutaneous administration of ProGP‐4 to BALB/c mice at a dose of 40–100 μg/day for up to 12 consecutive days induces both CD11c+ dendritic cells (DCs) and CD11c−/CD11b+ granulocytes in spleen, blood and lymph nodes of treated animals. Peak numbers of all cell populations were observed on day 7 of treatment, with CD11c+ DCs representing approximately 8% of total splenocytes at that time. Approximately 40–50% of these CD11c+ cells were also able to endocytose and process the exogenous fluorescent antigen DQ‐BSA. As a test of their therapeutic utility, freshly prepared CD11c+ DCs were pulsed with a defined tumor‐associated peptide epitope (murine p53232–240) and injected as a vaccine into BALB/c mice bearing day 7 established CMS4 sarcomas. Similarly prepared DCs were injected again 1 week later. Based on our results, we conclude that (i) both peptide‐pulsed CD11c+ DCs (harvested directly from ProGP‐4 treated mice) and pulsed bone marrow‐derived DCs effectively slow the growth of or mediate the regression (in 25 of 89 [28%] cases) of CMS4 tumors, and (ii) nonpulsed DCs mediated minimal or no therapeutic effect. These data support the ability of ProGP‐4 to enhance the peripheral frequencies of DCs that exhibit therapeutic efficacy when applied as a vaccine to treat tumor‐bearing animals.

Evaluation of SAS1B as a target for antibody-drug conjugate therapy in the treatment of pancreatic cancer

Successful therapeutic options remain elusive for pancreatic cancer. The exquisite sensitivity and specificity of humoral and cellular immunity may provide therapeutic approaches if antigens specific for pancreatic cancer cells can be identified. Here we characterize SAS1B (ovastacin, ASTL, astacin-like), a cancer-oocyte antigen, as an attractive immunotoxin target expressed at the surface of human pancreatic cancer cells, with limited expression among normal tissues. Immunohistochemistry shows that most pancreatic cancers are SAS1Bpos (68%), while normal pancreatic ductal epithelium is SAS1Bneg. Pancreatic cancer cell lines developed from patient-derived xenograft models display SAS1B cell surface localization, in addition to cytoplasmic expression, suggesting utility for SAS1B in multiple immunotherapeutic approaches. When pancreatic cancer cells were treated with an anti-SAS1B antibody-drug conjugate, significant cell death was observed at 0.01-0.1 μg/mL, while SAS1Bneg human keratinocytes were resistant. Cytotoxicity was correlated with SAS1B cell surface expression; substantial killing was observed for tumors with low steady state SAS1B expression, suggesting a substantial proportion of SAS1Bpos tumors can be targeted in this manner. These results demonstrate SAS1B is a surface target in pancreatic cancer cells capable of binding monoclonal antibodies, internalization, and delivering cytotoxic drug payloads, supporting further development of SAS1B as a novel target for pancreatic cancer.