Phase I/II trial of immunogenicity of a human papillomavirus (HPV) type 16 E7 protein–based vaccine in women with oncogenic HPV-positive cervical intraepithelial neoplasia

Purpose: Infection with oncogenic human papillomavirus (HPV) and HPV-16 in particular is a leading cause of anogenital neoplasia. High-grade intraepithelial lesions require treatment because of their potential to progress to invasive cancer. Numerous preclinical studies have demonstrated the therapeutic potential of E7-directed vaccination strategies in mice tumour models. In the present study, we tested the immunogenicity of a fusion protein (PD-E7) comprising a mutated HPV-16 E7 linked to the first 108 amino acids of Haemophilus influenzae protein D, formulated in the GlaxoSmithKline Biologicals adjuvant AS02B, in patients bearing oncogenic HPV-positive cervical intraepithelial neoplasia (CIN). Methods: Seven patients, five with a CIN3 and two with a CIN1, received three intramuscular injections of adjuvanted PD-E7 at 2-week intervals. Three additional CIN1 patients received a placebo. CIN3 patients underwent conization 8 weeks postvaccination. Cytokine flow cytometry and ELISA were used to monitor antigen-specific cellular and antibody responses from blood taken before and after vaccine or placebo injection. Results: Some patients had preexisting systemic IFN- CD4⁺ (1/10) and CD8⁺ (5/10) responses to PD-E7. Vaccination, not placebo injection, elicited systemic specific immune responses in the majority of the patients. Five vaccinated patients (71%) showed significantly increased IFN- CD8⁺ cell responses upon PD-E7 stimulation. Two responding patients generated long-term T-cell immunity toward the vaccine antigen and E7 as well as a weak H. influenzae protein D (PD)–directed CD4⁺ response. All the vaccinated patients, but not the placebo, made significant E7- and PD-specific IgG. Conclusions: The encouraging results obtained from this study performed on a limited number of subjects justify further analysis of the efficacy of the PD-E7/AS02B vaccine in CIN patients.     

A DNA vaccine co-expressing antigen and an anti-apoptotic molecule further enhances the antigen-specific CD8+ T-cell immune response

We have shown that DNA encoding the anti-apoptotic protein Bcl-xL enhances E7-specific CD8+ T-cell responses and DNA encoding pro-apoptotic protein caspase-3 suppresses E7-specific CD8+ T-cell responses when co-administered intradermally via gene gun with DNA encoding human papillomavirus type 16 (HPV-16) E7 linked to the sorting signal of the lysosome-associated membrane protein type 1 (LAMP-1). E7 and LAMP-1 are linked to form the chimeric Sig/E7/LAMP-1 (SEL). Because co-administration does not ensure delivery of both constructs to a single cell, we used pVITRO, a mammalian expression vector with double promoters, to ensure expression of both molecules in the same cell. We vaccinated C57BL/6 mice with pVITRO-SEL-Bcl-xL, pVITRO-SEL-mtBcl-xL, pVITRO-SEL, or pVITRO-SEL-caspase-3 intradermally via gene gun and intramuscularly via injection. We demonstrated that vaccination with pVITRO achieved similar results to a co-administration strategy: that Bcl-xL enhanced the E7-specific CTL response and caspase-3 suppressed the E7-specific CTL response. In addition, we found intradermal vaccination elicited significantly higher numbers of E7-specific CD8+ T cells compared to intramuscular vaccination. Thus, intradermal vaccination with a pVITRO vector combining an anti-apoptotic strategy (Bcl-xL) and an intracellular targeting strategy (SEL) further enhances the E7-specific CD8+ T-cell response and guarantees co-expression of both encoded molecules in transfected cells.T.W.K. and C.-F.H. contributed equally to this work.     

Electrofusion of syngeneic dendritic cells and tumor generates potent therapeutic vaccine

Antigen presentation by dendritic cells (DCs) has the potential to elicit therapeutic immune responses against malignant tumors. One strategy utilizing DC-tumor fusion hybrids as cancer vaccine is particularly attractive because of polyclonal presentation of a diverse array of unaltered tumor antigens. We have recently developed a large-scale electrofusion technique for generating DC-tumor heterokaryons and demonstrated their superb immunogenicity. Here, employing the weakly immunogenic MCA205 sarcoma, a single vaccination with electrofusion hybrids eradicated tumors established in the lung, skin, and brain. Immunotherapy required intra-lymphoid vaccine delivery and co-administration of adjuvants such as OX-40R antibody. Tumor eradication was immunologically specific and involved the participation of both CD4 and CD8 T cells. Consistent with DC's functionality of MHC-restriction, the use of syngeneic DCs for fusion was an obligatory requirement. Fusion with allogeneic DCs completely lacked therapeutic effects. These findings provide a strong impetus for treating cancer patients with similarly generated DC-tumor hybrids.     

Long-term treatment with low doses of interleukin-2 and interferon-α: immunological effects in advanced renal cell cancer

We aimed to determine the immunological effects of low doses of recombinant interleukin-2 (rIL-2) and recombinant interferon-α (rIFN-α) in patients bearing advanced renal cell carcinoma. Methods: Twenty-seven patients received therapeutic cycles consisting of subcutaneous rIL-2 for 5 days per week and intramuscular rIFN-α twice weekly, for 4 consecutive weeks. The cycle was repeated indefinitely at regular 4-month intervals, for all patients. rIL-2 (1 × 10⁶ IU/m²) was administered every 12 h on days 1 and 2 and once a day on days 3–5 of each week; rIFN-α (1.8 × 10⁶ IU/m²) was given on days 3 and 5. In the enrolled patients, total and differential white blood cell counts, phenotypic analysis of some lymphocyte subsets, and soluble IL-2 receptor (sIL-2R), were investigated before and after each of the first six cycles of therapy (about 24 months of follow-up). Results: The cycles of immunotherapy induced a significant increase of total lymphocytes (37%, P < 0.001), eosinophils (222%, P < 0.001), CD25+ cells (27%, P=0.004), sIL-2R (174%, P < 0.001) and natural killer (NK) cells (CD3-CD56+) (61%, P < 0.001); the subset that expresses CD56 with high density (CD56+ bright) expanded more (233%, P < 0.001) than the subset expressing the same marker with low density (CD56+ dimmer) (15%, P=0.043). Unlike the previous subsets, the treatment decreased significantly T-lymphocytes with NK cell marker (CD3+ CD56+) (28%, P=0.011). No significant differences of effectiveness were found among the subsequent treatment cycles, except for CD25+ cells and sIL-2R (P=0.036 and P=0.005, respectively): the increase induced by immunotherapy was maximum after the first cycle and decreased progressively thereafter. Conclusions: Long-term repeated cycles of low-dose immunotherapy induced repeated and significant expansion of one of the most important lymphocyte subsets for the non-MHC-restricted immune response to the tumour mass: CD3–CD56+ cells. Received: 8 November 2000 / Accepted: 11 January 2001     

Observed localization of the long-term cultured rat adherent natural killer cells in mammary tumor tissues

 Adherent natural killer (A-NK) cells were isolated from splenic lymphocytes and treated with long-term culture in the presence of recombinant interleukin-2 (rIL-2). Immunocytochemical and flow-cytometric analysis revealed that most of the A-NK cells strongly expressed lymphocyte-function-associated antigen 1 (LFA-1α, and LFA-1β) throughout the incubation. All A-NK cells from 8- to 150-day cultures, particularly those cultured for 8 days, showed significant cytolytic activity against all targets. Analysis of the tissue distribution of the injected [³H] uridine-labeled A-NK cells demonstrated that, in the first 3 h, most (over 60%) cells localized in the lungs, and that most cells remained temporally within the cavities of blood capillaries of the lungs and moved gradually into lymphoid and other tissues. Peritumoral injection of various kinds of adjuvant, particularly Freund's complete adjuvant (FCA) plus bacillus Calmetee-Guérin (BCG), resulted in a marked accumulation of [³H]A-NK cells in mammary tumor tissues 24 h after injection, and simultaneously in the formation of vessels resembling high-endothelium venules (HEV), infiltration of LFA-1⁺ lymphocytes and expression of the ICAM-1 molecule on the tumor cells in the sites of tumor tissues. When 30 × 10⁶ A-NK cells were intravenously administered, significant retardation of tumor growth and prolongation of survival of tumor-bearing rats were observed in the groups that received the prior injection of adjuvants, especially FCA + BCG and Freund's incomplete adjuvant (FIA) + BCG. The suppressive effect of combination therapy on tumor growth was blocked effectively by the injection of anti-ICAM-1 mAb. These results indicate that the prior injection of proper adjuvant into the peritumoral region is effective for the selective accumulation or infiltration of A-NK cells into the sites of tumor tissues, and results in the marked retardation of tumor growth. Received: 18 May 1999 / Accepted: 4 October 1999     

Initiation of humoral and cellular immune responses in patients with refractory Hodgkin's disease by treatment with an anti-CD16/CD30 bispecific antibody

Fifteen patients with refractory Hodgkin's disease were treated in a dose-escalation trial with the bispecific monoclonal antibody (bi-mAb) HRS-3/A9, which is directed against the Fcγ receptor III (CD16 antigen) and the Hodgkin's-associated CD30 antigen. Treatment consisted of four cycles of four bi-mAb infusions given over 1 h every 3–4 days at different dose levels ranging from 1 mg/m² to 64 mg/m². Measurable serum levels (above 0.1 μg/ml) of circulating bi-mAb could be detected in patients treated with doses above 4 mg/m², reaching peak levels of 9.5 μg/ml immediately after the end of antibody infusion on the highest dose level. Bi-mAb elimination corresponded to second-order kinetics with a terminal half-life time (t _1/2,β) of 28–32 h. Bi-mAb treatment induced the occurrence of human anti-(mouse Ig) antibodies (HAMA) in 6 out of 13 patients initially testing negative. All 6 patients not only developed anti-isotypic anti-(mouse Ig) but also anti-idiotypic and anti-anti-idiotypic antibodies. While no consistent changes of peripheral blood cell counts, or of any lymphocyte subpopulation including natural killer (NK) cells, has been observed, 4 out of 6 evaluable patients treated with doses of at least 4 mg/m² showed an increase of NK cell activity within 2 weeks after treatment, which lasted for a maximum of 12 weeks. Circulating amounts of soluble CD30 antigen could be detected in the serum of 6 patients. However, like the results and time courses of all the other immunological parameters evaluated, this was not predictive for treatment outcome. Received: 16 September 1999 / Accepted: 6 January 2000     

Signaling events involved in anti-CD20-induced apoptosis of malignant human B cells

Anti-CD20 monoclonal antibodies have been successfully employed in the clinical treatment of non-Hodgkin's lymphomas in both unmodified and radiolabeled forms. Previous publications have demonstrated that the antitumor effects of unmodified anti-CD20 mAb are mediated by several mechanisms including antibody-dependent cellular cytotoxicity, complement-mediated cell lysis, and induction of apoptosis by CD20 cross-linking. In this report, we demonstrate induction of apoptosis by three anti-CD20 monoclonal antibodies [1F5, anti-B1, and C2B8 (Rituximab)]. The magnitude of apoptosis induction was greater with the chimeric Rituximab antibody than with the murine 1F5 and anti-B1 antibodies. Apoptosis could be enhanced with any of the antibodies by cross-linking with secondary antibodies (or Fc-receptor-bearing accessory cells). The signaling events involved in anti-CD20-induced apoptosis were investigated, including activation of protein tyrosine kinases, increases in intracellular Ca²⁺ concentrations, caspase activation, and cleavage of caspase substrates. Our results indicate that anti-CD20-induced apoptosis can be attenuated by PP1, an inhibitor of protein tyrosine kinases Lck and Fyn, chelators of extracellular or intracellular Ca²⁺, and inhibitors of caspases, suggesting that anti-CD20-induced apoptosis may involve modulation of these signaling molecules. We also demonstrated that varying the expression of Bcl-2 did not affect the magnitude of anti-B1-induced apoptosis, possibly because of the sequestering effects of other Bcl-2 family members, such as Bad. These studies identify several of the signal-transduction events involved in the apoptosis of malignant B cells that transpire following ligation of CD20 by anti-CD20 antibodies in the presence of Fc-receptor-expressing cells or secondary goat anti-(mouse Ig) antibodies and which may contribute to the tumor regressions observed in mouse models and clinical trials. Received: 27 May 1999 / Accepted: 1 October 1999     

The effect of T1 and T2 cytokines on the cytotoxic T cell response to mannan-MUC1

MUC1 is a mucin over-expressed in breast cancer and a proposed target for immunotherapy. By immunising mice with MUC1 conjugated to mannan (M-FP), CD8⁺ MHC-class-I restricted cytotoxic T lymphocytes (CTL), of high CTL precursor (CTLp) frequency (1/8000) and with significant tumour protection, can be induced. The effect of various cytokines [interleukin-2 (IL-2), IL-4, IL-6, IL-7, interferon γ (IFNγ), and granulocyte/macrophage-colony-stimulating factor (GM-CSF)] on the MUC1 CTL immune response was investigated (a) by measuring the frequencies of CTLp in mice immunised with vaccinia virus constructs containing recombinant cytokines and M-FP, or (b) by immunising cytokine- or cytokine-receptor-knockout (−/−) mice with M-FP. Vaccinia virus (VV) constructs containing recombinant cytokines were used either individually or in combination in vivo with M-FP immunisation. M-FP immunisations combined with VV-IL-2, VV-IL-7 and VV-GM-CSF, and combinations of VV-IFNγ + VV-IL-2, VV-IFNγ + VV-IL-4 or VV-GM-CSF + VV-IL-7 increased CTLp frequencies up to threefold (1/17 666: M-FP + VV-GM-CSF + VV-IL-7) compared to M-FP (1/77 500) alone. By contrast, M-FP combined with VV-IL-4 decreased the CTLp frequency threefold whereas VV-IL-6 and VV-IFNγ had no effect. Studies in cytokine- and cytokine-receptor-gene-knockout (−/−) mice demonstrated that mice that are IL-2 −/− and IL-7 receptor −/− produce the same CTLp response to M-FP as do control mice, whereas responses in the IL-6 −/−, IL-10 −/− and IFNγ−/− mice were marginally improved and responses to M-FP in IL-4 −/− and tumour necrosis factor receptor 2 −/− mice were weaker. In spite of the increase in CTLp frequency, this was not reflected in an in vivo tumour model. Tumour challenges using MUC1⁺ P815 cells, demonstrated that the addition of cytokines had little additive effect on the already effective tumour-regression capabilities of M-FP alone. Received: 24 September 1998 / Accepted: 21 September 1999     

Inhibition ofras oncogene: A novel approach to antineoplastic therapy

The most frequently detected oncogene alterations, both in animal and human cancers, are the mutations in the ras oncogene family. These oncogenes are mutated or overexpressed in many human tumors, with a high incidence in tumors of the pancreas, thyroid, colon, lung and certain types of leukemia. Ras is a small guanine nucleotide binding protein that transduces biological information from the cell surface to cytoplasmic components within cells. The signal is transduced to the cell nucleus through second messengers, and it ultimately induces cell division. Oncogenic forms of p21(ras) lead to unregulated, sustained signaling through downstream effectors. The ras family of oncogenes is involved in the development of both primary tumors and metastases making it a good therapeutic target. Several therapeutic approaches to cancer have been developed pointing to reducing the altered gene product or to eliminating its biological function: (1) gene therapy with ribozymes, which are able to break down specific RNA sequences, or with antisense oligonucleotides, (2) immunotherapy through passive or active immunization protocols, and (3) inhibition of p21(ras) farnesylation either by inhibition of farnesyl transferase or synthesis inhibition of farnesyl moieties.