Cellular Immune Parameters Associated with Improved Survival in Breast Cancer Patients after Immunization with a HER2-Specific Vaccine

Transcription

1 Cellular Immune Parameters Associated with Improved Survival in Breast Cancer Patients after Immunization with a HER2-Specific Vaccine Tumor Vaccine Group University of Washington John Strickler November 6, 29

2 Introduction Recent clinical trials of cancer vaccines have failed to demonstrate a correlation of immune response with short-term clinical outcomes The TVG has performed six major clinical trials studying the clinical response of over 2 breast cancer patients to vaccines targeting the human epidermal growth factor receptor 2 (HER2) HER2 immunization induced varying levels of tumor specific cellular immunity as well as epitope spreading

3 Study Aims Determine the effect of the following immunologic parameters on long-term survival following vaccination: Presence of a T cell response to HER2 Magnitude of T cell response to HER2 Presence of epitope spreading

4 Patient Population Table 1. Patient Characteristics Protocol 1 Protocol 118 (Peptide vaccine - ECD, ICD, ECD/ICD) (Peptide vaccine - ECD/ICD) N=48 No. of Patients % N=19 No. of Patients % p-value Characteristic Race Caucasian African-American 2 4 p=.366 Age, years Median (range) 48.8 (25-85) 49.2 (33-76) p=.281 Gender Female Male 1 2 Disease Stage III IV p=.8 Hormone Status Not ER/PR ER and/or PR p=.542 HER-2/neu Status by IHC Unknown 1 2 p=.174 Trastuzumab Ever No Yes p<.1 Concurrent Trastuzumab No 48 1 Yes 19 1 p<.1 Abbreviations: ER, estrogen receptor; PR, progesterone receptor; HER-2, human epidermal growth factor receptor 2

5 1 The presence or absence of a HER2 peptide or protein specific T cell response after immunization did not predict improved survival 8 % Sur v iv al 6 4 Peptide/Protein + (n=55) 2 p=.2222 Peptide/Protein - (n=9) Figure 1 Months

6 The magnitude of the HER2 peptide or protein specific T cell response elicited with immunization is associated with improved survival 1 8 % Su r v iv al 6 4 Peptide/Protein Response > Median (n=33) 2 p=.112 Peptide/Protein Response < Median (n=33) Figure 2 Months

7 Epitope Spreading: Background Definition: T cell responses initiated by a single antigenic epitope evolve into multiepitopic responses* First described in models of autoimmunity Observed in multiple clinical vaccine trials, including melanoma, breast cancer, ovarian cancer, and lung cancer * Butterfield et al, Determinant Spreading Associated with Clinical Response in Dendritic Cell-based Immunotherapy for Malignant Melanoma, Clinical Cancer Research. Vol 9, , March 23.

8 The development of epitope spreading with vaccination is associated with improved survival 1 8 % Su r v iv a l p<.1 ES - (n=19) ES + (n=45) Figure 3 Months

9 2 The highest magnitude HER2 protein specific T cell responses occur in patients who develop epitope spreading with vaccination Stimulation Index to HER2 ICD p<.1 Figure 4 No (N=12) Epitope Spreading Yes (N=43)

10 Epitope Spreading Correlates to Survival in Multivariate Analysis Variable Table 2. Univariate and Multivariate Cox Proportional Hazard Models Category UNIVARIATE ANALYSIS (N=67) HR (95% CI) DISEASE VARIABLES MULTIVARIATE ANALYSIS (N=54) HR (95% CI) Age continuous.99 ( ) ( ).765 Stage III IV 2.99 ( ) ( ).197 Disease Status CR Stable 2.64 ( ) ( ).114 PD 7.78 ( ) ( ).8 Hormone Status (ER/PR) No/Yes 1.8 ( ) ( ).895 # Mets Sites continuous 1.64 ( ).2.89 ( ).783 VACCINATION VARIABLES Vaccinating peptide response No/Yes.42 ( ) ( ).957 Vaccinating protein response No/Yes.86 ( ) ( ).246 Vaccinating peptide/protein response No/Yes.42 ( ).174 Collinear - dropped Max vaccinating peptide response continuous.96 ( ) ( ).378 Max vaccinating protein response continuous.95 ( ) ( ).4 Max vaccinating peptide/protein response continuous.96 ( ) ( ).348 Epitope spreading No/Yes.25 (.1-.6).2.17 (.4-.72).17 P P

11 Conclusions Certain measures of antigen specific immunity, elicited with vaccination, may be markers associated with clinical outcome In multivariate analysis, only epitope spreading elicited with active immunization was an independent predictor of overall survival Development of epitope spreading following vaccination may be a critical parameter to monitor as a potential indicator of beneficial response Vaccine development should focus on strategies designed to induce epitope spreading

12 References 1. Disis, M.L., et al., Generation of Te cell immunity of the HER-2/neu protein after active immunization with HER-2/neu peptide based vaccine. J. Clin Onc, 22. 2(11): Webster, D., et al., A Phase I/II study of a HER-2/neu peptide vaccine plus concurrent trastuzumab. Proc. Amer. Soc. Clin. Onc., 26 Poster 3. Disis, M.L., et al., Generation of immunity to the HER-2/neu oncogenic protein in patients with breast and ovarian cancer using a peptide-based vaccine. Clin Cancer Res, (6): p Salazar,L.G.,etal.,Kinetics of tumor-specific T-cell response development after active immunization in patients with HER-2/neu overexpressing cancers. Clin Immunol, (3): p Coveler, A.L., et al., Common adjuvant breast cancer therapies do not inhibit cancer vaccine induced T cell immunity. Breast Cancer Res Treat, 28 Jan 31 [Epub ahead of print]. 6. Butterfield et al, Determinant Spreading Associated with Clinical Response in Dendritic Cell-based Immunotherapy for Malignant Melanoma. Clin Cancer Res, 23. 9: p

13 Acknowledgements Nora Disis Danelle Wallace Vivian Goodell Tumor Vaccine Group: Lupe Salazar Doreen Higgins Jennifer Childs Kathleen Tietje Yushe Dang Meredith Slota

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15 Appendix

16 Cellular immune parameters associated with improved long term survival in advanced stage breast cancer patients after active immunization with a HER2 specific vaccine JH Strickler, D Wallace, V Goodell, LG Salazar, D Higgins, J Childs, K Tietje, J Link, Y Dang, M Slota, J Waisman, and ML Disis Center for Translational Medicine in Women s Health, Tumor Vaccine Group, University of Washington, Seattle, WA INTRODUCTION Several recent reports of clinical trials of cancer vaccines have failed to demonstrate a correlation of immune response with clinical outcome. The development of epitope spreading with vaccination is associated with improved survival 1 RESULTS Survival was not related to the development of a T cell response to a vaccinating peptide and/or protein (p=.2222) (Figure 1). We have conducted several studies of vaccinating against HER2 in breast cancer patients with minimal residual disease. HER2 immunization induced both tumor specific cellular immunity as well as epitope spreading. We questioned which, if any, cellular immune parameters were associated with improved survival. PATIENTS AND METHODS Patient population: Patient population: One-hundred and six patients with Stage III, or IV HER2 overexpressing breast, ovarian, or lung cancer with stable or completeremissionwereenrolledintwophasei-iistudiesofthreeher2peptide vaccines targeting ECD, ICD, or both ECD and ICD (ECD/ICD) [1, 2]. The studies were approved by both the United States Food and Drug Administration and the University of Washington Institutional Review Board. Patients received intradermal (id.) vaccinations monthly for six months. Blood was obtained for assessment of immunologic responses at baseline, monthly, and during long-term follow-up timepoints. Samples from all 16 patients were available for analysis of HER2 specific T cell response by a tritiated thymadine based modified limiting dilution (mlda) that has been previously described [3]. Subjects were included in the analysis if they had breast cancer and received at least three vaccinations (n=67). We chose 3 vaccinations as a criterion for the ability to be immunized as previous studies by our group have demonstrated 3 immunizations will confer immunity in the majority of patients capable of response [4]. The eligibility criteria of the two studies differed in stage (Protocol 118 enrolled only Stage IV breast cancer patients while Protocol 1 enrolled both Stage III and Stage IV). Furthermore, Protocol 118 allowed the use of concurrent trastuzumab therapy with HER2 immunization. Despite these significant differences we believe the populations are comparable as all patients demonstrated the ability to respond to recall antigens despite advanced stage indicating immune competence. Furthermore, published studies by our group have demonstrated that the concurrent use of trastuzumab with immunization did not impact either the ability to generate an antigen specific T cell response nor the magnitude of the immune response as compared to matched patients receiving a similar vaccine without concurrent trastuzumab [5]. Evaluation of T cell response: Tritiated thymidine incorporation measuring antigen specific proliferation was performed as previously reported [3]. HER2 recombinant proteins (ECD/ICD), immunizing peptides, and non-immunizing peptides (as a reflection of intramolecular epitope spreading) were used for analysis. Data were expressed as a standard stimulation index (SI) that is defined as the mean CPM of 24 experimental wells divided by the mean CPM of the control wells (no antigen). Any SI >2. was considered a positive T cell response based on an analysis of a reference population of age matched control donors. The median level of HER2 specific T cell response was an SI of 5. and, thus, the magnitude of the T cell response was defined as greater or less than the median. Epitope spreading was defined as a positive (SI >2.) post-vaccination response to non-vaccinating peptides or proteins. Statistical methods: Differences in patient characteristics were evaluated using Chi-squared or Mann-Whitney tests. The Mann-Whitney test of equality of medians was performed for ICD responses across epitope spreading. The ICD was used to examine the relationship between magnitude of the T cell response and epitope spreading as trastuzumab treatment resulted in a significant number of patients in Protocol 118 having pre-existent immunity to the HER2 ECD. Overall survival was defined as the time elapsed between beginning initial treatment (vaccination) and death. Data for patients without death were censored at time of last contact. Immunologic variables were considered time-dependent, and patients without epitope spreading (n=19), were censored at the time of their last blood draw. Patients were followed a median of 1.7 years (range 1 month years), mean 3.6 years. Thirty deaths were observed in this cohort of sixty-seven women. Unadjusted Cox proportional hazard models examined univariate associations with mortality for categorical/dichotomous variables, and trends for continuous variables. The multivariate Cox proportional hazard model included all variables found to be significant in univariate analysis at the alpha level of.25 or less, and all clinically significant variables, such as age and stage, regardless of significance. There were sixty-seven patients included in the univariate versus fifty-four in the multivariate analysis due to missing data. Differences in the hazard models was assessed using the Gehan-Breslow-Wilcoxon Test. All analyses were performed using Stata 1. (StataCorp, College Station, TX) or GraphPad Prism 5.1 (GraphPad Software, Inc., San Diego, CA). % Su r v iv al The magnitude of the HER2 peptide or protein specific T cell response elicited with immunization is associated with improved survival 1 % Su r v iv a l The presence or absence of a HER2 peptide or protein specific T cell response after immunization did not predict improved survival p=.2222 Peptide/Protein - (n=9) Figure 1 Months Peptide/Protein Response > Median (n=33) Peptide/Protein + (n=55) 2 Peptide/Protein Response < Median p=.112 (n=33) Figure 2 Months Stimulation Index to HER2 ICD % Su r v iv a l p<.1 ES - (n=19) ES + (n=45) Figure 3 Months The highest magnitude HER2 protein specific T cell responses occur in patients who develop epitope spreading with vaccination Figure 4 p<.1 No (N=12) Yes (N=43) Epitope Spreading Survival was significantly associated with the magnitude of the T cell response to a vaccinating peptide and/or protein (p=.112) (Figure 2). Survival was significantly associated with the development of epitope spreading following vaccination (p<.1) (Figure 3). Regardless of stage, subjects with epitope spreading had significantly higher HER2 protein specific responses (mean SI=3.89) than subjects without epitope spreading (mean SI=1.12), p<.1 (Figure 4). Multivariate analysis indicated that epitope spreading was an independent variable (p=.17, HR=.17) for prediction of overall survival in this population (Table 2). CONCLUSIONS These data suggest that certain measures of antigen specific immunity, elicited with vaccination, may be markers associated with clinical outcome. In multivariate analysis, however, epitope spreading elicited with active immunization was the only immune marker which was an independent predictor of overall survival. Development of epitope spreading following vaccination may be a critical parameter to monitor as a potential indicator of beneficial response. Vaccine development should focus on strategies designed REFERENCES to induce epitope spreading. 1. Disis, M.L., etal., Generation of Te cell immunity of the HER-2/neu protein after active immunization with HER-2/neu peptide based vaccine. J. Clin Onc, 22. 2(11): Webster, D., et al., A Phase I/II study of a HER-2/neu peptide vaccine plus concurrent trastuzumab. Proc. Amer. Soc. Clin. Onc., 26 Poster 3. Disis, M.L., et al., Generation of immunity to the HER-2/neu oncogenic protein in patients with breast and ovarian cancer using a peptide-based vaccine. Clin Cancer Res, (6): p Salazar, L.G., et al., Kinetics of tumor-specific T-cell response development after active immunization in patients with HER-2/neu overexpressing cancers. Clin Immunol, (3): p Coveler, A.L., etal., Common adjuvant breast cancer therapies do not inhibit cancer vaccine induced T cell immunity. Breast Cancer Res Treat, 28 Jan 31 [Epub ahead of print]. ACKNOWLEDGEMENTS This research was supported by the Gateway for Cancer Research Foundation and the National Cancer Institute awards R1 CA75163 and K24 CA85218.

17 Summary of Available Data Protocol Vaccine Tumor Stage- (avg) n= 1 HER-2/neu peptide + GM-CSF 12 HER-2/neu peptide + Flt3 Ligand 14 HER-2/neu peptide + Flt3 Ligand GM-CSF 15 HER-2/neu protein + GM-CSF Breast, lung (1), ovarian III-29, IV-45 (3.6) 74* Prostate C, D 21 Breast, ovarian III, IV (3.4) 11 Breast, ovarian II-2, III-7, IV- (2.3) 16 EGFRvIII peptide Prostate, ovarian III, IV (3.8) DNA plasmid encoding HER-2/neu ICD 118 HER-2/neu peptide + GM-CSF in patient receiving Herceptin Breast III, IV (3.2) 39 Breast IV- 2 (4.) 2** * Excludes patients vaccinated with 9-MER vaccine ** 21 patients enrolled, 1 patients currently have ICD-S.I. data

18 Epitope spreading leads to increased T cell response at all stages Protocols 1 and 118: Vaccination with HER-2/neu peptide + GM-CSF ICD Stimulation Index Epitope Spreading (n= 43) No Epitope Spreading (n= 13) III Tumor Stage IV Protocol 1 CTL/ECD/ICD vaccines, and 118 (CTL). Must have received baseline labs and at least 3 vaccinations

19 More Vigorous Response to Tumor Vaccination in Earlier Stage Disease Stimulation Index Stage II (n= 2) Stage III (n= 25) Stage IV (n= 38) S.E.M. 25 ICD Tetanus Control PHA Control Protocol 1 CTL/ECD/ICD vaccines, 15 (ICD pr), and 118 (CTL). Must have received baseline labs and at least 3 vaccinations

20 More Vigorous Response to Tumor Vaccination in Earlier Stage Disease Stimulation Index C.I.= 95% Stage II (n= 2) Stage III (n= 25) Stage IV (n= 38) 2 II III IV

21 Antibody Response Remains Stable As Tumor Stage Increases Protocols 1, 15 and 118* Maximum Anti-Tumor Antibody Response (mcg/ml) II III IV Stage * Where n= 2 for stage 2, n=36 for stage 3, and n=65 for stage 4. Includes all vaccination types, no exclusion criteria.

22 Epitope spreading falls as stage increases Protocols 1, 118: Vaccination with HER-2/neu peptide + GM-CSF 1 Epitope Spreading (%) III (n= 18) IV (n= 38) Tumor Stage