Elke Jaeger

Generation of cytotoxic T‐cell responses with synthetic melanoma‐associated peptides in vivo: Implications for tumor vaccines with melanoma‐associated antigens

Peptide epitopes derived from differentiation antigens of the melanocyte lineage have been identified in human melanomas and normal cultured melanocytes as targets for MHC‐restricted cytotoxic T lymphocytes (CTL). Characterization of multiple CTL‐defined antigenic determinants and the presence of corresponding precursor CTL open perspectives for the development of antigen‐based vaccines. In the present study, we determined the CTL reactivity against melanoma‐associated peptides derived from Melan A/MART‐I, tyrosinase and gp100/Pme117 in 10 HLA‐A2* melanoma patients and 10 healthy individuals. Then, we examined the immunological effects and toxicity of intradermal inoculation of synthetic melanoma‐associated peptides. Six patients with advanced melanoma received weekly intradermal injections of 6 melanoma‐associated peptides and the influenza matrix peptide as a control for 4 consecutive weeks. DTH reactions were observed in 5/6 patients at the injection sites of the tyrosinase signal peptide and of the influenza matrix peptide. No toxic side effects were observed. Changes in CTL reactivity after peptide vaccination were assessed by an MLPC assay for each peptide. Generation of peptide‐specific CTL was documented against Melan A/MART‐I‐derived peptide epitopes, the tyrosinase signal peptide and the influenza matrix peptide after vaccination. A decreasing CTL response against the internal tyrosinase peptide was documented in 1 patient through the course of vaccination and a decrease in DTH reactions. No major tumor regressions were observed. Two patients with rapidly progressive disease before vaccination have shown disease stabilization since vaccinations started. In conclusion, our results demonstrate that peptide alone injected intradermally may generate antigen‐specific DTH reactions and an increase of antigen‐specific CTL reactivity.

Phase II Trial of Biweekly Infusional Fluorouracil, Folinic Acid, and Oxaliplatin in Patients With Advanced Gastric Cancer

PURPOSE To evaluate the toxicity and activity of infusional fluorouracil (FU), folinic acid (FA), and oxaliplatin, administered every 2 weeks in patients with metastatic gastric cancer. PATIENTS AND METHODS Forty-one previously untreated patients with measurable adenocarcinoma of the stomach were eligible for the study. Patients received FU 2.6 g/m(2) (24-hour continuous infusion), FA 500 mg/m(2) (2-hour intravenous infusion), and oxaliplatin 85 mg/m(2) (2-hour intravenous infusion) every 2 weeks for 6 weeks. Treatment was continued until progression of disease was observed. RESULTS All patients were assessable for toxicity and 37 of 41 patients were assessable for response. Patient characteristics were: sex (male, 28; female,13), median age 60 years (range, 20 to 77 years), and median Eastern Cooperative Oncology Group performance status of 1. Response was evaluated every 6 weeks. Of 37 assessable patients, one complete and 15 partial remissions were observed (overall response rate, 43%). Stable disease was observed in 12 patients (32%) and progressive disease in nine patients (24%). The median overall survival was 9.6 months. WHO grade 3 or 4 hematologic toxicities included neutropenia in two patients (4.9%) and thrombocytopenia in one patient (2.4%). Other WHO grade 3 or 4 toxicities included diarrhea in three patients (7.3%) and vomiting in two patients (4.9%). There were no cases of grade 3 peripheral neuropathy and no treatment-related deaths. CONCLUSION Biweekly fluorouracil, folinic acid, and oxaliplatin is active and well-tolerated in patients with advanced gastric cancer. Response rates, time to progression, and overall survival were comparable to those achieved with other combination chemotherapy regimens, including FOLFOX6, with significantly less toxicity.

Pharmacogenetic Analyses of a Phase III Trial in Metastatic Gastroesophageal Adenocarcinoma With Fluorouracil and Leucovorin Plus Either Oxaliplatin or Cisplatin: A Study of the Arbeitsgemeinschaft Internistische Onkologie

PURPOSE To evaluate the association of germ-line polymorphisms of genes that may impact treatment outcome of platinum and fluorouracil combination chemotherapy in advanced gastric cancer (AGC). PATIENTS AND METHODS Blood samples of 156 patients enrolled onto a phase III study comparing fluorouracil, leucovorin, and oxaliplatin with fluorouracil, leucovorin, and cisplatin were collected. Polymorphisms within genes of TS, MTHFR, MTR, OPRT, XPD, ERCC1, XRCC1, XPA, GSTP1, GSTT1, and GSTM1 were genotyped using polymerase chain reaction-based techniques. RESULTS Median overall survival (OS) was 11.8 months (95% CI, 9.75 to 13.79 months) and median progression-free survival (PFS) was 5.8 months (95% CI, 4.99 to 6.61 months). The TS-3R/+6 haplotype (P = .004), the GSTT1 deletion polymorphism (P = .015), and genotypes of OPRT-Gly213Ala (P = .003) and XRCC1-Arg399Gln (P = .023) could be identified as independent predictors of OS. For PFS analyses, the TS-3R/+6 haplotye (P = .003) and MTR-A2756G (P = .01) were identified as independent positive predictors. The association between the GSTT1 deletion polymorphism and PFS showed only borderline significance (P = .053). Treatment related hematotoxicity in terms of grade 3/4 leukopenia was lowest among TS-3R/+6 haplotype carriers (P = .037). Grade 3/4 neutropenia was directly associated with the MTR-2756G/G genotype (P = .011), GSTP1-105Ile/Ile genotype (P = .02), and with the ERCC1-118T/8092C-haplotype (P = .042). In addition, significant associations between GSTP1-105Ile/Ile genotype and neurotoxicity and between the XPD-Asn312/751Gln haplotype and nephrotoxicity could be identified (P = .028 and P = .005, respectively). CONCLUSION These findings underline the hypothesis that germ-line polymorphisms may play an important role in individualizing chemotherapy in AGC and deserve further prospective evaluation in AGC patients.

Induction of immunogenicity of a human renal-cell carcinoma cell line by TAP1-gene transfer

Reduced expression of the major‐histocompatibility‐complex(MHC)‐class‐I antigens has been demonstrated in renal‐cell carcinoma (RCC), and appeared to be associated with deficiencies in the expression and function of different components of the MHC‐class‐I‐antigen‐processing pathway and poor recognition by cytotoxic T‐lymphocytes (CTL). In order to investigate the role of peptide transporters for the immunogenic phenotype of RCC, tumor cells were stably transfected with the human TAP1A gene. While the TAP1 transfectants showed heterogeneous TAP1‐transgene expression pattern of mRNA and protein, high TAP1 expression and a TAP‐controlled increase in MHC‐class‐I surface expression could be achieved in selected transfectants. IFN‐γ up‐regulates the expression of MHC‐class‐I antigens and TAP1 both in control and in TAP1‐transfected RCC cells to a similar level. No additive effect of TAP1 over‐expression was observed in TAP1 transfectants. Although no enhanced CTL‐mediated lysis was obtained, cytokine release was substantially increased in response to TAP1‐transfected RCC cells, but not to control cells. Furthermore, TAP1 transfectants were able to stimulate the proliferation of allogeneic T cells. These studies suggest that abnormalities of MHC‐class‐I surface expression due to dysfunctional peptide transporters contribute to the immune escape phenotype of RCC cells and that the immune tolerance of RCC could be altered by TAP1‐gene transfer. Int. J. Cancer 81:125–133, 1999.

Spontaneous CD8 T Cell Responses against the Melanocyte Differentiation Antigen RAB38/NY-MEL-1 in Melanoma Patients

The melanocyte differentiation Ag RAB38/NY-MEL-1 was identified by serological expression cloning (SEREX) and is expressed in the vast majority of melanoma lesions. The immunogenicity of RAB38/NY-MEL-1 has been corroborated previously by the frequent occurrence of specific Ab responses in melanoma patients. To elucidate potential CD8 T cell responses, we applied in vitro sensitization with overlapping peptides spanning the RAB38/NY-MEL-1 protein sequence and the reverse immunology approach. The identified peptide RAB38/NY-MEL-150–58 exhibited a marked response in ELISPOT assays after in vitro sensitization of CD8 T cells from HLA-A∗0201+ melanoma patients. In vitro digestion assays using purified proteasomes provided evidence of natural processing of RAB38/NY-MEL-150–58 peptide. Accordingly, monoclonal RAB38/NY-MEL-150–58-specific T cell populations were capable of specifically recognizing HLA-A2+ melanoma cell lines expressing RAB38/NY-MEL-1. Applying fluorescent HLA-A2/RAB38/NY-MEL-150–58 multimeric constructs, we were able to document a spontaneously developed memory/effector CD8 T cell response against this peptide in a melanoma patient. To elucidate the Ag-processing pathway, we demonstrate that RAB38/NY-MEL-150–58 is produced efficiently by the standard proteasome and the immunoproteasome. In addition to the identification of a RAB38/NY-MEL-1-derived immunogenic CD8 T cell epitope, this study is instrumental for both the onset and monitoring of future RAB38/NY-MEL-1-based vaccination trials.

Melanocyte differentiation antigen RAB38/NY-MEL-1 induces frequent antibody responses exclusively in melanoma patients

Expression pattern and immunogenicity are critical issues that define tumor antigens as diagnostic markers and potential targets for immunotherapy. The development of SEREX (serological analysis of recombinant expression libraries) has provided substantial progress in the identification of tumor antigens eliciting both cellular and humoral immune responses in cancer patients. By SEREX, we have previously identified RAB38/NY-MEL-1 as a melanocyte differentiation antigen that is highly expressed in normal melanocytes and melanoma tissues but not in other normal tissues or cancer types. In this study, we further demonstrate that RAB38/NY-MEL-1 is strongly immunogenic, leading to spontaneous antibody responses in a significant proportion of melanoma patients. The immune response occurs solely in malignant melanoma patients and was not detected in patients with other diseases, such as vitiligo, affecting melanocytes. Fine analysis of the spontaneous anti-RAB38/NY-MEL-1 antibody response reveals a polyclonal B cell recognition targeting various epitopes, although a dominant immunogenic region was preferentially recognized. Interestingly, our data indicate that this recognition is not rigid in the course of a patient’s response, as the dominant epitope changes during the disease evolution. Implications for the understanding of spontaneous humoral immune responses are discussed.

LUD01–014: Phase 1/2 study of chimeric monoclonal antibody cG250 in combination with vinblastine in patients with advanced renal cell carcinoma (ARCC)

2531 Background: To asses the safety and efficacy of the combination of cG250 and vinblastine in patients (pts) with ARCC. METHODS Eighteen pts with ARCC of clear cell histology and measurable disease were included. Pts received intravenous doses of cG250, 10 mg/m2, and vinblastine, 5 mg/m2, weekly for 8 weeks (wks) followed by a 2-wk rest period (one cycle). Pts were evaluated for toxicity and the development of human antichimeric antibody (HACA) prior to each treatment, and for response after each cycle. Treatment was continued unless pts had disease progression or limiting toxicity. RESULTS All pts were evaluable for toxicity and 16/18 for response. Pts characteristics were: gender (male-10, female-8); median age 63 (48-84); median Karnofsky performance status 90%. Nine pts received 1 cycle; 7/18 pts received ≥2 and 4/18 ≥3 cycles. WHO III/IV toxicities were anemia and neutropenia in 2/18 pts (11%) and 5/18 pts (28%), respectively. Other toxicities included WHO I/II fatigue in 6/18 pts (33%) and I/II neuropathy in 2/18 pts (11%). Of 18 pts, 4 pts are still on study and 14 have withdrawn (9 for PD during the 1st cycle, 3 for PD diagnosed after the 2nd cycle, and 2 at their own discretion). HACA were not developed. SD was observed in 7/16 (44%), and PD in 9 evaluable pts (56%). In 3 pts SD lasted 12+, 12.8+, and 17.8 months. For assessable pts followed longer than 12 months (10 pts), the 1-year progression free survival (PFS) rate was 30%. Median survival has not been reached yet. CONCLUSIONS cG250 in combination with vinblastine is feasible and safe. A high rate of SD with prolonged PFS was observed that warrants further confirmation in a randomized phase II trial. No significant financial relationships to disclose.

Abstract A006: Frameshift peptide neoantigens as vaccine targets in microsatellite-unstable cancers

Background: Deficient DNA mismatch repair (MMR) boosts the accumulation of frameshift mutations in genes encompassing coding microsatellites (cMS). This results in translation of proteins with mutation-induced frameshift peptide (FSP) neoantigens rendering MMR-deficient microsatellite-unstable (MSI) cancers highly immunogenic. Patients with MSI cancers and healthy individuals affected by Lynch syndrome, an inherited prediposition for MSI cancers, develop specific immune responses against these neoantigens. MSI cancers are unique in tumor immunology, because they express a defined set of long neoantigens that result from functionally relevant driver mutations and therefore are shared by the majority of MSI cancers. Consequently, MSI cancers in Lynch syndrome are an ideal model to evaluate the concept of cancer vaccines, which, with the increasing knowledge about mutational antigens in a wide variety of cancer types, can potentially be applied to many human cancer types. We here report the results of a clinical phase I/IIa trial as the first step to translate this concept into the clinical application. Methods: The vaccination protocol comprised 3 cycles of 4 subcutaneous applications of FSP antigens (frameshift variants of the coding microsatellite-containing genes AIM2, HT001, TAF1B) mixed with Montanide ISA-51 VG over a 6 month period. Inclusion criteria were history of MSI-H colorectal cancer (UICC stage III or IV) and completion of standard chemotherapy. Phase I of the trial evaluated safety and toxicity as the primary endpoint (6 patients), phase IIa addressed the induction of cellular and humoral immune responses (16 patients). Results: Significant induction of FSP-specific immune responses against one or more FSP antigens was observed in all patients vaccinated per protocol. No vaccination-induced systematic severe adverse events occurred. Few patients had stage IV disease and were evaluable according to RECIST. One heavily pretreated patient with bulky metastases showed a stable disease and stable CEA levels over 7 months under the study treatment. Conclusions: Vaccination with FSPs is well tolerated and leads to the induction of humoral and cellular immune responses. FSP vaccination represents a promising novel approach for treatment of MSI cancer patients and for tumor prevention in Lynch syndrome, allowing the evaluation of the concept of preventive cancer vaccines in an ideal model scenario of a defined high-risk patient population. Note: This abstract was not presented at the conference. Citation Format: Magnus von Knebel Doeberitz, Matthias Kloor, Miriam Reuschenbach, Claudia Pauligk, Mohammad-Reza Rafiyan, Salah-Eddin Al Batran, Julia Karbach, Mirjam Tariverdian, Elke Jaeger. Frameshift peptide neoantigens as vaccine targets in microsatellite-unstable cancers [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A006.

Abstract A12: Vicoryx: A phase I/IIa clinical trial using a p16INK4a derived peptide as vaccine in patients with advanced human papillomavirus-associated cancer

Objectives: The tumor suppressor p16 INK4a is strongly overexpressed in HPV-associated neoplasia, whereas in normal tissues barely any p16 INK4a expression is detectable. Targeting this HPV type-independent antigen by vaccination could represent an interesting complementary therapeutic approach to recently developed HPV-E6/E7-based therapeutic or secondary preventive vaccines. We performed a phase I/IIa peptide vaccination trial to monitor toxicity and immunogenicity of p16 INK4a vaccination in patients with advanced HPV-associated cancers. Patients and Methods: 21 patients with p16 INK4a -overexpressing, HPV DNA-positive advanced cancers (16 cervical, 5 head and neck) were included. The protocol comprised 12 applications of a synthetic 27mer p16 INK4a peptide mixed with Montanide® ISA-51 VG over a six months period. Objectives of the study were clinical safety and changes of humoral and cellular immune responses against the p16 INK4a peptide. T cell responses were monitored by interferon-gamma ELISpot and antibodies by ELISA. Conclusions: No vaccine-related toxicity was observed in any of the patients. One patient (head and neck cancer) completed the entire study protocol with stable disease for now 18 months after the last vaccination.11 patients had progressing disease and were excluded from the study after 4 to 12 weeks. 9 patients continue to be vaccinated. While at baseline only one patient had pre-existing T cell responses (CD4) against the p16 INK4a peptide, p16 INK4a -reactive T cells (CD4) were successfully induced in at least four patients after 4 to 12 vaccine doses. None of the patients had pre-existing p16 INK4a antibodies, but 3 patients developed increasing p16 INK4a peptide-specific antibody titers after the 5th dose. This is the first study demonstrating that p16 INK4a peptide vaccination is safe and well tolerated. The results show that spontaneous immune responses against p16 INK4a are rare, but can be induced by p16 INK4a peptide vaccination. Further studies are needed to assess clinical efficacy of the approach. Please refer to the protocol of this trial under http://clinicaltrials.gov/show/NCT01462838. Citation Format: Miriam Reuschenbach, Julia Karbach, Franziska Faulstich, Madeleine Sauer, Matthias Kloor, Mohammad-Reza Rafiyan, Claudia Pauligk, Salah AlBatran, Elke Jaeger, Magnus von Knebel Doeberitz. Vicoryx: A phase I/IIa clinical trial using a p16INK4a derived peptide as vaccine in patients with advanced human papillomavirus-associated cancer. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr A12.

Abstract CN01-03: Frameshift peptide vaccination: Towards cancer prevention in Lynch syndrome

Lynch syndrome is one of the most common hereditary cancer syndromes. It is caused by germ line mutations of DNA mismatch repair genes and predisposes affected individuals to the development of colorectal cancer and extracolonic malignancies with a lifetime risk of over 50%. Lynch syndrome-associated cancers show the high-level microsatellite instability (MSI-H) phenotype and are characterized by signs of a pronounced local anti-tumoral immune response, suggesting that the immune system plays an active role in the surveillance and natural history of these cancers. The progression of MSI-H cancers is triggered by mutations in microsatellite sequences within gene-encoding regions, which can cause shifts of the translational reading frame and lead to the generation of immunogenic frameshift peptides (FSP) at the carboxy-terminal end of the respective proteins. FSP-specific immune responses have been observed frequently in MSI-H cancer patients and Lynch syndrome mutation carriers. We have therefore initiated a clinical phase I/IIa vaccination trial that evaluates vaccination with a combination of three FSP antigens in patients with MSI-H colorectal cancer, UICC stage III and IV. Preliminary data demonstrate that no treatment-associated severe adverse events have been observed after FSP vaccination. Moreover, significant T cell and humoral immune responses against FSP antigens were induced in the majority of vaccinated patients. The observation of strong FSP-specific immune responses in vaccinated patients suggests that FSP-based vaccination may represent a novel option for the treatment of MSI-H colorectal cancer patients. Moreover, FSP vaccination may be used for tumor prevention in Lynch syndrome mutation carriers in the future. Citation Format: Matthias Kloor, Miriam Reuschenbach, Julia Karbach, Reza Rafiyan, Salah-Eddin Al-Batran, Claudia Pauligk, Elke Jaeger, Magnus von Knebel Doeberitz. Frameshift peptide vaccination: Towards cancer prevention in Lynch syndrome. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr CN01-03.