The Landscape of Early Clinical Gene Therapies outside of Oncology.

The field of cell and gene therapy (GT) is expanding rapidly and there is undoubtedly a wave of enthusiasm and anticipation for what these treatments could achieve next. Here we assessed the worldwide landscape of GT assets currently in early clinical development (clinical trial phase 1/2 or about to enter clinical trial). We included all gene therapies, i.e., strategies that modify an individual's protein make-up by introducing exogenous nucleic acid or nucleic acid modifiers, regardless of delivery. Unmodified cell therapies, oncology therapies (reviewed elsewhere), and vaccine programs (distinct therapeutic strategy) were not included. Using a December 31, 2018 cutoff date, we identified 336 gene therapies being developed for 138 different indications covering 165 genetic targets. In all, we found that the early clinical GT landscape comprises a very disparate group of drug candidates in terms of indications, organizations, and delivery methods. We also highlight interesting trends, revealing the evolution of the field toward in vivo therapies and adeno-associated virus vector-based delivery systems. It will be interesting to witness what proportion of this current list effectively translates into new medicines.

Polyfunctional anti-human epidermal growth factor receptor 3 (anti-HER3) antibodies induced by HER3 vaccines have multiple mechanisms of antitumor activity against therapy resistant and triple negative breast cancers.

BACKGROUND: Upregulation of human epidermal growth factor receptor 3 (HER3) is a major mechanism of acquired resistance to therapies targeting its heterodimerization partners epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2), but also exposes HER3 as a target for immune attack. We generated an adenovirus encoding full length human HER3 (Ad-HER3) to serve as a cancer vaccine. Previously we reported the anti-tumor efficacy and function of the T cell response to this vaccine. We now provide a detailed assessment of the antitumor efficacy and functional mechanisms of the HER3 vaccine-induced antibodies (HER3-VIAs) in serum from mice immunized with Ad-HER3. METHODS: Serum containing HER3-VIA was tested in complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) assays and for its effect on HER3 internalization and degradation, downstream signaling of HER3 heterodimers and growth of metastatic HER2+ (BT474M1), HER2 therapy-resistant (rBT474), and triple negative (MDA-MB-468) breast cancers. RESULTS: HER3-VIAs mediated CDC and ADCC, HER3 internalization, interruption of HER3 heterodimer-driven tumor signaling pathways, and anti-proliferative effects against HER2+ tumor cells in vitro and significant antitumor effects against metastatic HER2+ BT474M1, treatment refractory HER2+ rBT474 and triple negative MDA-MB-468 in vivo. CONCLUSIONS: In addition to the T cell anti-tumor response induced by Ad-HER3, the HER3-VIAs provide additional functions to eliminate tumors in which HER3 signaling mediates aggressive behavior or acquired resistance to HER2-targeted therapy. These data support clinical studies of vaccination against HER3 prior to or concomitantly with other therapies to prevent outgrowth of therapy-resistant HER2+ and triple negative clones.

A randomized phase II study of immunization with dendritic cells modified with poxvectors encoding CEA and MUC1 compared with the same poxvectors plus GM-CSF for resected metastatic colorectal cancer.

OBJECTIVE: To determine whether 1 of 2 vaccines based on dendritic cells (DCs) and poxvectors encoding CEA (carcinoembryonic antigen) and MUC1 (PANVAC) would lengthen survival in patients with resected metastases of colorectal cancer (CRC). BACKGROUND: Recurrences after complete resections of metastatic CRC remain frequent. Immune responses to CRC are associated with fewer recurrences, suggesting a role for cancer vaccines as adjuvant therapy. Both DCs and poxvectors are potent stimulators of immune responses against cancer antigens. METHODS: Patients, disease-free after CRC metastasectomy and perioperative chemotherapy (n = 74), were randomized to injections of autologous DCs modified with PANVAC (DC/PANVAC) or PANVAC with per injection GM-CSF (granulocyte-macrophage colony-stimulating factor). Endpoints were recurrence-free survival overall survival, and rate of CEA-specific immune responses. Clinical outcome was compared with that of an unvaccinated, contemporary group of patients who had undergone CRC metastasectomy, received similar perioperative therapy, and would have otherwise been eligible for the study. RESULTS: Recurrence-free survival at 2 years was similar (47% and 55% for DC/PANVAC and PANVAC/GM-CSF, respectively) (χ P = 0.48). At a median follow-up of 35.7 months, there were 2 of 37 deaths in the DC/PANVAC arm and 5 of 37 deaths in the PANVAC/GM-CSF arm. The rate and magnitude of T-cell responses against CEA was statistically similar between study arms. As a group, vaccinated patients had superior survival compared with the contemporary unvaccinated group. CONCLUSIONS: Both DC and poxvector vaccines have similar activity. Survival was longer for vaccinated patients than for a contemporary unvaccinated group, suggesting that a randomized trial of poxvector vaccinations compared with standard follow-up after metastasectomy is warranted. (NCT00103142).

Novel adenoviral vector induces T-cell responses despite anti-adenoviral neutralizing antibodies in colorectal cancer patients.

First-generation, E1-deleted adenovirus subtype 5 (Ad5)-based vectors, although promising platforms for use as cancer vaccines, are impeded in activity by naturally occurring or induced Ad-specific neutralizing antibodies. Ad5-based vectors with deletions of the E1 and the E2b regions (Ad5 [E1-, E2b-]), the latter encoding the DNA polymerase and the pre-terminal protein, by virtue of diminished late phase viral protein expression, were hypothesized to avoid immunological clearance and induce more potent immune responses against the encoded tumor antigen transgene in Ad-immune hosts. Indeed, multiple homologous immunizations with Ad5 [E1-, E2b-]-CEA(6D), encoding the tumor antigen carcinoembryonic antigen (CEA), induced CEA-specific cell-mediated immune (CMI) responses with antitumor activity in mice despite the presence of preexisting or induced Ad5-neutralizing antibody. In the present phase I/II study, cohorts of patients with advanced colorectal cancer were immunized with escalating doses of Ad5 [E1-, E2b-]-CEA(6D). CEA-specific CMI responses were observed despite the presence of preexisting Ad5 immunity in a majority (61.3 %) of patients. Importantly, there was minimal toxicity, and overall patient survival (48 % at 12 months) was similar regardless of preexisting Ad5 neutralizing antibody titers. The results demonstrate that, in cancer patients, the novel Ad5 [E1-, E2b-] gene delivery platform generates significant CMI responses to the tumor antigen CEA in the setting of both naturally acquired and immunization-induced Ad5-specific immunity.

Polyclonal HER2-specific antibodies induced by vaccination mediate receptor internalization and degradation in tumor cells.

INTRODUCTION: Sustained HER2 signaling at the cell surface is an oncogenic mechanism in a significant proportion of breast cancers. While clinically effective therapies targeting HER2 such as mAbs and tyrosine kinase inhibitors exist, tumors overexpressing HER2 eventually progress despite treatment. Thus, abrogation of persistent HER2 expression at the plasma membrane to synergize with current approaches may represent a novel therapeutic strategy. METHODS: We generated polyclonal anti-HER2 antibodies (HER2-VIA) by vaccinating mice with an adenovirus expressing human HER2, and assessed their signaling effects in vitro and anti-tumor effects in a xenograft model. In addition, we studied the signaling effects of human HER2-specific antibodies induced by vaccinating breast cancer patients with a HER2 protein vaccine. RESULTS: HER2-VIA bound HER2 at the plasma membrane, initially activating the downstream kinases extracellular signal-regulated protein kinase 1/2 and Akt, but subsequently inducing receptor internalization in clathrin-coated pits in a HER2 kinase-independent manner, followed by ubiquitination and degradation of HER2 into a 130 kDa fragment phosphorylated at tyrosine residues 1,221/1,222 and 1,248. Following vaccination of breast cancer patients with the HER2 protein vaccine, HER2-specific antibodies were detectable and these antibodies bound to cell surface-expressed HER2 and inhibited HER2 signaling through blocking tyrosine 877 phosphorylation of HER2. In contrast to the murine antibodies, human anti-HER2 antibodies induced by protein vaccination did not mediate receptor internalization and degradation. CONCLUSION: These data provide new insight into HER2 trafficking at the plasma membrane and the changes induced by polyclonal HER2-specific antibodies. The reduction of HER2 membrane expression and HER2 signaling by polyclonal antibodies induced by adenoviral HER2 vaccines supports human clinical trials with this strategy for those breast cancer patients with HER2 therapy-resistant disease.

Co-delivery of antigen and IL-12 by Venezuelan equine encephalitis virus replicon particles enhances antigen-specific immune responses and antitumor effects.

We recently demonstrated that Venezuelan equine encephalitis virus-based replicon particle (VRPs) encoding tumor antigens could break tolerance in the immunomodulatory environment of advanced cancer. We hypothesized that local injection of VRP-expressing interleukin-12 (IL-12) at the site of injections of VRP-based cancer vaccines would enhance the tumor-antigen-specific T cell and antibody responses and antitumor efficacy. Mice were immunized with VRP encoding the human tumor-associated antigen, carcinoembryonic antigen (CEA) (VRP-CEA(6D)), and VRP-IL-12 was also administered at the same site or at a distant location. CEA-specific T cell and antibody responses were measured. To determine antitumor activity, mice were implanted with MC38-CEA-2 cells and immunized with VRP-CEA with and without VRP-IL-12, and tumor growth and mouse survival were measured. VRP-IL-12 greatly enhanced CEA-specific T cell and antibody responses when combined with VRP-CEA(6D) vaccination. VRP-IL-12 was superior to IL-12 protein at enhancing immune responses. Vaccination with VRP-CEA(6D) plus VRP-IL-12 was superior to VRP-CEA(6D) or VRP-IL-12 alone in inducing antitumor activity and prolonging survival in tumor-bearing mice. Importantly, local injection of VRP-IL-12 at the VRP-CEA(6D) injection site provided more potent activation of CEA-specific immune responses than that of VRP-IL-12 injected at a distant site from the VRP-CEA injections. Together, this study shows that VRP-IL-12 enhances vaccination with VRP-CEA(6D) and was more effective at activating CEA-specific T cell responses when locally expressed at the vaccine site. Clinical trials evaluating the adjuvant effect of VRP-IL-12 at enhancing the immunogenicity of cancer vaccines are warranted.

Phase 1 clinical trial of HER2-specific immunotherapy with concomitant HER2 kinase inhibition [corrected].

BACKGROUND: Patients with HER2-overexpressing metastatic breast cancer, despite initially benefiting from the monoclonal antibody trastuzumab and the EGFR/HER2 tyrosine kinase inhibitor lapatinib, will eventually have progressive disease. HER2-based vaccines induce polyclonal antibody responses against HER2 that demonstrate enhanced anti-tumor activity when combined with lapatinib in murine models. We wished to test the clinical safety, immunogenicity, and activity of a HER2-based cancer vaccine, when combined with lapatinib. METHODS: We immunized women (n = 12) with metastatic, trastuzumab-refractory, HER2-overexpressing breast cancer with dHER2, a recombinant protein consisting of extracellular domain (ECD) and a portion of the intracellular domain (ICD) of HER2 combined with the adjuvant AS15, containing MPL, QS21, CpG and liposome. Lapatinib (1250 mg/day) was administered concurrently. Peripheral blood antibody and T cell responses were measured. RESULTS: This regimen was well tolerated, with no cardiotoxicity. Anti-HER2-specific antibody was induced in all patients whereas HER2-specific T cells were detected in one patient. Preliminary analyses of patient serum demonstrated downstream signaling inhibition in HER2 expressing tumor cells. The median time to progression was 55 days, with the majority of patients progressing prior to induction of peak anti-HER2 immune responses; however, 300-day overall survival was 92% (95% CI: 77-100%). CONCLUSIONS: dHER2 combined with lapatinib was safe and immunogenic with promising long term survival in those with HER2-overexpressing breast cancers refractory to trastuzumab. Further studies to define the anticancer activity of the antibodies induced by HER2 vaccines along with lapatinib are underway. TRIAL REGISTRY: ClinicalTrials.gov NCT00952692.

New perspectives on zoledronic acid in breast cancer: potential augmentation of anticancer immune response.

A small subset of T cells (gamma-delta T cells) is able to recognize phosphoantigens that are overexpressed in some cancer cells and may selectively target and kill cancer cells with high levels of phosphoantigen. Moreover, nitrogen-containing bisphosphonates, such as zoledronic acid, are able to induce accumulation of specific phosphoantigens in some cancer cells. A recent preclinical study showed that gamma-delta T cells effectively targeted and killed zoledronic acid-treated estrogen-receptor-positive breast cancer cells. These new data provide growing insight into a potential mechanism of action for some of the anticancer activity demonstrated by zoledronic acid in breast cancer clinical trials.

Immunomodulatory gene therapy prevents antibody formation and lethal hypersensitivity reactions in murine pompe disease.

Infantile Pompe disease progresses to a lethal cardiomyopathy in absence of effective treatment. Enzyme-replacement therapy (ERT) with recombinant human acid alpha-glucosidase (rhGAA) has been effective in most patients with Pompe disease, but efficacy was reduced by high-titer antibody responses. Immunomodulatory gene therapy with a low dose adeno-associated virus (AAV) vector (2 x 10(10) particles) containing a liver-specific regulatory cassette significantly lowered immunoglobin G (IgG), IgG1, and IgE antibodies to GAA in Pompe disease mice, when compared with mock-treated mice (P < 0.05). AAV-LSPhGAApA had the same effect on GAA-antibody production whether it was given prior to, following, or simultaneously with the initial GAA injection. Mice given AAV-LSPhGAApA had significantly less decrease in body temperature (P < 0.001) and lower anaphylactic scores (P < 0.01) following the GAA challenge. Mouse mast cell protease-1 (MMCP-1) followed the pattern associated with hypersensitivity reactions (P < 0.05). Regulatory T cells (Treg) were demonstrated to play a role in the tolerance induced by gene therapy as depletion of Treg led to an increase in GAA-specific IgG (P < 0.001). Treg depleted mice were challenged with GAA and had significantly stronger allergic reactions than mice given gene therapy without subsequent Treg depletion (temperature: P < 0.01; symptoms: P < 0.05). Ubiquitous GAA expression failed to prevent antibody formation. Thus, immunomodulatory gene therapy could provide adjunctive therapy in lysosomal storage disorders treated by enzyme replacement.

Synergism from combined immunologic and pharmacologic inhibition of HER2 in vivo.

The monoclonal antibody trastuzumab and the EGFR/HER2 tyrosine kinase inhibitor lapatinib improve the clinical outcome of patients with HER2-overexpressing breast cancer. However, the majority of metastatic cancers will eventually progress, suggesting the need for other therapies. Because HER2 overexpression persists, we hypothesized that the anti-HER2 immune response induced by cancer vaccines would be an effective strategy for treating trastuzumab- and lapatinib-refractory tumors. Furthermore, we hypothesized that the antibody response could synergize with lapatinib to enhance tumor inhibition. We developed a recombinant adenoviral vector expressing a kinase-inactive HER2 (Ad-HER2-ki) to use as a cancer vaccine. Vaccine-induced polyclonal HER2-specific antiserum was analyzed for receptor internalization and signaling effects alone and in combination with lapatinib. Ad-HER2-ki vaccine-induced potent T cell and antibody responses in mice and the vaccine-induced polyclonal HER2-specific antiserum mediated receptor internalization and degradation much more effectively than trastuzumab. Our in vitro studies demonstrated that HER2 vaccine-induced antibodies effectively caused a decrease in HER2 expression, but when combined with lapatinib caused significant inhibition of HER2 signaling, decreased pERK and pAKT levels and reduced breast tumor cell proliferation. In addition, a known mechanism of resistance to lapatinib, induction of survivin, was inhibited. The combination of Ad-HER2-ki plus lapatinib also showed superior antitumor efficacy in vivo. Based on these results, we feel clinical studies using this approach to target HER2-overexpressing breast cancer, including trastuzumab- and lapatinib-resistant tumors is warranted.

Depletion of human regulatory T cells specifically enhances antigen-specific immune responses to cancer vaccines.

CD4(+)CD25(high)FoxP3(+) regulatory T (Treg) cells limit antigen-specific immune responses and are a cause of suppressed anticancer immunity. In preclinical and clinical studies, we assessed the immune consequences of FoxP3(+) Treg-cell depletion in patients with advanced malignancies. We demonstrated that a CD25(high) targeting immunotoxin (denileukin diftitox) depleted FoxP3(+) Treg cells, decreased Treg-cell function, and enhanced antigen-specific T-cell responses in vitro. We then attempted to enhance antitumor immune responses in patients with carcinoembryonic antigen (CEA)-expressing malignancies by Treg-cell depletion. In a pilot study (n = 15), denileukin diftitox, given as a single dose or repeated dosing, was followed by immunizations with dendritic cells modified with the fowlpox vector rF-CEA(6D)-TRICOM. By flow cytometric analysis, we report the first direct evidence that circulating CD4(+)CD25(high)FoxP3(+) Treg cells are depleted after multiple doses of denileukin diftitox. Earlier induction of, and overall greater exposure to, the T-cell response to CEA was observed in the multiple-dose group, but not the single-dose group. These results indicate the potential for combining Treg-cell depletion with anticancer vaccines to enhance tumor antigen-specific immune responses and the need to explore dose and schedule of Treg depletion strategies in optimizing vaccine efforts.

Induction of Wilms' tumor protein (WT1)-specific antitumor immunity using a truncated WT1-expressing adenovirus vaccine.

PURPOSE: Wilms' tumor protein (WT1) is overexpressed in most leukemias and many solid tumors and is a promising target for tumor immunotherapy. WT1 peptide-based cancer vaccines have been reported but have limited application due to HLA restriction of the peptides. We sought to vaccinate using adenoviral (Ad) vectors encoding tumor-associated antigens such as WT1 that can stimulate tumor-associated antigen-specific immunity across a broad array of HLA types and multiple class I and class II epitopes. EXPERIMENTAL DESIGN: We developed a novel Ad vector encoding a truncated version of WT1 (Ad-tWT1) lacking the highly conserved COOH terminus zinc finger domains and tested its ability to stimulate WT1-specific immune responses and antitumor immunity in two murine models of WT1-expressing tumors. RESULTS: Despite encoding a transcription factor, we found that Ad-tWT1-transduced murine and human dendritic cells showed cytoplasmic expression of the truncated WT1 protein. In addition, vaccination of C57BL/6 mice with Ad-tWT1 generated WT1-specific cell-mediated and humoral immune responses and conferred protection against challenge with the leukemia cell line, mWT1-C1498. Moreover, in a tumor therapy model, Ad-tWT1 vaccination of TRAMP-C2 tumor-bearing mice significantly suppressed tumor growth. CONCLUSIONS: This is the first report of a WT1-encoding Ad vector that is capable of inducing effective immunity against WT1-expressing malignancies. Based on these findings, Ad-tWT1 warrants investigation in human clinical trials to evaluate its applications as a vaccine for patients with WT1-expressing cancers.

Harmonization guidelines for HLA-peptide multimer assays derived from results of a large scale international proficiency panel of the Cancer Vaccine Consortium.

PURPOSE: The Cancer Vaccine Consortium of the Cancer Research Institute (CVC-CRI) conducted a multicenter HLA-peptide multimer proficiency panel (MPP) with a group of 27 laboratories to assess the performance of the assay. EXPERIMENTAL DESIGN: Participants used commercially available HLA-peptide multimers and a well characterized common source of peripheral blood mononuclear cells (PBMC). The frequency of CD8+ T cells specific for two HLA-A2-restricted model antigens was measured by flow cytometry. The panel design allowed for participants to use their preferred staining reagents and locally established protocols for both cell labeling, data acquisition and analysis. RESULTS: We observed significant differences in both the performance characteristics of the assay and the reported frequencies of specific T cells across laboratories. These results emphasize the need to identify the critical variables important for the observed variability to allow for harmonization of the technique across institutions. CONCLUSIONS: Three key recommendations emerged that would likely reduce assay variability and thus move toward harmonizing of this assay. (1) Use of more than two colors for the staining (2) collect at least 100,000 CD8 T cells, and (3) use of a background control sample to appropriately set the analytical gates. We also provide more insight into the limitations of the assay and identified additional protocol steps that potentially impact the quality of data generated and therefore should serve as primary targets for systematic analysis in future panels. Finally, we propose initial guidelines for harmonizing assay performance which include the introduction of standard operating protocols to allow for adequate training of technical staff and auditing of test analysis procedures.

Precision and linearity targets for validation of an IFNgamma ELISPOT, cytokine flow cytometry, and tetramer assay using CMV peptides.

BACKGROUND: Single-cell assays of immune function are increasingly used to monitor T cell responses in immunotherapy clinical trials. Standardization and validation of such assays are therefore important to interpretation of the clinical trial data. Here we assess the levels of intra-assay, inter-assay, and inter-operator precision, as well as linearity, of CD8+ T cell IFNgamma-based ELISPOT and cytokine flow cytometry (CFC), as well as tetramer assays. RESULTS: Precision was measured in cryopreserved PBMC with a low, medium, or high response level to a CMV pp65 peptide or peptide mixture. Intra-assay precision was assessed using 6 replicates per assay; inter-assay precision was assessed by performing 8 assays on different days; and inter-operator precision was assessed using 3 different operators working on the same day. Percent CV values ranged from 4% to 133% depending upon the assay and response level. Linearity was measured by diluting PBMC from a high responder into PBMC from a non-responder, and yielded R2 values from 0.85 to 0.99 depending upon the assay and antigen. CONCLUSION: These data provide target values for precision and linearity of single-cell assays for those wishing to validate these assays in their own laboratories. They also allow for comparison of the precision and linearity of ELISPOT, CFC, and tetramer across a range of response levels. There was a trend toward tetramer assays showing the highest precision, followed closely by CFC, and then ELISPOT; while all three assays had similar linearity. These findings are contingent upon the use of optimized protocols for each assay.

Dendritic cell vaccines.

Dendritic cells are antigen-presenting cells that have been shown to stimulate tumor antigen-specific T cell responses in preclinical studies. Consequently, there has been intense interest in developing dendritic cell based cancer vaccines. A variety of methods for generating dendritic cells, loading them with tumor antigens, and administering them to patients have been described. In recent years, a number of early phase clinical trials have been performed and have demonstrated the safety and feasibility of dendritic cell immunotherapies. A number of these trials have generated valuable preliminary data regarding the clinical and immunologic response to DC-based immunotherapy. The emphasis of dendritic cell immunotherapy research is increasingly shifting toward the development of strategies to increase the potency of dendritic cell vaccine preparations.

Investigation of HIFU-induced anti-tumor immunity in a murine tumor model.

BACKGROUND: High intensity focused ultrasound (HIFU) is an emerging non-invasive treatment modality for localized treatment of cancers. While current clinical strategies employ HIFU exclusively for thermal ablation of the target sites, biological responses associated with both thermal and mechanical damage from focused ultrasound have not been thoroughly investigated. In particular, endogenous danger signals from HIFU-damaged tumor cells may trigger the activation of dendritic cells. This response may play a critical role in a HIFU-elicited anti-tumor immune response which can be harnessed for more effective treatment. METHODS: Mice bearing MC-38 colon adenocarcinoma tumors were treated with thermal and mechanical HIFU exposure settings in order to independently observe HIFU-induced effects on the host's immunological response. In vivo dendritic cell activity was assessed along with the host's response to challenge tumor growth. RESULTS: Thermal and mechanical HIFU were found to increase CD11c+ cells 3.1-fold and 4-fold, respectively, as compared to 1.5-fold observed for DC injection alone. In addition, thermal and mechanical HIFU increased CFSE+ DC accumulation in draining lymph nodes 5-fold and 10-fold, respectively. Moreover, focused ultrasound treatments not only caused a reduction in the growth of primary tumors, with tumor volume decreasing by 85% for thermal HIFU and 43% for mechanical HIFU, but they also provided protection against subcutaneous tumor re-challenge. Further immunological assays confirmed an enhanced CTL activity and increased tumor-specific IFN-gamma-secreting cells in the mice treated by focused ultrasound, with cytotoxicity induced by mechanical HIFU reaching as high as 27% at a 10:1 effector:target ratio. CONCLUSION: These studies present initial encouraging results confirming that focused ultrasound treatment can elicit a systemic anti-tumor immune response, and they suggest that this immunity is closely related to dendritic cell activation. Because DC activation was more pronounced when tumor cells were mechanically lysed by focused ultrasound treatment, mechanical HIFU in particular may be employed as a potential strategy in combination with subsequent thermal ablations for increasing the efficacy of HIFU cancer treatment by enhancing the host's anti-tumor immunity.

Long term disease-free survival and T cell and antibody responses in women with high-risk Her2+ breast cancer following vaccination against Her2.

BACKGROUND: The HER2-inhibiting antibody trastuzumab, in combination with chemotherapy, significantly improves survival of women with resected, HER2-overexpressing breast cancers, but is associated with toxicities including a risk of cardiomyopathy. Additionally, the beneficial effect of trastuzumab is expected to decrease once the drug is discontinued. We proposed to address these concerns by using cancer vaccines to stimulate HER2 intracellular domain (ICD)-specific T cell and antibody responses. METHODS: Subjects with stage II (> or = 6 +LN), III, or stage IV breast cancer with > 50% HER2 overexpressing tumor cells who were disease-free after surgery and adjuvant therapy were eligible. Vaccines consisted of immature, cultured DC (n = 3), mature cultured DC (n = 3), or mature Flt3-ligand mobilized peripheral blood DC (n = 1) loaded with ICD, or tetanus toxoid, keyhole limpet hemocyanin or CMV peptide as controls, and were administered intradermally/subcutaneously four times at 3 week intervals. ICD-specific T cell and antibody responses were measured. Cardiac function was determined by MUGA or ECHO; long term disease status was obtained from patient contact. RESULTS: All seven patients successfully underwent DC generation and five received all 4 immunizations. There were no toxicities greater than grade 1 or ejection fraction decrements below normal. Delayed-type hypersensitivity (DTH) reactions at the injection site occurred in 6/7 patients and HER2 specificity was detected by cytokine flow cytometry or ELISPOT in 5 patients. At more than 5 years of follow-up, 6/7 had detectable anti-ICD antibodies. One patient experienced a pulmonary recurrence at 4 years from their study immunizations. This recurrence was resected and they are without evidence of disease. All patients are alive and disease-free at 4.6-6.7 years of follow-up. CONCLUSION: Although this was a small pilot study, the well-tolerated nature of the vaccines, the lack of cardiac toxicity, significant immunogenicity, and a 100% 4.5-year survival rate suggest that vaccination with HER2 ICD protein-containing DC is appropriate for further study in this population. TRIAL REGISTRATION: ClinicalTrials.gov NCT00005956.

Immunotherapeutic targeting of Wilms' tumor protein.

The expression of Wilms' tumor protein (WT1)-derived peptides on malignant cell surfaces and recognition of those peptides by cellular and humoral immune responses suggest that WT1 may be a promising potential target antigen in immunotherapeutic trials. With a high frequency of expression in hematopoietic as well as solid tumors, WT1 is a broadly applicable target. Both in vivo mouse model and in vitro human studies have demonstrated the ability of WT1-specific cytotoxic T-lymphocytes to lyse WT1-expressing malignancies without harming normal tissue. WT1-peptide vaccination, in combination with adjuvants, has demonstrated the ability to activate WT1-specific immune responses and evidence of clinical activity. Because peptide-based vaccines are human leukocyte antigen-restricted, other more broadly applicable strategies are now being developed to activate WT1-specific immune responses, including the use of WT1-specific viral vectors.

Maximizing the retention of antigen specific lymphocyte function after cryopreservation.

The ability to cryopreserve lymphocytes in peripheral blood mononuclear cells (PBMC) to retain their function after thawing is critical to the analysis of cancer immunotherapy studies. We evaluated a variety of cryopreservation strategies with the aim of developing an optimized protocol for freezing and thawing PBMC to retain viability and function. We determined several factors which do not affect cell viability after cryopreservation such as shipping frozen samples on dry ice, the length of time and speed at which samples are washed and centrifuged after thawing, and the number of cells frozen per container. Different media additives, however, did impact the viability of the cells after thawing. There was a significant reduction in the viability of the cells after freezing when using human AB serum compared to all other additives tested (p<0.000). A second critical parameter was the temperature of the media used to wash the cells after removal from the cryotubes. When the media was cooled to 4 degrees C prior to washing, the mean viability was 69.7+/-12.5%, at 25 degrees C 92.55+/-3.1%, and at 37 degrees C 95.11+/-2.5%. Finally, we used an optimized cryopreservation protocol with different media additives to determine if functional T cell responses to tetanus toxoid could be preserved. There was a statistically significant correlation between the tetanus specific stimulation index (S.I.) of the non-cryopreserved PBMC and SI obtained from cells frozen with media containing human serum albumin as compared to other additives such as dextran or fetal bovine serum.

Dendritic cell-based immunotherapy.

Dendritic cells (DCs) play a crucial role in the induction of antigen-specific T-cell responses, and therefore their use for the active immunotherapy of malignancies has been studied with considerable interest. More than a decade has passed since the publication of the first clinical data of DC-based vaccines, and through this and subsequent studies, a number of important developmental insights have been gleaned. These include the ideal source and type of DCs, the discovery of novel antigens and methods of loading DCs, the role of DC maturation, and the most efficient route of immunization. The generation of immune responses against tumor antigens after DC immunization has been demonstrated, and favorable clinical responses have been reported in some patients; however, it is difficult to pool the results as a whole, and thus the body of data remains inconclusive, in part because of varying DC preparation and vaccination protocols, the use of different forms of antigens, and, most importantly, a lack of rigorous criteria for defining clinical responses. As such, the standardization of clinical and immunologic criteria utilized, as well as DC preparations employed, will allow for the comparison of results across multiple clinical studies and is required in order for future trials to measure the true value and role of this treatment modality. In addition, issues regarding the optimal dose and clinical setting for the application of DC vaccines remain to be resolved, and recent clinical studies have been designed to begin to address these questions.