Background A. HER2 is a tumor antigen in human adenocarcinomas . The HER2 oncogenic protein consists of a cysteine rich extracellular ligand binding domain, a short transmembrane domain, and cytoplas mic protein tyrosine kinase domain. (2,3) Binding of ligand to the extracellular domain (ECD) leads to dimerization that stimulates the intrinsic tyrosine kinase activity of the receptor and triggers autophosphorylation of specific tyrosi ne residues within the intracellular cytoplasmic domain (ICD). These phosphorylated residues then serve as anchoring sites for signaling molecules involved in the regulation of intracellular signaling cascades (2) and, thus, cell growth. HER2 is a self -protein expressed in a variety of tissues of epithelial origin and plays a fundamental role in cellular proliferation and differentiation during fetal development. In adults, the HER2 gene is present as a single copy in normal cells; howev er, amplification of the gene and resultant protein overexpression is seen in various cancers including breast, ovarian, colon, uterine, gastric, prostate, and adenocarcinoma of the lung. Furthermore, the overexpression of HER2 is implicated in the malignant transformation of breast cancer (4 -6) and is a biologically relevant protein in the pathogenesis of several other epithelial- based tumors, for example leading to the development of hormone resistance in prostate cancer. (7) Relapse is a significant clinical problem for breast cancer patients whose tumors overexpress HER2. (8) HER2 is also a tumor antigen. Patients whose tumors overexpress the HER2 protein can have both low level antibody and T cell i mmunity directed against the protein. (9) Generating an active immune response directed against the HER2 protein has several potential clinical advantages. Vaccination, if effective, would stimulate immunologic memory and could result in the prevention of relapse after standard therapy. 6304p Protocol 118 04/06/2016 B. Patients with HER2 over expressing tumors can be safely immunized with HER2 derived peptides and generate a HER2 specific CTL response. The CTL immune response has been considered the primary effect or arm involved in mediating an anti -tumor immune response. Experimental vaccination strategies designed to stimulate tumor specific CTL responses in vivo vaccinate individuals with tumor cells or viruses recombinant for tumor antigens that can infect via ble cells so that proteins are expressed inside the cell, processed and presented in the class I MHC antigen processing pathway. An alternative strategy uses soluble peptide that is identical or similar to naturally processed peptides present in class I M HC molecules as the vaccine along with an adjuvant. HLA -A2 binding peptide p369- 377, derived from the protein sequence of HER2 ECD, has been used extensively in clinical trials. In an initial clinical study, HLA -A2 positive patients with metastatic HER2 overexpressing breast, ovarian, or colorectal carcinomas were immunized with 1 mg of p369- 377 admixed in incomplete Freund’s adjuvant (IFA) every 3 weeks. (10) Peptide specific CTL were isolated and expanded from the peripheral blood of p atients after 2 or 4 immunizations. The CTL could lyse HLA matched peptide pulsed target cells but could not lyse HLA matched tumors expressing the HER2 protein. More recently, a similar study was performed immunizing patients with p369 -377 using GM -CSF as an adjuvant. (11) GM-CSF is a recruitment and maturation factor for skin dendritic cells (DC), Langerhans cells (LC) and theoretically provides more efficient presentation of peptide epitopes than standard adjuvants such as IFA. HLA -A2 patients with HER2 -overexpressing cancers received 6 monthly vaccinations with 500 mcg of HER2 peptide, p369- 377 peptide, admixed with 100 mcg of GM -CSF. The patients had either stage III or IV breast or ovarian cancer. Immune responses to the p369 -377 were examined using an IFN -gamma ELIspot assay. Prior to vaccination, the median precursor frequency, defined as precursors/10 6 PBMC, to p369- 377 was not detectable. Following vaccination, HER2 peptide -specific precursors developed to p369- 377 in just 2 of 4 evaluable subjects. The responses were short -lived and not detectable at 5 months after the final vaccination. Immunocompetence was evident as patients had detectable T cell responses to tetanus toxoid and influenza. These results demonstrate that HER2 MHC class I epitopes can induce HER2 peptide -specific IFN -gamma - producing CD8+ T cells. However, the magnitudes of the responses were low, as well as short -lived. Theoretically, the addition of CD4+ T cell help would allow the generation of lasting immunity. Interestingly, immunizing patients with putative T -helper epitopes from HER2 that have HER2 HLA -A2 binding motifs embedded in their natural sequence resulted in the generation of peptide -specific CTL capable of lysing HER2 overexpressing tumor and immune responses were durable. (12) In that trial, 19 HLA -A2 patients with HER2 -overexpressing cancers received the vaccine, twelve patients developed both HER2 specific CD4+ and CD8+ T cell responses, and the level of HER2 immunity w as similar to immunity to KLH, a foreign protein used as a control immunization (Fig. 1). Importantly, the immune responses were detectable for greater than 1 year after the final vaccination in selected patients. These results demonstrate that HER2 MHC class II epitopes containing encompassed MHC class I epitopes are able to induce long -lasting HER2 -specific IFN -gamma - producing CD8 T cells. Indeed, when a comparison was made between the clinical trial that used a HER2 HLA - A2 binding peptide alone vs. im munizing to achieve CTL using a class II peptide with a class I peptide embedded in the natural sequence, the latter immunization strategy was necessary to maintain both high magnitude and long lived CD8+ T cell immunity (Fig. 2). We will evaluate this va ccine, composed of HER2 T helper epitopes which encompass HLA -A2 CD8+ epitopes of HER2 in combination with trastuzumab to augment HER2 specific CTL T cell immunity in a combination immunotherapy approach. C. Immunization with a HER2 peptide based vaccin e, designed to elicit CTL, is associated with the development of immunologic memory and epitope spreading . Epitope spreading was first described in autoimmune disease (13) and has been associated with both MHC Class I - and MHC Class II -restricted responses . An additional finding of the study described above (12) as well as a larger clinical trial vaccinating patients with a variety of putative class II binding HER2 epitopes (14) was that epitope spreading was observed in 84% of patients and significantly correlated with the generation of HER2 protein- specific T cell immunity (p=.03). Epitope spreading represents the generation of an immune response to a particular portion of an immunogenic protein and the natural spread of that immunity to other areas of the protein or even to other antigens present in the environment. (15,16) In this study, epitope spreading reflected the extension of a significant T cell immune response to portions of t he HER2 protein that were not contained in the patient’s vaccine (Fig. 3). Theoretically, a broadening of the immune response may represent endogenous processing of 6304p Protocol 118 04/06/2016 antigen at sites of inflammation initiated by a specific T cell response or “driver clone” . (17) The initial immune response can create a microenvironment at the site of the tumor that enhances endogenous immune effector cells present locally. These immune cells, e.g. APC and T cells present antigen and create a CTL response to tumor antigen present in the body. It may be that the development of epitope spreading is a better predictor of vaccine efficacy than the magnitude of the immune response generated. (18) We have recently begun to assess the presence of long term immunity in previously immunized patients. Figure 4 demonstrates persistent detectable HER2 specific immunity and epitope spreading present in a patient over 4 years after receiving her last immunization with the HER2 CTL vaccine. This is o ne of the first demonstrations of persistent long term immunity developing in a cancer patient after the administration of a cancer vaccine. The mechanisms responsible for the maintenance of memory T cells are not entirely known. Studies have shown that memory cells are more metabolically active than naïve T cells suggesting that memory cells may be continuously signaled by extrinsic factors. Such signaling most likely involves some degree of contact with persistent antigen. As most of the patients in our vaccine trials are advanced stage patients who have been treated to maximal response and invariably have minimal residual disease, it may be that this persistent microscopic tumor stimulates the persistence of immunity. Overall survival in patients wit h HER2+ Stage IV breast cancer is well defined. (19) A recent trial randomized over 400 stage IV breast cancer patients to receive chemotherapy alone vs. chemotherapy plus trastuzumab as primary therapy for their disease. The median overall survival for the patients receiving chemotherapy alone was 20 months and for those receiving the combination antibody and chemotherapy was 29 months (Fig. 5). Patients from the HER2 peptide based vaccine studies (12) have now been fol lowed a median of 5 years (Fig. 6). Six of the 9 (67%) patients who completed immunization with the CTL vaccine are still alive. The 74% overall survival (OS) Kaplan -Meyer statistic on this subset is limited because of the small number of patients, howev er, the historical control data for stage IV patients with distant metastasis suggests a benefit to vaccination. Although HER2 overexpressing ovarian cancer patients treated with tr astuzumab alone are eligible for this study, based on our previous trials enrolling breast and ovarian cancer patients we anticipate over 80% of patients enrolled will be Stage IV breast cancer patients. T hus, immunization of patients with advanced stage disease who have been optimally treated to prevent relapse is a clinical g oal of vaccination with the HER2 CTL vaccine. D. Addition of trastuzumab to active immunization with a HER2 CTL peptide based vaccine may result in augmented immunity and the generation of a more vigorous CTL response . Treatment of advanced HER2 overex pressing breast cancer has evolved since our first study was initiated. A common regimen for relapsed stage IV HER2 overexpressing breast cancer is treatment with trastuzumab and chemotherapy for 4 months or until maximal response and trastuzumab monother apy continued for a year. Patients with de novo stage IV disease often receive a longer cycle of chemotherapy with continued trastuzumab for 1 year. For this reason, vaccines given in the setting of stage IV disease need to be administered concurrent wit h trastuzumab if patients are to be immunized in a minimal residual disease state. Trastuzumab, when administered prior to or concurrent with doxorubicin results in a higher incidence of cardiac toxicity tha n what is anticipated with doxorubicin alone. (20) For patients with minimal prior exposure to anthracycline the risk of cardiac dysfunction, defined as decrease in left ventricular ejection fraction (LVEF), was 1%. For patients with more extensive prior doxorubicin exposure the risk of cardiac dysfunction with trastuzumab was 7%. If trastuzumab was given concurrent with doxorubicin the risk of cardiac dysfunction was 29%. The etiology of the cardiac dysfunction related to trastuzumab administration is unknown, but the temporal relat ionship to concurrent doxorubicin exposure suggests a link with anthracycline induced myocardial injury. As this will be the first study evaluating the concurrent administration of trastuzumab and active immunization targeting HER2 we will evaluate any potentially toxicities closely with particular emphasis on cardiac status. There is a good rationale to suggest that trastuzumab concurrent to vaccination may actually augment HER2 specific CTL immunity. Recent studies, by our collaborator, Dr. Helga Ber nhard, have demonstrated that “sensitization” of HER2 overexpressing tumor cells with trastuzumab, in vitro , will enhance the function of CTL specific for HER2. Lytic activity of HER2 specific CTL derived from unprimed patients has been low, in part, beca use HER2 is a self antigen and T cells may display a low affinity T cell receptor due to induction of tolerance. (21-23) On this basis, the lytic potential of HER2 specific CTL could, theoretically, be improved by 6304p Protocol 118 04/06/2016 further increasing the n umber of HLA class I bound peptides on tumor cells with the help of trastuzumab, an inhibitory antibody against HER2. Upon binding of trastuzumab, the HER2 receptor is internalized and degraded, subsequently inhibiting HER2 mediated signal transduction an d tumor cell growth. (24,25) As antibody -induced degradation of HER2 is likely to be accompanied with increased numbers of HER2 peptides presented with HLA molecules, Bernhard and colleagues questioned whether trastuzumab- treated tumor ce lls were more susceptible to CTL -mediated lysis. HER2 reactive CTL clones lysed class I -matched, HER2 overexpressing tumor cells more efficiently after treatment with trastuzumab (Fig. 7). (1) Thus, combination of trastuzumab with the HE R2 CTL vaccine may even further enhance the generation of a HER2 specific CTL response and potentially translate into improved survival for advanced stage breast and ovarian cancer patients when used in the adjuvant setting. 3. Rationale Data from a pha se I study of a HER2 CTL peptide -based vaccine demonstrated the safety of the vaccine when adminis tered as a single immunotherape utic agent. Moreover, in addition to patients developing antigen specific T cell responses after vaccination, 5 -year follow -up of the Stage IV breast cancer patients enrolled on study suggests a potential survival advantage for the patients immunized. Recent studies have demonstrated that “sensitization” of HER2 overexpressing tumor cells with trastuzumab, in vitro , will enhance the function of CTL specific for HER2. Thus, a combination of trastuzumab with the HER2 CTL vaccine may even further enhance the generation of a HER2 specific CTL response. Before moving forward with an efficacy study, however, safety of concomitant adm inistration of vaccine and trastuzumab must be demonstrated. This proposal outlines a phase I -II study of a combined immunotherapeutic approach for the generation of HER2 specific CTL in vivo. 4.