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• W2345970825 abstract "The tumor vaccine MGN1601 was designed and developed for treatment of metastatic renal cell carcinoma (mRCC). MGN1601 consists of a combination of fourfold gene-modified cells with the toll-like receptor 9 agonist dSLIM, a powerful connector of innate and adaptive immunity. Vaccine cells originate from a renal cell carcinoma cell line (grown from renal cell carcinoma tissue), express a variety of known tumor-associated antigens (TAA), and are gene modified to transiently express two co-stimulatory molecules, CD80 and CD154, and two cytokines, GM-CSF and IL-7, aimed to support immune response. Proof of concept of the designed vaccine was shown in mice: The murine homologue of the vaccine efficiently (100%) prevented tumor growth when used as prophylactic vaccine in a syngeneic setting. Use of the vaccine in a therapeutic setting showed complete response in 92% of mice as well as synergistic action and necessity of the components. In addition, specific cellular and humoral immune responses in mice were found when used in an allogeneic setting. Immune response to the vaccine was also shown in mRCC patients treated with MGN1601: Peptide array analysis revealed humoral CD4-based immune response to TAA expressed on vaccine cells, including survivin, cyclin D1, and stromelysin. The tumor vaccine MGN1601 was designed and developed for treatment of metastatic renal cell carcinoma (mRCC). MGN1601 consists of a combination of fourfold gene-modified cells with the toll-like receptor 9 agonist dSLIM, a powerful connector of innate and adaptive immunity. Vaccine cells originate from a renal cell carcinoma cell line (grown from renal cell carcinoma tissue), express a variety of known tumor-associated antigens (TAA), and are gene modified to transiently express two co-stimulatory molecules, CD80 and CD154, and two cytokines, GM-CSF and IL-7, aimed to support immune response. Proof of concept of the designed vaccine was shown in mice: The murine homologue of the vaccine efficiently (100%) prevented tumor growth when used as prophylactic vaccine in a syngeneic setting. Use of the vaccine in a therapeutic setting showed complete response in 92% of mice as well as synergistic action and necessity of the components. In addition, specific cellular and humoral immune responses in mice were found when used in an allogeneic setting. Immune response to the vaccine was also shown in mRCC patients treated with MGN1601: Peptide array analysis revealed humoral CD4-based immune response to TAA expressed on vaccine cells, including survivin, cyclin D1, and stromelysin. Tumor vaccines restore the immune system’s intrinsic ability to recognize tumor cells. Cell-based tumor vaccines are classified as dendritic cell (DC)-, T-cell- or tumor cell-based vaccines. For the latter one, tumor-specific effects were shown including activation of T cells and strengthening of CD8+T-cell responses by direct antigen activation and cross-priming, development of memory cells, and increase of antibody-based response.1Ogi C Aruga A Immunological monitoring of anticancer vaccines in clinical trials.Oncoimmunology. 2013; 2: e26012Crossref PubMed Scopus (33) Google Scholar,2Vujanovic L Butterfield LH Melanoma cancer vaccines and anti-tumor T cell responses.J Cell Biochem. 2007; 102: 301-310Crossref PubMed Scopus (31) Google Scholar Cell-based tumor vaccines own a huge reservoir of tumor-related antigens and are, therefore, able to address a broad repertoire of T cells. Plurality of antigens on cell-based vaccines hampers immune escape of the tumor cells by selective antigen loss. However, individual antigens of the cell-based vaccines evoke a rather weak immune response underlining the necessity to strengthen their immunogenicity by multiple gene modifications. Preparation of multiple gene-modified cells requires high-efficient expression vectors. Recently, MIDGE (minimalistic immunogenically defined gene expression) DNA vectors were developed for clinical use3Endmann A Baden M Weisermann E Kapp K Schroff M Kleuss C et al.Immune response induced by a linear DNA vector: influence of dose, formulation and route of injection.Vaccine. 2010; 28: 3642-3649Crossref PubMed Scopus (22) Google Scholar, 4Schakowski F Gorschlüter M Junghans C Schroff M Buttgereit P Ziske C et al.A novel minimal-size vector (MIDGE) improves transgene expression in colon carcinoma cells and avoids transfection of undesired DNA.Mol Ther. 2001; 3: 793-800Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 5Schmidt M Volz B Wittig B MIDGE vectors and dSLIM immunomodulators: DNA-based molecules for gene therapeutic strategies.in: Knäblein J Müller RH Modern Biopharmaceutical. WILEY-VCH Verlag GmbH&Co. KGaA, Weinheim2005: 1-29Crossref Scopus (4) Google Scholar allowing the generation of multiple gene-modified cells with only minimal amount of foreign DNA. MIDGE vectors display a linear covalently closed topology with single-stranded loops and are biotechnologically manufactured from plasmids. Their small size of about 1,200 bp plus coding sequence is based on their exclusive content of the expression cassette consisting of the CMV promoter, the selected coding sequence, and a poly(A) signal. The prevention of genes for resistance to antibiotics, of replication origins, and other functional elements improves their overall safety profile and ensures that application of MIDGE vectors does not add conflicting potential to public health issues. Transient gene modification of cells helps to maintain their natural expression profiles by minimizing adaptation processes following otherwise stable transfection. Additionally, waiving clonal selection preserves the heterogeneity of cells representing all subtypes outgrown from original tumor tissue. Preservation of the antigen repertoire of the source cell line during the manufacturing process to vaccine cells is important because this repertoire is a main criterion for selection of source cells. Besides the selection of cell line, vector, and gene modification, the identification of an additional immunomodulator as multiplier of the tumor-specific immune response is crucial. Toll-like receptor 9 (TLR-9) agonists are powerful connectors of innate and adaptive immunity and therefore supposed to be ideally suited to strengthen tumor vaccines.6Bode C Zhao G Steinhagen F Kinjo T Klinman DM CpG DNA as a vaccine adjuvant.Expert Rev Vaccines. 2011; 10: 499-511Crossref PubMed Scopus (564) Google Scholar dSLIM (double stem loop immunomodulator) is a noncoding dumbbell-shaped and covalently closed DNA molecule with non-methylated CG motifs acting via TLR-9 (refs. 7Kapp K Kleuss C Schroff M Wittig B Genuine immunomodulation with dSLIM.Mol Ther Nucleic Acids. 2014; 3: e170Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 8Schmidt M Anton K Nordhaus C Junghans C Wittig B Worm M Cytokine and Ig-production by CG-containing sequences with phosphorodiester backbone and dumbbell-shape.Allergy. 2006; 61: 56-63Crossref PubMed Scopus (34) Google Scholar, 9Schmidt M Hagner N Marco A König-Merediz SA Schroff M Wittig B Design and structural requirements of the potent and safe TLR-9 agonistic immunomodulator MGN1703.Nucleic Acid Ther. 2015; 25: 130-140Crossref PubMed Scopus (41) Google Scholar). Currently, dSLIM is evaluated in clinical trials for the treatment of solid tumors.10Schmoll HJ Wittig B Arnold D Riera-Knorrenschild J Nitsche D Kroening H et al.Maintenance treatment with the immunomodulator MGN1703, a Toll-like receptor 9 (TLR9) agonist, in patients with metastatic colorectal carcinoma and disease control after chemotherapy: a randomised, double-blind, placebo-controlled trial.J Cancer Res Clin Oncol. 2014; 140: 1615-1624Crossref PubMed Scopus (72) Google Scholar,11Weihrauch MR Richly H von Bergwelt-Baildon MS Becker HJ Schmidt M Hacker UT et al.Phase I clinical study of the toll-like receptor 9 agonist MGN1703 in patients with metastatic solid tumours.Eur J Cancer. 2015; 51: 146-156Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar Renal cell carcinoma (RCC) is well known for its response to immune therapies.12Biswas S Eisen T Immunotherapeutic strategies in kidney cancer–when TKIs are not enough.Nat Rev Clin Oncol. 2009; 6: 478-487Crossref PubMed Scopus (43) Google Scholar,13Pal SK Hu A Figlin RA A new age for vaccine therapy in renal cell carcinoma.Cancer J. 2013; 19: 365-370Crossref PubMed Scopus (16) Google Scholar Launch of targeted therapies has improved treatment of patients with metastatic RCC. However, RCC, especially at metastatic stage, remains a life-threatening condition with high medical need for effective treatment. The aim of the current work was to develop a tumor vaccine for an improved treatment of metastatic renal cell carcinoma (mRCC). During our previous development of tumor vaccines, cells derived from autologous tumor tissue were gene modified to discretely express IL-2, IL-7, IL-12, or granulocyte-macrophage colony-stimulating factor (GM-CSF).14Micka B Trojaneck B Niemitz S Lefterova P Kruopis S Huhn D et al.Comparison of non-viral transfection methods in melanoma cell primary cultures.Cytokine. 2000; 12: 828-833Crossref PubMed Scopus (9) Google Scholar, 15Möller P Sun Y Dorbic T Alijagic S Makki A Jurgovsky K et al.Vaccination with IL-7 gene-modified autologous melanoma cells can enhance the anti-melanoma lytic activity in peripheral blood of patients with a good clinical performance status: a clinical phase I study.Br J Cancer. 1998; 77: 1907-1916Crossref PubMed Scopus (91) Google Scholar, 16Sun Y Jurgovsky K Möller P Alijagic S Dorbic T Georgieva J et al.Vaccination with IL-12 gene-modified autologous melanoma cells: preclinical results and a first clinical phase I study.Gene Ther. 1998; 5: 481-490Crossref PubMed Scopus (161) Google Scholar The use of twofold gene-modified vaccine cells secreting IL-7 and GM-CSF presented a new milestone in autologous vaccine development.17Wittig B Märten A Dorbic T Weineck S Min H Niemitz S et al.Therapeutic vaccination against metastatic carcinoma by expression-modulated and immunomodified autologous tumor cells: a first clinical phase I/II trial.Hum Gene Ther. 2001; 12: 267-278Crossref PubMed Scopus (90) Google Scholar In this study, 50% of treated patients showed clinical response to treatment with at least disease stabilization. Encouraged by these results and driven by the increased knowledge and immunological understanding of tumor vaccines,18Parmiani G Pilla L Maccalli C Russo V Autologous versus allogeneic cell-based vaccines?.Cancer J. 2011; 17: 331-336Crossref PubMed Scopus (18) Google Scholar additional gene modification of vaccine cells (besides cytokines) with co-stimulatory molecules CD80 (ref. 19Köchling J König-Merediz SA Stripecke R Buchwald D Korte A Von Einsiedel HG et al.Protection of mice against Philadelphia chromosome-positive acute lymphoblastic leukemia by cell-based vaccination using nonviral, minimalistic expression vectors and immunomodulatory oligonucleotides.Clin Cancer Res. 2003; 9: 3142-3149PubMed Google Scholar) and CD154 (refs. 20Liu KJ Lu LF Cheng HT Hung YM Shiou SR Whang-Peng J et al.Concurrent delivery of tumor antigens and activation signals to dendritic cells by irradiated CD40 ligand-transfected tumor cells resulted in efficient activation of specific CD8+ T cells.Cancer Gene Ther. 2004; 11: 135-147Crossref PubMed Scopus (13) Google Scholar,21Loskog A Tötterman TH CD40L - a multipotent molecule for tumor therapy.Endocr Metab Immune Disord Drug Targets. 2007; 7: 23-28Crossref PubMed Scopus (49) Google Scholar) was chosen. These modifications were supposed to further improve immune response to the vaccine antigens by mimicking features of antigen presenting cells (APC) and T cells and result in fourfold gene-modified cells. The concept of fourfold gene-modified cells was evaluated in mice, using a murine vaccine consisting of murine tumor cells and expressing murine cytokines and co-stimulatory molecules in combination with the TLR-9 agonist dSLIM. Proof of concept of the antitumor efficacy was shown as well as cellular and humoral immune responses. Multiple toxicological studies did not reveal any safety concerns. The next developmental step was the identification a human cell line for generation of a human vaccine. B25MOL cells were selected as source material for the vaccine due to their success in an autologous treatment schedule of an mRCC patient (complete response for 7 years, survival for 13 years) and their broad expression of tumor-associated antigens (TAA). Fourfold (CD80, CD154, GM-CSF, and IL-7) gene-modified vaccine cells were combined with the TLR-9 agonist dSLIM—resulting in MGN1601. Expression characteristics of vaccine cells were analyzed. The immune responses shown in murine studies were confirmed in translational analyses with blood samples from patients treated within the ASET (Assess Safety and Efficacy of the Tumor Vaccine MGN1601) study with MGN1601. In order to evaluate the efficacy of the designed vaccine for treatment of RCC, a murine homologue of the vaccine was manufactured. This vaccine consisted of fourfold gene-modified murine RCC cells (Renca), combined with dSLIM. First, the immunotherapeutic concept was assessed by prophylactic vaccination in a syngeneic mouse model. Balb/c mice were treated four times with the murine homologue before tumor growth was initiated by inoculation of Renca cells (Figure 1a,b). All mice of the vaccinated group survived, and none of them developed a tumor at any time of the experiment (Figure 1a). In contrast, 70% of mice treated with phosphate-buffered saline (PBS) developed a tumor, and none of the tumor-bearing mice survived the experimental period due to tumor growth. In vaccinated mice, tumor-free survival as well as total survival were significantly increased (P < 0.001; Figure 1b). These data unequivocally proved that prophylactic vaccination with the newly designed vaccine protects mice from tumor development after inoculation of Renca cells. The newly designed concept was further evaluated by therapeutic vaccination using different variants of the vaccine (murine homologue). Balb/c mice first received Renca cells for tumor inoculation. One week later, treatment was started (Figure 1c,d and Supplementary Table S1). Treatment was repeated three times (n = 12 mice/group). One important goal of the study was to evaluate the efficacy of experimental variants of the vaccine to inhibit tumor growth and thus the necessity of the components within the therapeutic concept. Complete vaccine (“complete”) was compared to the efficacy of vaccine cells without the immunomodulator dSLIM (“tf Renca” ≙ fourfold gene-modified Renca cells), to irradiated Renca cells (“Renca” ≙ without gene modification, without dSLIM), and to dSLIM as single component (“dSLIM”). Mean tumor volume at the end of the study or at the date of death of a mouse was 1,370 mm3 for mice receiving PBS (Figure 1c), whereas mice treated with the murine homologue showed an average tumor volume of 69 mm3 (11 mice without tumor and 1 mouse with 832 mm3 tumor volume). Experimental variants of the vaccine used resulted in mean tumor volumes between those of PBS- and vaccine-treated mice. Tumor volumes of mice treated with “complete vaccine” or with “tf Renca” were statistically significant lower (P < 0.001) compared to those of PBS-treated mice. Furthermore, tumor volumes of mice treated with “dSLIM” as monotherapy were significant lower (P < 0.05), whereas tumor volumes of mice treated with “Renca” showed no significant reduction. These data are summarized in Supplementary Table S1. Tumor growth in tumor-bearing mice of all five treatment groups was compared over the time course of the study (Figure 1d and Supplementary Figure S1). All vaccinated animals showed reduced tumor growth compared to PBS-treated mice. However, the complete vaccine achieved the most prominent effects. Furthermore, the survival rate of tumor-bearing mice was highest (92%) for mice treated with the complete vaccine, whereas all tumor-bearing mice treated with PBS died during the study due to tumor-related reasons. Survival data of mice in the other three treatment groups ranked between those of PBS and complete vaccine groups: “tf Renca”: 75%, “Renca”: 50%, and “dSLIM”: 17%. Mice that had been classified as tumor bearers during the study but finished the study without measurable tumor size were classified as “complete remission.” Eleven out of 12 tumor-bearing mice of the complete vaccine group were free of tumor at the end of the study, resulting in 92% complete remission rate. In contrast, none of the PBS-treated mice showed any reduction of tumor volume at any time point. The variants of the vaccine showed intermediate complete remission rates: “tf Renca”: 66%, “Renca”: 50%, and “dSLIM”: 17%. In summary, the newly designed therapeutic concept was identified as superior over other tested experimental variants underlining the relevance and potential synergistic effects of the vaccine cells, their gene modification, and the TLR-9 agonist dSLIM. Next, the immunomodulatory effects of the vaccine were addressed. Again, the murine homologue of the vaccine was repeatedly applied (8 to 13 times) once weekly to NMRI mice (Naval Medical Research Institute mouse strain, allogeneic to Renca cells). Skin sections of the injection sites of the vaccine were analyzed for the presence of T cells (CD4+ or CD8+ cells) and APC (CD86+ cells) in NMRI mice. A bright staining intensity for CD4+ and CD8+ T cells, as well as for APC (CD86+ cells) was seen in mice treated repeatedly (eight times) with the vaccine, while skin sections of application sites of PBS-treated mice did not stain for any of the analyzed cell populations (Figure 2a and Supplementary Figure S2). These findings indicate the recruitment of T cells and APC by the vaccine to the injection sites. Sera of vaccinated mice were analyzed for Renca-specific antibodies after eight applications of the vaccine (Figure 2b). Sera of all vaccinated mice clearly recognized Renca cell lysates (titer 11,000 to 35,000), while sera of PBS-treated mice did not react at all, indicating high CD4+-dependent B-cell responses to the vaccine. Mice were vaccinated repeatedly (13 times) with either low dose or high dose of the vaccine (murine homologue) in an allogeneic setting. Thereafter, spleen cells were isolated, re-stimulated with Renca cells ex vivo, and analyzed for specific cytotoxicity against Renca cells as target (Figure 2c). Highest Renca-specific cytotoxicity of spleen cells was found in the high-dose group, although all examined groups showed pronounced lytic activities for Renca cells if compared to spleen cells from PBS-treated mice. Cytotoxicity of spleen cells was maintained even 4 weeks after the last high-dose application (recovery group), indicating a sustained CD8+-based immune response against the vaccine cells. To assess the safety of the tumor vaccine, single- and repeated-dose toxicological studies were performed using either MGN1601 in rats (heterologous model) or the murine homologue in mice (homologous model, allogeneic setting) according to regulatory requirements. In the heterologous setting, the single administration of up to a 500-fold excess regarding the human dose (per kg body weight) as well as the repeated application (five times) of up to a 60-fold excess did not result in any relevant toxicological findings. In the homologous model, the murine vaccine was applied to NMRI mice. Repeated application (13 times) of up to a 3,000-fold excess regarding the human dose resulted in only minor observations representing most probably immune activation due to the anticipated pharmacodynamic effects of the vaccine. Observations included a reversible increase of 35% (statistically significant with P < 0.01, only for female mice) in absolute and relative spleen weight (Supplementary Figure S5) as well as lymphoid hyperplasia of the lymph nodes. Additionally, histopathology of the injection sites revealed infiltration by mononuclear cells and granulomatous inflammation. No signs for systemic toxicity of MGN1601 or its murine homologue were observed. After proof of concept of the designed vaccine was shown in mice, the next aim was the transition of the murine to a human vaccine for mRCC patients. This translational effort required the identification of a suitable cell line. In order to assure a comprehensive presentation of TAA by the vaccine cells, the cell line used as source for vaccine preparation was carefully selected. Selection was done on the basis of 17 cell lines each established from tumor material of patients with RCC obtained during nephrectomy. These cell lines had been used for the preparation of autologous vaccines. Based on propagation characteristic and clinical response, cell line B25MOL was chosen for further development of an allogeneic tumor vaccine. B25MOL cells originate from tumor material of a female patient with clear cell mRCC (stage IV with multiple metastases (pT3a pTx G3)). Tumor tissue showed outgrowth of cells after 6 weeks of cultivation, and cells exhibited stable propagation. No clonal selection was performed. After four autologous vaccinations with gene-modified B25MOL cells expressing IL-7 and GM-CSF over a course of 6 weeks, the patient showed complete clinical response (lasting for 7 years and resulting in 13 years of survival) with disappearance of all metastases indicating immunologically favored characteristics of these cells. A master cell bank of B25MOL cells was manufactured, stored, and certified for use in humans. Stability of transcriptome was analyzed by comparison of RNA of B25MOL cells after initial and late round of seeding of a master cell bank aliquot (Figure 3a). From 32,000 genes analyzed, only 5 changed expression more than threefold between passage 20 and passage 40. Three of these genes were not annotated at the time of analysis and two coded for small nucleolar RNA (SNORA36C and SNORA36A) involved in the modification of 18S rRNA. mRNA data of the cells used as source for the vaccine showed expression of TAA (see Supplementary Table S2). Congruency of mRNA data and protein expression data was evaluated for 242 surface proteins, easily accessible for fluorescence-activated cell sorting. Besides a few exceptions, correlation was roughly given. Out of the analyzed antigens, 102 were clearly expressed (geometric mean value higher than threefold of respective isotype control). From those, about a dozen are described as immunogenic or are even in use as components of tumor vaccines, including CD40, CD44, CD54 (ICAM), CD73, CD95 (FAS), CD105 (TGF-β receptor complex, endoglin), CD146 (MCAM), CD151, CD227 (MUC1), CD326 (EpCAM), CD340 (Her-2/neu), EGFR, and Met (Figure 3b). Four different MIDGE vectors each coding for the expression of CD80, CD154, GM-CSF, or IL-7 were used for transient gene modification of B25MOL cells. Thereafter, gene-modified B25MOL cells were gamma-irradiated to prevent further propagation of cells. Typically, about 50% of vaccine cells expressed CD80 as well as CD154 (Figure 4a) on their cell surface. Concomitantly, vaccine cells secreted the cytokines GM-CSF and IL-7. Both accumulated in the culture supernatants and were quantified to 38,035 ± 9,266 (GM-CSF) and 3,815 ± 703 (IL-7) pg/1 × 106 cells/4 hours. Secretion data of cytokines are given as mean (± SD) of eight batches (N = 8), representing >2 × 1010 vaccine cells. Concentration of GM-CSF was about 10 times higher than that of IL-7, indicating different secretory potentials for the two cytokines by the vaccine cells. A 6-day culture of vaccine cells confirmed the higher secretory capacity of GM-CSF (Figure 4b). The concentration of IL-7 in the supernatant increased for about 1 day and was stable thereafter, while the concentration of GM-CSF increased pronounced within the first 24 hours and moderate thereafter. Furthermore, the expression of CD80 on the surface of living cells remained almost constant over several days. Due to the irradiation of gene-modified B25MOL cells, the percentage of living cells constantly dropped after a lag phase of about 10 hours until day 14 when no viable cell was detected. Expression of cytokines was additionally analyzed in vivo. For this, a murine homologue of the vaccine (murine Renca cells, gene-modified with murine forms of CD80, CD154, GM-CSF, and IL-7, in combination with dSLIM) was manufactured. In vitro expression data were comparable to MGN1601 (Figure 4c, data of cytokine secretion: 25,295 pg/4 hours × 1 × 106 cells (± 10,852) for GM-CSF and 660 pg/4 hours × 1 × 106 cells (± 143) for IL-7 (N = 7), representing about 5 × 109 vaccine cells). The murine homologue was injected s.c. to mice in an allogeneic setting. The concentration of the murine cytokines GM-CSF and IL-7 at the injection sites was analyzed in biopsy homogenates at various time points after application of the vaccine (Figure 4d). Cytokine concentration increased after application for up to 8 hours and decreased thereafter. Fluctuation in individual cytokine concentration is most probably due to the fact that biopsies do not perfectly co-localize with the site of highest cytokine concentration. The concentration of GM-CSF in vivo was clearly higher (10–20 times) than that of IL-7, confirming the in vitro secretion characteristics. B25MOL cells own a unique repertoire of potentially immunogenic TAA. Maintenance of this profile during their manufacturing process to vaccine cells is an important goal. Comparison of expression of cell surface proteins from B25MOL cells and engineered vaccine cells is shown in Figure 5. Geometric mean values of antigens on B25MOL cells and on vaccine cells are mainly similar, indicating constant expression of the analyzed surface proteins. Especially highly expressed proteins (geometric mean values > 100) show only marginal changes in their surface expression. Besides the expected increase in expression of CD80 and CD154 two ABC transporters show higher expression (7.4-fold for CD338 and 2.7-fold for CD243) as well as CD15, SSEA-1, and disialoganglioside. Reduced expression was only noted for CD201 and MUC1 (CD227). Both molecules got lost from the cell surface during manufacturing of B25MOL cells to vaccine cells, probably due to either a shedding process because their soluble forms are described22Julian J Dharmaraj N Carson DD MUC1 is a substrate for gamma-secretase.J Cell Biochem. 2009; 108: 802-815Crossref PubMed Scopus (32) Google Scholar,23Lillehoj EP Han F Kim KC Mutagenesis of a Gly-Ser cleavage site in MUC1 inhibits ectodomain shedding.Biochem Biophys Res Commun. 2003; 307: 743-749Crossref PubMed Scopus (46) Google Scholar or an increase in intracellular compartments. Comparison of expression of selected surface-TAA in B25MOL cells and vaccine cells is shown in Figure 5b. Expression of TAA is highly preserved on vaccine cells indicating the maintenance of the characteristics of B25MOL cells in engineered vaccine cells. Furthermore, expression data of mRNA analyzed by microarrays remain mainly on similar levels in the vaccine cells compared to B25MOL cells (K. Heinrich, personal communication). After assuring that the human vaccine expresses the analyzed TAA, co-stimulatory molecules, and cytokines, the translational course was continued, and a phase 1/2 clinical trial (ASET) was performed to assess safety, efficacy, and immunological effects of MGN1601 in mRCC cancer patients24Grünwald V Weikert S Schmidt-Wolf IGH Hauser S Magheli A Schroff M Schmidt M Wittig B ASET study: Long-term follow-up data of patients with metastatic renal cell carcinoma treated with MGN1601.J Clin Oncol. 2014; 32: LBA399Crossref Google Scholar (V. Grünwald, S. Weikert, I.G.H. Schmidt-Wolf, S. Hauser, A. Magheli, K. Kapp et al., personal communication). Here, the translational analyses regarding CD4-based humoral immune responses are in the focus in order to strengthen the developmental process from mice to men. In short, 19 heavily pretreated patients with mRCC were included to receive eight doses of MGN1601 within 12 weeks (intended-to-treat)—with 10 of them completing the treatment per protocol (PP). Application of MGN1601 to patients was safe and well tolerated. Notably, the PP-treated patients showed significantly increased overall survival (P < 0.05, median overall survival at final study analysis of 115.3 weeks) over the intended-to-treat population.24Grünwald V Weikert S Schmidt-Wolf IGH Hauser S Magheli A Schroff M Schmidt M Wittig B ASET study: Long-term follow-up data of patients with metastatic renal cell carcinoma treated with MGN1601.J Clin Oncol. 2014; 32: LBA399Crossref Google Scholar Here, the humoral immune response of MGN1601-treated patients against TAA expressed on vaccine cells was evaluated. For this purpose, sera of the PP-treated patients from the ASET study were analyzed for their binding properties to 17 known TAA—each represented on an array by overlapping peptides (Table 1). The serum of each patient showed binding to peptides either not recognized by their respective pre-immune serum or with a clearly increased intensity after MGN1601 treatment (shown for a single patient in Supplementary Figure S3). In average, serum of each patient recognized 37 out of 4,399 peptides (±29, N = 10).Table 1Analysis of patients’ sera by peptide array: identification of shared immune responsesaSera of all PP-treated patients (N = 10) from the ASET study were collected before the first vaccination and after eight vaccinations (week 12) and analyzed for binding intensity to 17 antigens. Each antigen was represented by successive 15-mer peptides with an overlap of 13 amino acids. Each peptide was spotted in duplicate to the array. Immune response to peptides was identified when three criteria were fulfilled: high binding intensit" @default.
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• W2345970825 title "Design and characterization of the tumor vaccine MGN1601, allogeneic fourfold gene-modified vaccine cells combined with a TLR-9 agonist" @default.
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• W2345970825 doi "https://doi.org/10.1038/mto.2015.23" @default.
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