HER2 Vaccine Research Articles

Abstract OT3-1-02: Phase II randomized study of combination immunotherapy with or without Polysaccharide Krestin (PSK®) concurrently with a HER2 ICD peptide-based vaccine and trastuzumab in patients with stage IV breast cancer

Abstract Background: Endogenous immunity in patients with HER2+ metastatic breast cancer (MBC) is likely dampened by an immune-suppressive tumor microenvironment and not sufficient to control tumor growth. Thus, most patients have disease relapse after achieving complete remission with standard therapies. Immunomodulation directed at enhanced stimulation of tumor specific immunity could result in immunologic eradication of residual HER2+ tumor cells and prevent BC relapse. We have shown PSK to be a potent TLR-2 agonist that stimulates both innate and adaptive immunity in a BC mouse model. Additionally, we have shown combination immunotherapy with HER2 peptide vaccines and trastuzumab (TRAZ) to be safe and able to elicit HER2 specific Th1 immunity and epitope spreading (ES) which has been associated with survival in vaccinated patients. Lastly, decreased serum TGF-β elicited by HER2 vaccination correlates with Th1 ES and may serve as a biomarker to predict cancer vaccine efficacy. We hypothesize that PSK, when given with TRAZ can augment vaccine induced HER2 specific TH1 immunity and prevent disease relapse in patients with optimally treated HER2+ MBC. Trial design: Phase II randomized two-arm clinical trial. Patients will be enrolled and randomly assigned in equal numbers to 1 of 2 arms (15 patients/arm) as follows: Arm 1:HER2 ICD vaccine, TRAZ and placebo or Arm 2:HER2 ICD vaccine, TRAZ and PSK. All patients will receive 3 monthly HER2 ICD vaccines plus TRAZ and 4 months of concomitant PSK or placebo. Serial blood draws for immunologic monitoring will be done. Eligibility criteria: Patients with Stage IV HER2+ BC who have been treated with definitive therapy and are: (1) without evidence of disease or have stable-bone only disease, (2) receiving TRAZ monotherapy, and (3) without clinically significant autoimmune disease. Patients must have normal LVEF per MUGA scan or echocardiogram. Aims: (1) Evaluate safety of PSK when given with a HER2 vaccine and TRAZ (2) Evaluate the effect of PSK on serum TGF-β levels when given with a HER2 vaccine and TRAZ and (3) Evaluate the effect of PSK on intermolecular ES when given with a HER2 vaccine and TRAZ. A secondary objective is to evaluate progression free survival (PFS) and overall survival (OS). Statistical methods: (1) Toxicity will be determined by clinical and chemical parameters and grading will be done per CTEP CTCAE 4.0.; (2) Evaluation of TGF-β levels, pre and post-PSK treatment will be assessed with linear regression models; and analysis of multiple post-baseline measurements will be performed using generalized estimating equations; (3) A positive antigen-specific immune response will be defined as a precursor frequency >1:20,000 antigen-specific peripheral blood mononuclear cells. Differences in the levels of HER2 immunity will be evaluated between arms using a two-tailed T test. The degree of ES in each arm will be evaluated with generalized linear modeling; (4) Large differences in PFS and OS observed between groups will be noted and described. Target accrual: 30 patients. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr OT3-1-02.

Abstract P2-15-02: The efficacy of recruitment and retention strategies for research subjects in an early phase investigator-initiated breast cancer trial.

Abstract Background: One of the biggest challenges faced by investigators is the implementation of effective strategies to improve the recruitment and retention of research participants. This is especially true for investigator-initiated, federally funded (e.g. NIH and DOD), early phase clinical trials that involve the treatment of serious diseases such as metastatic breast cancer (MBC). These studies may face additional barriers to participation since patients have often already undergone maximal treatment and are usually not in a financial position that for allows the travel and lodging necessary to receive further investigative treatment. Moreover, efficacy and toxicology in early phase clinical trials are unknown. Thus, when study budget constraints do not allow monetary incentives to participation, it is difficult to provide motivation for patients to enroll and remain adherent to the protocol requirements. However, many MBC patients are motivated to join clinical trials for altruistic purposes alone, and evidence supports that the researcher-patient relationship may be the most important factor in clinical trial participation. Recognizing that many patients are willing to participate if provided the appropriate resources despite limited monetary incentives, we developed a system to improve patient recruitment and retention to our studies, which are primarily federally funded. We report here on the strategies developed and used by our group to recruit and retain patients in a federally-funded investigator-initiated phase I/II vaccine study in MBC patients. Methods: This study was funded by the NIH/NCI and involved infusion of HER2 specific T cells in HER2+ MBC patients after completing in vivo priming with a HER2 vaccine. It required 11 visits to Seattle, Washington. Working with agencies that offer free services to patients enrolled in clinical trials, a list of available resources was compiled and a visit flowchart with specific information on travel and lodging resources (e.g. Angel Flights and ACS sponsorship), local transportation and entertainment was developed. During screening, patients were given the list of resources and trial information. An email system was used to quickly communicate and follow-up with patients. Eligible patients were given the visit flowchart to help with their planning of study visits. An enrollment packet was provided at the first visit with a calendar to keep track of the visit schedule. Coordination of care between the patient's primary oncologist and the research staff was maintained throughout the study. Results: 17 of 19 patients enrolled were not from Washington State. Two out-of-state patients withdrew early from the trial for reasons unrelated to disease progression or toxicity; one subject completed 8 visits and enrolled in another study and the other completed 2 visits and discontinued the trial to resume chemotherapy. Conclusion: We have developed a successful system to enroll and retain patients in a trial requiring multiple study visits. Development and implementation of site-specific standard procedures are critical to improve study participation and retention, especially when patients receive no financial benefit. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-15-02.

Concurrent HER2 Vaccination and Inhibition of Kinase Activity: Safety and Immunogenicity

Passive immunotherapy with the monoclonal antibody trastuzumab and tyrosine kinase activity inhibition with lapatinib are HER2-targeted therapies used in the clinic for the treatment of HER2-overexpressing breast cancers. Unfortunately, the therapeutic efficacy of both these therapies is abolished by primary and acquired tumor resistance. Active immunotherapy against HER2, which, thanks to trastuzumab, is a clinically validated tumor-associated antigen, might provide an alternative therapeutic strategy for HER2-overexpressing breast cancers. This Phase I study of HER2 immunotherapy with concomitant lapatinib treatment in 12 patients with metastatic breast cancer resistant to trastuzumab demonstrates the feasibility and safety of concurrent vaccination against HER2 and inhibition of HER1 and HER2 kinases. However, it is inconclusive regarding the effect of lapatinib on the immune responses induced by dHER2/AS15; vaccination triggered variable levels of anti-HER2 antibodies in all the patients, but a HER2-specific T-cell response was detected in one patient only. Since the presence of Tregs in these patients was not assessed, it remains unclear whether lapatinib and/or Tregs account for the near absence of a T-cell response.

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

IntroductionSustained 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.MethodsWe 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.ResultsHER2-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.ConclusionThese 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.

OT1-02-10: Phase I-II Study of HER2 Vaccination with Poly(I) • Poly(C12U) (Ampligen®) as an Adjuvant in Optimally Treated Breast Cancer Patients.

Abstract Background Despite improved response rates and overall survival, many HER2+ breast cancer (BC) patients have disease relapse suggesting residual microscopic disease. HER2 vaccines given with adjuvants that can enhance, sustain, and skew antigen immunogenicity toward a Th1 phenotype could induce robust tumor-specific Th1 immunity resulting in immunologic eradication of residual tumor cells and potentially prevent relapse. One such adjuvant is Ampligen which is highly selective as a TLR-3 agonist. Our pre-clinical studies show a dose effect in the tumor prevention efficacy of Ampligen when given as an adjuvant with vaccines. We hypothesize HER2 peptide vaccination given with standard adjuvant 100mcg GMCSF and Ampligen can induce a higher incidence and magnitude of protective HER2−specific Th1 immunity than with GMCSF alone. Trial design: Phase I-II randomized 2-stage HER2 vaccine study. Stage I will enroll 40 patients (10/arm) into one of 4 Ampligen dose arms (4, 20, 79, or 495 mcg + HER2 vaccine). The Ampligen “maximum biologic dose” (MBD), the dose with the highest incidence/magnitude of immune response and lowest incidence of toxicity will be defined. Stage II will enroll 48 patients (24/arm) receiving Ampligen MBD + HER2 vaccine + GMCSF or HER2 vaccine + GMCSF to evaluate if Ampligen MBD increases the incidence and magnitude of immunity vs HER2 vaccine + GMCSF alone. Patients will be enrolled sequentially and randomized equally into all arms via a permuted block design. Patients will receive 3 monthly vaccines. Toxicity and immune response will be assessed. Aims: 1) To evaluate toxicity and define the MBD of Ampligen as an adjuvant with HER2 vaccination 2) determine if Ampligen MBD when combined with GMCSF as adjuvant and HER2 vaccination increases incidence/magnitude of HER2 Th1 immunity compared to standard GMCSF alone. Eligibility criteria: Stage II–IV HER2+ BC patients who: 1) have completed definitive standard treatment, and in clinical remission 2) 14 days post chemotherapy and steroids 3) have adequate blood counts 4) are off trastuzumab 5) have no active autoimmune disease. Statistical methods: In aim 1, we expect mild toxicity between the 4 dose arms, thus lack of efficacy based on incidence of immune response will be evaluated. Six responses must be observed within a dose arm to move forward based on historical 60% response rate (RR) with standard GMCSF (probability of continuing if true RR is 40% and 70% is 0.17, 0.85, respectively). In aim 2, 24 patients/arm provides 80% power to detect 40% difference in incidence of immune response between the 2 groups (Pearson chi-square test, two-sided alpha of 0.05) and 82% power to assess a 0.85 SD unit difference in change between control and MBD, based on a 2-sample t-test (p=0.05) and effect size defined as the difference in the means divided by the common SD. Incidences of HER2 Th1 immunity will be compared across treatment arms via Pearson chi-square test; magnitude of immune response will be compared across groups via linear regression model. Study Accrual: Target accrual is 88 patients: Stage 1 (n=40) and Stage II (n=48). There has been no accrual at this time. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr OT1-02-10.

OT3-01-18: Combination Immunotherapy with Trastuzumab and the HER2 Vaccine E75 in Low and Intermediate HER2−Expressing Breast Cancer Patients To Prevent Recurrence.

Abstract Background In a phase II trial, the HER2−derived E75 vaccine administered with the immunoadjuvant GM-CSF has been shown to reduce breast cancer recurrence in the adjuvant setting, with a greater benefit seen in patients with low levels of HER2 expression (IHC 1+ or 2+). There has also been recent suggestion that patients with low HER2 expression may also benefit from trastuzumab. Preclinical testing of the combination of trastuzumab and the E75 vaccine has shown a synergism with combinational therapy. Finally, from our phase II trials of cytotoxic T cell-eliciting peptide vaccines, sequential treatment with trastuzumab and HER2 vaccination has resulted in no recurrences in 30 patients with a median follow-up of 48 months. Based on these results, we have designed a trial to evaluate the ability of the combination of trastuzumab and the E75 vaccine to prevent breast cancer recurrence. Trial Design: This study will be a multi-center, prospective, randomized, single-blinded, phase II trial evaluating trastuzumab + E75+GM-CSF (immunoadjuvant) vs. trastuzumab + GM-CSF alone (no E75) in the adjuvant setting in breast cancer patients. Eligible patients include node positive (or node negative if negative for both ER and PR) disease-free breast cancer patients with low or intermediate levels of HER2 expression (IHC 1+ or 2+) and adequate cardiac function (LVEF >50%). Patients must be HLA-A2/A3+ (E75 is HLA-A2/A3-restricted). Patients will be enrolled after completing standard of care multi-modal therapy and randomized between the two treatment arms with stratification by HER2 expression (1+ or 2+) and nodal status (N0, N1, N2, or N3). Vaccinations (E75+GM-CSF or GM-CSF alone) will be administered as six monthly intradermal inoculations concurrently with trastuzumab therapy. The primary efficacy endpoint is to compare disease-free survival (DFS) between treatment arms at 24 months. Secondary objectives will include evaluation of cardiac toxicity from combination therapy (periodic cardiac assessment with MUGA or ECHO), DFS at 36 months, and immunologic responses to vaccination. From previously published experience with trastuzumab, we expect a recurrence rate of 15% in trastuzumab (plus GM-CSF) treated patients and anticipate that the combination of trastuzumab with E75+GM-CSF will reduce this recurrence rate to 5%. In order to show a statistical difference between these recurrence rates, we plan to enroll 150 patients per treatment arm (300 total) with a type I error rate of 5% and 80% power to detect the primary endpoint. Trial accrual is anticipated to begin in January 2012, with a two year period of enrollment followed by a three year follow-up period. Conclusion: We hypothesize that combination adjuvant immunotherapy with trastuzumab and E75 vaccination will result in a greater reduction in breast cancer recurrence than trastuzumab therapy alone and have designed a multi-center, prospective, randomized, single-blinded, phase II trial evaluating the efficacy of this immunotherapy combination. Contact Information: This trial is sponsored by Genentech and RXi Pharmaceuticals through the Henry M. Jackson Foundation. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr OT3-01-18.

OT2-05-05: Phase I/II Study of Adoptive T Cell Therapy Following In Vivo Priming with a HER2 Peptide-Based Vaccine in Patients with Stage IV Breast Cancer.

Abstract Background Adoptive T cell therapy has evolved from preclinical setting to a potentially feasible treatment strategy for advanced breast cancer (BC). However, the ability to expand tumor antigen specific T cells ex vivo has been one of the major hurdles that has limited clinical translation of adoptive T cell therapy. Tumor specific T cells are rare in unprimed patients and generating large bulk cultures from rare precursor frequencies is difficult. We have found immunizing HER2+ patients to increase tumor specific T cell precursor frequencies to the levels of a vaccinated foreign antigen markedly improves the ability to generate large numbers of tumor specific T cells in vitro. We hypothesize that T cell expansion strategies that are facilitated by prior immunization will be clinically useful in the treatment of advanced BC. Design: A Phase I/II non-randomized, single arm study. Priming with a HER2 ICD vaccine will be performed at 1 week intervals for a total of 3 vaccines. Patients will undergo leukapheresis 2 weeks after the 3rd vaccine to collect PBMC for T cell expansion. Patients will be pre-treated with cyclophosphamide 24 hours prior to 1st T cell infusion and then receive up to 3 dose-escalating infusions of T cells given 7–10 days apart. Three HER2 vaccine booster immunizations will then be administered at 1, 2, and 4 months after the final T cell infusion. Follow-up for persistent and continued immunity will then ensue. Aims: To evaluate the safety of infusing escalating doses of HER2 specific T cells into patients with advanced HER2+ BC using ex vivo expanded autologous T cells, to investigate to what extent HER2 specific T cell immunity can be boosted or generated in individuals after infusion of HER2 specific T cells, to evaluate how long T cell immune augmentation persists in vivo after adoptive transfer of HER2 specific T cells and subsequent booster immunizations. Criteria: Patients with HER2+ Stage IV BC who: have been maximally treated and not achieved a complete remission, have stable or slowly progressive disease, HER2+, and have adequate LVEF. Statistical Methods: Toxicity will be determined by chemical and clinical parameters evaluated at various time points. If the true probability of a DLT is 0.11, then the probability of observing 0 DLT's in 20 patients is 0.097. If the true probability of a DLT is 0.18, then the probability of observing ≤1 DLT's in 20 patients is 0.102, and if the true probability of a DLT is 0.27, then the probability of observing ≤2 DLT's in 20 patients is 0.064. Therefore, with low observed rates of DLT (≤10%), we can be reasonably confident (∼90%) with 20 patients that the true DLT rate is < 0.27. Immunologic response, defined as the successful boosting of precursor frequency with infusion of HER2−specific T cells, will be evaluated by assessing the change in T cell level from baseline. To assess the durability of the T cell response, we are primarily interested in estimating the proportion of patients whose T cells persist at a level the same or greater as the level after the final T cell infusion as long as 6 months following the final booster vaccine. Accrual: Target=20/Actual=14 Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr OT2-05-05.

The ubiquitin-like protein, ISG15, is a novel tumor-associated antigen for cancer immunotherapy

The recent announcement of the first FDA-approved therapeutic vaccine for prostate cancer, Sipuleucel-T, is a watershed moment for the field of tumor immunotherapy. However, while Sipuleucel-T provides a powerful tool to clinicians for the most prevalent form of cancer in men, there remains an unmet need for a similar therapeutic strategy against breast cancer, the most prevalent cancer in women. While current breast cancer vaccines in development target several antigens, the most prevalent is the tumor-associated antigen, HER2. Initial results with HER2 vaccines appear promising in terms of efficacy; however, the lack of HER2 overexpression by a majority of breast tumors and the safety concerns associated with current HER2-targeted immunotherapy suggest that additional therapeutic strategies would be beneficial. Recently, several studies have identified ISG15 as a molecule highly expressed in numerous malignancies. ISG15 is a small ubiquitin-like protein regulated by type-I interferon and classically associated with viral defense. Elevated ISG15 expression in breast cancer is especially well documented and is independent of HER2, progesterone receptor, and estrogen receptor status. Additionally, high ISG15 expression in breast cancer correlates with an unfavorable prognosis and poor responses to traditional treatment strategies such as chemotherapy and radiation. To overcome these challenges, we employ a novel strategy to specifically target tumor-associated ISG15 expression with immunotherapy. We demonstrate that vaccination against ISG15 results in significant CD8-mediated reductions in both primary and metastatic mammary tumor burden. These results validate ISG15 as a tumor-associated antigen for cancer immunotherapy.

Abstract PL2-1: Drug Development — Targeting HER2 beyond Trastuzumab

Abstract The use of trastuzumab in the treatment of early and advanced stage HER2 overexpressing (HER2+) breast cancers represents a significant advance in the treatment of breast cancer. In the advanced stage setting, the development of therapeutic resistance, either primary or secondary/acquired, is a clinical problem limiting the clinical efficacy of trastuzumab. This discussion will include alternative approaches to the treatment of HER2+ breast cancers, and strategies to enhance the clinical efficacy of trastuzumab by overcoming the development of therapeutic resistance. Elucidating mechanisms of resistance provides an opportunity to develop combination strategies based on scientific rationale to overcome or prevent its development. For example, the IGF1/IGF-1R signaling axis, which is frequently co-expressed in HER2+ breast cancers promotes trastuzumab resistance in preclinical models. IGF-1R inhibitors overcomes trastuzumab resistance in preclinical models, providing a strong rationale for evaluating the combination in clinical trials. Similarly, the pro-survival PI3K-Akt signaling network is frequently deregulated in breast cancers as a consequence of PI3KCA gain-of-function mutations or loss of the tumor suppressor PTEN. In preclinical models, deregulated PI3K signaling mediates therapeutic resistance to trastuzumab, and small molecule PI3K inhibitors have been shown to overcome trastuzumab resistance. Other mechanisms of resistance to trastuzumab include the presence of heregulin (HRG), a soluble growth factor ligand to HER3 and HER4, which promotes the formation of HER2/HER3 heterodimers, a complex that potently activates PI3K signaling. Other HER2 antibodies e.g. pertuzumab, are more effective at blocking the effects of HRG on HER2/HER3 dimerization. Another mechanism of resistance is truncation of HER2, resulting in a receptor that lacks the extracellular domain that contains the trastuzumab binding site. Strategies to prevent HER2 truncation have been examined preclinically. HER2 signaling has also been targeted using small molecules that compete with ATP for binding at the catalytic kinase domain of HER2. Lapatinib, an oral, reversible inhibitor of HER2 and EGFR tyrosine kinases is approved in combination with capecitabine for treating advanced stage HER2+ breast cancers that progressed on prior trastuzumab-based therapies. The dilemma is that the majority of women treated with lapatinib develop therapeutic resistance within 12 months of initiating therapy. Mechanisms of therapeutic resistance to lapatinib have been identified including (i) de-repression of estrogen receptor (ER) signaling, and (ii) activation of the pro-survival mediator NF-kB; the role of PTEN loss and the presence of PI3K activating mutations in the development of lapatinib resistance remains controversial. This insight has led to rational combinations to overcome or perhaps even delay the development of lapatinib resistance e.g. approval of letrozole plus lapatinib in HER2+/ER+ breast cancers. And finally, other HER2 targeted approaches will be discussed, including hsp90 antagonists, HER2 antibody conjugates (e.g. T-DM1), HER2 vaccines, and combinations of antibody-based and HER2 kinase inhibitors. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PL2-1.

Reply to F. Bellati et al

The antitumor activity of trastuzumab in human epidermal growth factor receptor 2 (HER2) –positive breast cancer has been ascribed to both immunologic and nonimmunologic mechanisms. It is unlikely that any one of these mechanisms alone is responsible for the antitumor activity of trastuzumab. We thank Bellati et al for pointing out the importance of the adaptive immune response as another mechanism involved in mediating trastuzumab antitumor activity. We agree with Bellati et al that HER2-based vaccines represent a promising therapeutic approach in the treatment of breast cancer. Our group is conducting an ongoing study combining lapatinib with a HER2-directed vaccine to further understand the role of the immune system in HER2-directed therapy antitumor activity (ClinicalTrials .gov Identifier: NCT00952692). As we have previously shown in preclinical models, total HER2 blockade using anti-HER2 antibodies generated in response to various HER2 vaccines in combination with lapatinib results in enhanced antitumor activity compared with either treatment alone. These experiments were conducted in cell culture without the addition of immune effector cells, which further implicates the role of nonimmunologic mechanisms in the antitumor activity. The clinical validation of these studies was recently demonstrated in a randomized phase III clinical trial in women with advanced stage HER2-positive breast cancer who had progressed on trastuzumab-containing therapy, where we showed the clinical benefit of combining trastuzumab with lapatinib compared with lapatinib treatment alone. This should dispel some of the concern about possible antagonistic effects of combining trastuzumab with small molecule inhibitors of ErbB2 tyrosine kinase activity. In addition, the article referenced in the correspondence of Bellati et al described the binding of lapatinib to epidermal growth factor receptor by x-ray crystallography, not HER2. To our knowledge, similar x-ray crystallographic studies of lapatinib interaction with HER2 have not been reported. Finally, we completely agree with Bellati et al and Winer and Burstein in their Editorial that greater insight into predictors of response to targeted therapies, such as trastuzumab, is needed. Quantitative HER2 status alone has clearly not been sufficient to predict for clinical response to trastuzumab. To more effectively treat individuals with HER2-positive breast cancer, a greater understanding of the tumor profile and patient immunological status that predict for clinical response to trastuzumab and potentially other HER2-targeted therapies is needed.

Molecular therapy of breast cancer: progress and future directions

Breast cancer is a major cause of death in Western women, with a 10% lifetime risk of the disease. Most breast cancers are estrogen-dependent. Molecular therapies for breast cancer have developed rapidly in the past few decades and future treatment strategies are being investigated. The selective estrogen receptor (ER) modulator tamoxifen, which until now has served as a standard therapy, functions not only as an estrogen antagonist but also as an estrogen agonist in terms of bone maintenance. Aromatase inhibitors have performed well in international trials and have become a new standard therapy for estrogen-dependent breast cancer. The systematic study of estrogen activation pathways suggests that the enzymes steroid sulfatase and 17beta-hydroxysteroid dehydrogenase type 1, which both have pivotal roles in estrogen biosynthesis, are promising targets; the results of a phase I trial of steroid sulfatase inhibitors are encouraging. The activity of the human epidermal growth factor receptor (HER) pathway correlates negatively with that of the ER. HER2 is overexpressed in 22% of all breast cancers. In the decade since HER2 began being targeted, the monoclonal antibody trastuzumab has been used as well as pertuzumab and HER2 vaccines. Among the estrogen-independent breast cancers, the basal-like subtype has low survival, and therapeutic improvement is a priority. Crosstalk between ER and HER2 signaling pathways means that combinatory therapies may hold the key to enhancement of treatment responses. Other molecular therapies involving functional genomics and RNA interference studies also hold promise.

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Abstract 2421: An adenoviral vaccine encoding full-length inactivated human HER2 exhibits potent immunogenicity and enhanced therapeutic efficacy without oncogenicity

Abstract While HER2 oncogene directed therapies such as lapatinib and trastuzumab confer clinical benefits, a majority of metastatic HER2+ patients eventually experience progressive disease; however, their tumors remain strongly HER2+. Thus, current therapeutic limitations combined with continued HER2 expression in patients makes immunotherapeutic vaccination against HER2 an attractive strategy. But as HER2 is an oncogene, it is imperative to develop HER2 vaccines with maximal immunologic potential, but minimal oncologic potential. To address these issues, we created recombinant adenoviral vectors expressing the extracellular domain of HER2 (Ad-HER2-ECD), ECD plus the transmembrane domain (Ad-HER2-ECD-TM) and full length HER2 inactivated for kinase function (Ad-HER2-ki) and determined their relative immunogenicity, anti-tumor effectiveness, as well as their oncogenic potential. As an immunotherapeutic vaccine, we found that that both Ad-HER2-ki and Ad-HER2-ECD-TM were highly effective in eliciting significant T-cell and antibody responses to HER2 in naïve mouse models compared to plasmid vaccination with HER2-ki constructs, thus validating the immunologic efficacy of the adenoviral platform. In contrast, we did not observe any induction of HER2 specific T-cell or antibody responses in HER2-ECD vaccinated animals. While the strong immune responses from both Ad-HER2-ki and Ad-HER2-ECD-TM vectors translated into significantly retarded tumor growth in naïve animals, our studies revealed that Ad-HER2ki vaccinated animals had the most significant HER2-specific T-cell responses as well as the most significant anti-tumor response. When a HER2+ tolerant mouse model was used, we observed that direct Ad-HER2-ki vaccination elicited only slightly diminished T-cell and antibody responses compared to HER2 naïve mice. Most significantly, we were able to demonstrate that Ad-HER2-ki vaccination of HER2+ tolerant mice elicits significant anti-HER2+ tumor responses. Subsequent investigation into the oncogenicity associated with strong overexpression of HER2-ki revealed no evidence for its oncogenic functionality in terms of phosphorylation or transcriptional signature in primary human cells. We also found no evidence for its oncogenicity in enabling enhanced cellular proliferation, anchorage-independent growth, or transformation in vivo. In sum, we found that vaccination with mutationally inactivated, non-oncogenic Ad-HER2-ki results in robust polyclonal immune responses to HER2 in tolerant models, which translate into strong and effective anti-tumor responses in vivo. Ad-HER2-ki is thus a safe and promising vaccine for evaluation in clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2421.

An Adenoviral Vaccine Encoding Full-Length Inactivated Human Her2 Exhibits Potent Immunogenicty and Enhanced Therapeutic Efficacy without Oncogenicity

Overexpression of the breast cancer oncogene HER2 correlates with poor survival. Current HER2-directed therapies confer limited clinical benefits and most patients experience progressive disease. Because refractory tumors remain strongly HER2+, vaccine approaches targeting HER2 have therapeutic potential, but wild type (wt) HER2 cannot safely be delivered in immunogenic viral vectors because it is a potent oncogene. We designed and tested several HER2 vaccines devoid of oncogenic activity to develop a safe vaccine for clinical use. We created recombinant adenoviral vectors expressing the extracellular domain of HER2 (Ad-HER2-ECD), ECD plus the transmembrane domain (Ad-HER2-ECD-TM), and full-length HER2 inactivated for kinase function (Ad-HER2-ki), and determined their immunogenicity and antitumor effect in wild type (WT) and HER2-tolerant mice. To assess their safety, we compared their effect on the cellular transcriptome, cell proliferation, anchorage-dependent growth, and transformation potential in vivo. Ad-HER2-ki was the most immunogenic vector in WT animals, retained immunogenicity in HER2-transgenic tolerant animals, and showed strong therapeutic efficacy in treatment models. Despite being highly expressed, HER2-ki protein was not phosphorylated and did not produce an oncogenic gene signature in primary human cells. Moreover, in contrast to HER2-wt, cells overexpressing HER2-ki were less proliferative, displayed less anchorage-independent growth, and were not transformed in vivo. Vaccination with mutationally inactivated, nononcogenic Ad-HER2-ki results in robust polyclonal immune responses to HER2 in tolerant models, which translates into strong and effective antitumor responses in vivo. Ad-HER2-ki is thus a safe and promising vaccine for evaluation in clinical trials.

Phase II Study of a HER-2/Neu (HER2) Intracellular Domain (ICD) Vaccine Given Concurrently with Trastuzumab in Patients with Newly Diagnosed Advanced Stage Breast Cancer.

Abstract HER2 is a tumor antigen in breast cancer and several trials have demonstrated that breast cancer patients can be immunized against this protein. We have developed HER2 peptide based vaccines that are aimed at eliciting CD4+ Th1 tumor antigen specific T cell responses. Th1 effectors provide immunologic memory, enhance cross priming which will allow the elaboration of tumor specific CD8+ T cells, and stimulate epitope spreading which we have shown to be a potential biomarker of clinical response. 52 patients will be enrolled with the primary objective to determine relapse free survival after active immunization. Eligible patients are newly diagnosed with Stage III (B or C) or Stage IV breast cancer and begin vaccination within 6 months of starting maintenance trastuzumab. This interim report will present data on the first 25 patients enrolled; 21 stage IV and 4 locally advanced patients. The vaccine is well tolerated with all adverse events (AE) being Grade I or 2. The most common AE is injection site reaction. Moreover, the combination of HER2 vaccination with trastuzumab did not result in additive cardiac toxicity in these patients. Immune responses were evaluated by IFN-gamma ELISPOT. To date, 88% of patients immunized developed significant immunity to the components of the ICD vaccine. The majority, 75%, developed robust immunity to the HER2 protein. Our group has recently demonstrated that a broadening of immunity throughout the HER2 protein, to components of the protein that weren't in the vaccine, i.e. epitope spreading, may be associated with improved survival in vaccinated patients. 63% of immunized patients demonstrated evidence of intramolecular epitope spreading. We questioned whether such high frequencies of homing Type 1 T cells might modulate the immunosuppressive tumor microenvironment, so we evaluated whether circulating serum immunosuppressive cytokines were impacted by immunization. TGF-beta is an immunosuppressive cytokine secreted by tumor stroma and regulatory T cells. We found that the levels of serum TGF-beta decreased significantly in the majority of patients after vaccination. We further analyzed the correlation between the change of serum levels of TGF-beta post vaccination and HER2 ICD vaccine-induced T cell responses. We found that the greater the magnitude of the HER2 specific T cell response, as demonstrated by IFN-gamma secretion, the greater the decrease in serum TGF-beta (p=0.0045, r=0.742). The correlation between the increased epitope spreading T cell response and decreased levels of TGF-beta was even more significant (p=0.0003). The median overall survival has not been reached with 100% of patients alive at this time. Relapse free survival data will be presented. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5102.

The HER-2 receptor and breast cancer: ten years of targeted anti-HER-2 therapy and personalized medicine.

The human epidermal growth factor receptor (HER-2) oncogene encodes a transmembrane tyrosine kinase receptor that has evolved as a major classifier of invasive breast cancer and target of therapy for the disease. The validation of the general prognostic significance of HER-2 gene amplification and protein overexpression in the absence of anti-HER-2 targeted therapy is discussed in a study of 107 published studies involving 39,730 patients, which produced an overall HER-2-positive rate of 22.2% and a mean relative risk for overall survival (OS) of 2.74. The issue of HER-2 status in primary versus metastatic breast cancer is considered along with a section on the features of metastatic HER-2-positive disease. The major marketed slide-based HER-2 testing approaches, immunohistochemistry, fluorescence in situ hybridization, and chromogenic in situ hybridization, are presented and contrasted in detail against the background of the published American Society of Clinical Oncology-College of American Pathologists guidelines for HER-2 testing. Testing issues, such as the impact of chromosome 17 polysomy and local versus central HER-2 testing, are also discussed. Emerging novel HER-2 testing techniques, including mRNA-based testing by real-time polymerase chain reaction and DNA microarray methods, HER-2 receptor dimerization, phosphorylated HER-2 receptors, and HER-2 status in circulating tumor cells, are also considered. A series of biomarkers potentially associated with resistance to trastuzumab is discussed with emphasis on the phosphatase and tensin homologue deleted on chromosome ten/Akt and insulin-like growth factor receptor pathways. The efficacy results for the more recently approved small molecule HER-1/HER-2 kinase inhibitor lapatinib are also presented along with a more limited review of markers of resistance for this agent. Additional topics in this section include combinations of both anti-HER-2 targeted therapies together as well as with novel agents including bevacizumab, everolimus, and tenespimycin. A series of novel HER-2-targeting agents is also presented, including pertuzumab, ertumaxomab, HER-2 vaccines, and recently discovered tyrosine kinase inhibitors. Biomarkers predictive of HER-2 targeted therapy toxicity are included, and the review concludes with a consideration of HER-2 status in the prediction of response to non-HER-2 targeted treatments including hormonal therapy, anthracyclines, and taxanes.

Genetic Regulation of the Response to Her-2 DNA Vaccination in Human Her-2 Transgenic Mice

Genetic regulation of immune reactivity to Her-2 vaccination and the consequent antitumor effect was tested in human Her-2 transgenic (Tg) mice of C57BL/6 (B6), BALB/c (BALB), and (B6x BALB) F1 (F1) background. Mice were electrovaccinated with Her-2 DNA with or without pretreatment with CD25 monoclonal antibody to remove CD25(hi) regulatory T cells. When CD25(+) T cells were intact, BALB Her-2 Tg mice were more responsive than the other two strains in both humoral and cellular immunities, and their tumor growth was significantly delayed. B6 Her-2 Tg mice responded poorly and F1 mice showed modest immune reactivity, but tumor growth did not change in either strain. Depletion of CD25(hi) T cells before vaccination significantly improved protection from tumor challenge in F1 Her-2 Tg mice. This was associated with elevated levels of Her-2 IgG1, IgG2a, and IgG2c antibodies, and some mice also showed IFN-gamma producing T-cell response. The same treatment induced modest improvement in B6 Her-2 Tg mice. In BALB Her-2 Tg mice, however, depletion of CD25(hi) T cells did not further improve antitumor efficacy. Although their Her-2-specific IgG1 and interleukin-5-secreting T cells increased, the levels of IgG2a and IFN-gamma-secreting T cells did not change. These results are the first to show genetic regulation of the response to a cancer vaccine and an unequal effect of removing CD25(hi) T cells on antitumor immunity. These results warrant individualized treatment plans for patients with heterogeneous genetic backgrounds and possibly differential intrinsic immune reactivity to tumor antigens.

Her-2 DNA versus cell vaccine: immunogenicity and anti-tumor activity

Direct comparison and ranking of vaccine formulations in pre-clinical studies will expedite the identification of cancer vaccines for clinical trials. Two human ErbB-2 (Her-2) vaccines, naked DNA and whole cell vaccine, were tested side-by-side in wild type and Her-2 transgenic mice. Both vaccines can induce humoral and cellular immunity to the entire repertoire of Her-2 epitopes. Mice were electro-vaccinated i.m. with a mixture of pGM-CSF and pE2TM, the latter encodes Her-2 extracellular and transmembrane domains. Alternatively, mice were injected i.p. with human ovarian cancer SKOV3 cells that have amplified Her-2. In wild type mice, comparable levels of Her-2 antibodies (Ab) were induced by these two vaccines. However, T cell immunity and protection against Her-2(+) tumors were superior in DNA vaccinated mice. In BALB Her-2 transgenic (Tg) mice, which were tolerant to Her-2, DNA and cell vaccines were administered after regulatory T cells (Treg) were removed by anti-CD25 mAb. Again, comparable levels of Her-2 Ab were induced, but DNA vaccines rendered greater anti-tumor activity. In B6xDR3 Her-2 Tg mice that expressed the autoimmune prone HLA-DR3 allele, higher levels of Her-2 Ab were induced by SKOV3 cell than by Her-2 DNA. But anti-tumor activity was still more profound in DNA vaccinated mice. Therefore, Her-2 DNA vaccine induced greater anti-tumor immunity than cell vaccine, whether mice were tolerant to Her-2 or susceptible to autoimmunity. Through such side-by-side comparisons in appropriate pre-clinical test systems, the more effective vaccine formulations will emerge as candidates for clinical trials.

602 POSTER An anti-idiotypic HER2 vaccine can reverse immunological tolerance to HER2 and induce anti-tumor immunity in huHER2 female transgenic mice

602 POSTER An anti-idiotypic HER2 vaccine can reverse immunological tolerance to HER2 and induce anti-tumor immunity in huHER2 female transgenic mice

DNA Vaccination Controls Her-2+ Tumors that Are Refractory to Targeted Therapies

Her-2/neu(+) tumor cells refractory to antibody or receptor tyrosine kinase inhibitors are emerging in treated patients. To investigate if drug resistant tumors can be controlled by active vaccination, gefitinib and antibody sensitivity of four neu(+) BALB/c mouse mammary tumor lines were compared. Significant differences in cell proliferation and Akt phosphorylation were observed. Treatment-induced drug resistance was associated with increased chromosomal aberrations as shown by spectral karyotyping analysis, suggesting changes beyond neu signaling pathways. When mice were immunized with pneuTM encoding the extracellular and transmembrane domains of neu, antibody and T-cell responses were induced, and both drug-sensitive and drug-resistant tumor cells were rejected. In T-cell-depleted mice, drug-sensitive tumors were still rejected by vaccination, but drug-refractory tumors survived in some mice, indicating their resistance to anti-neu antibodies. To further test if T cells alone can mediate tumor rejection, mice were immunized with pcytneu encoding full-length cytoplasmic neu that is rapidly degraded by the proteasome to activate CD8 T cells without inducing antibody response. All test tumors were rejected in pcytneu-immunized mice, regardless of their sensitivity to gefitinib or antibody. Therefore, cytotoxic T lymphocytes activated by the complete repertoire of neu epitopes were effective against all test tumors. These results warrant Her-2 vaccination whether tumor cells are sensitive or resistant to Her-2-targeted drugs or antibody therapy.