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SummaryBackground. Ataxia telangiectasia mutated (ATM) kinase orchestrates DNA double strand break (DSB) repair; ATM inhibitors may therefore enhance the therapeutic effect of DSB-inducing treatments such as radiotherapy (RT). M3541 is an orally administered selective inhibitor of ATM. Methods. This phase I dose-escalation study evaluated the maximum-tolerated dose (MTD), recommended phase II dose(s) (RP2D), safety, pharmacokinetics (PK) and antitumor activity of M3541 in combination with fractionated palliative RT in patients with solid tumors. Fifteen patients received palliative RT (30 Gy in 10 fractions) and escalating doses of M3541 (50–300 mg administered on RT fraction days) guided by a Bayesian 2-parameter logistic regression model with overdose control. Results. Doses of M3541 up to 300 mg/fraction day were well tolerated. One patient (200 mg group) experienced two dose-limiting toxicities (urinary tract infection, febrile neutropenia) that resolved with antibiotics. All patients reported ≥ 1 treatment-emergent adverse event (TEAE) but none led to treatment discontinuation. No grade ≥ 4 TEAEs were reported and there was no indication of a dose effect for any TEAE. Three patients (20.0%; 95% confidence interval 4.3–48.1) had confirmed complete or partial response. M3541 total plasma levels did not increase with dose following single or repeated dosing. No relationship was observed between dose and changes in the ratio of phosphorylated to total ATM or in immune cell counts. Conclusions. The MTD and RP2D could not be established as the study closed early due to the absence of a dose–response relationship and non-optimal PK profile. No further clinical development of M3541 was pursued. (Trial registration number ClinicalTrials.gov NCT03225105. Registration date July 21, 2017).

Purpose: Despite the great success of HER2 vaccine strategies in animal models, effective clinical results have not yet been obtained. We studied the feasibility of using DNA coding for chimeric rat/human HER2 as a tool to break the unresponsiveness of T cells from patients with HER2-overexpressing tumors (HER2-CP).Experimental Design: Dendritic cells (DCs) generated from patients with HER2-overexpressing breast (n = 28) and pancreatic (n = 16) cancer were transfected with DNA plasmids that express human HER2 or heterologous rat sequences in separate plasmids or as chimeric constructs encoding rat/human HER2 fusion proteins and used to activate autologous T cells. Activation was evaluated by IFN-γ ELISPOT assay, perforin expression, and ability to halt HER2+ tumor growth in vivo.Results: Specific sustained proliferation and IFN-γ production by CD4 and CD8 T cells from HER2-CP was observed after stimulation with autologous DCs transfected with chimeric rat/human HER2 plasmids. Instead, T cells from healthy donors (n = 22) could be easily stimulated with autologous DCs transfected with any human, rat, or chimeric rat/human HER2 plasmid. Chimeric HER2-transfected DCs from HER2-CP were also able to induce a sustained T-cell response that significantly hindered the in vivo growth of HER2+ tumors. The efficacy of chimeric plasmids in overcoming tumor-induced T-cell dysfunction relies on their ability to circumvent suppressor effects exerted by regulatory T cells (Treg) and/or interleukin (IL)-10 and TGF-β1.Conclusions: These results provide the proof of concept that chimeric rat/human HER2 plasmids can be used as effective vaccines for any HER2-CP with the advantage of being not limited to specific MHC. Clin Cancer Res; 20(11); 2910–21. ©2014 AACR.

PDF file - 270KB, Supplementary Figure S1. Phenotype of in vitro generated mDCs. Supplementary Figure S2. Efficiency of transfection. Supplementary Figure S3. HuHuT-, HuRT- and RHuT-DCs from HS elicit anti-HER2 CD8 response. Supplementary Figure S4. RHuT-DCs from HER2-CP elicit anti-HER2 CD8 T cell response. Supplementary Figure S5. HuHuT DCs affects Treg cells activity. Supplementary Figure S6. HuHuT-DCs activate CD8 and CD4 T cells from CTRL-CP. Supplementary Table S1. HER2-overexpressing cancer patients. Supplementary Table S2. HER2-negative cancer patients.

Background and Aims Analysis of serum or plasma from patients with IgA nephropathy (IgAN) has confirmed the presence of elevated levels of circulating immune complexes containing Gd-IgA1 (Czerkinsky 1986). New sensitive and reasonably specific noninvasive tests are emerging to guide the therapeutic strategy that is applicable to all stages of IgAN (Suzuki 2014). Here we are reporting the fit for purpose validation of an ELISA method for the quantitative determination of Gd-IgA1 in human serum samples to support biomarker investigations in clinical studies of Merck KGaA, Darmstadt. Method The assay was developed based on a commercially available immunoassay kit. The dynamic range of the calibration curve was determined from 1.56 ng/mL (LLOQ) to 100 ng/mL (ULOQ). With a minimum required dilution of 200-fold and standard assay volume of 50.0 μL, the range of the method in matrix was from 312 ng/mL to 20, 000 ng/mL. In assay validation phase, multiple validation parameters were evaluated, which included minimum required dilution (MRD), calibration curve, matrix effect, Intra- & Inter run accuracy & precision, selectivity, and parallelism. Additional validation parameters include sample stability (short/long term, freeze-thaw) and batch-to-batch comparison. Results All samples measured for intra & Inter - assay precision, accuracy, fulfilled the specifications according to the acceptance criteria. The selectivity was assessed using blank serum matrix from 10 individuals: the result indicated that matrix components in serum did not interfere with the detection of Gd-IgA1. Parallelism assessment was performed successfully for both samples from healthy donors and IgAN patient samples up to dilution factor (DF) 3200 (serum samples from healthy donors were determined up to DF 1600). All DF-corrected results within the assay range were determined with %CV ≤ 30.0%. Batch to batch comparison was assessed successfully based on the known shelf life of the kit. Short term stability using QC samples were given for up to 24hrs at room temperature. Freeze-thaw stability was given for up to 3 cycles at -20°C±5°C and -75°C±15°C. The investigations were performed according to general guidelines for method validation and applicable regulations. The results of investigated validation parameters fulfilled the requirements and recommendations, generally accepted for bioanalytical projects. Conclusion The present validation qualified the method for the quantitative determination of Gd-IgA1 in human serum samples from clinical studies.

Purpose: Despite the great success of HER2 vaccine strategies in animal models, effective clinical results have not yet been obtained. We studied the feasibility of using DNA coding for chimeric rat/human HER2 as a tool to break the unresponsiveness of T cells from patients with HER2-overexpressing tumors (HER2-CP). Experimental Design: Dendritic cells (DCs) generated from patients with HER2-overexpressing breast (n = 28) and pancreatic (n = 16) cancer were transfected with DNA plasmids that express human HER2 or heterologous rat sequences in separate plasmids or as chimeric constructs encoding rat/human HER2 fusion proteins and used to activate autologous T cells. Activation was evaluated by IFN-γ ELISPOT assay, perforin expression, and ability to halt HER2+ tumor growth in vivo. Results: Specific sustained proliferation and IFN-γ production by CD4 and CD8 T cells from HER2-CP was observed after stimulation with autologous DCs transfected with chimeric rat/human HER2 plasmids. Instead, T cells from healthy donors (n = 22) could be easily stimulated with autologous DCs transfected with any human, rat, or chimeric rat/human HER2 plasmid. Chimeric HER2-transfected DCs from HER2-CP were also able to induce a sustained T-cell response that significantly hindered the in vivo growth of HER2+ tumors. The efficacy of chimeric plasmids in overcoming tumor-induced T-cell dysfunction relies on their ability to circumvent suppressor effects exerted by regulatory T cells (Treg) and/or interleukin (IL)-10 and TGF-β1. Conclusions: These results provide the proof of concept that chimeric rat/human HER2 plasmids can be used as effective vaccines for any HER2-CP with the advantage of being not limited to specific MHC. Clin Cancer Res; 20(11); 2910–21. ©2014 AACR.

Relocated in transformation/papillary thyroid carcinoma, RET/PTC3 (RP3) is a fusion oncogene that causes a form of papillary thyroid cancer (PTC). In addition to driving transformation, the constitutively active kinase precociously phosphorylates itself as well as other intracellular proteins, thereby providing tumor-specific targets for the adaptive immune system. PTCs are known to have the ability to escape the immune system driving a dysregulation of the activity of several cell populations involved in the immune response. Indeed we found that mice immunized with RP3+/MHC class II+ cells produce dramatically fewer IFNg-secreting CD4+ T cells compared to mice immunized with RP3-/MHC class II+ counterparts as measured in the spleens. We demonstrated the immunogenicity of RP3 with reactivity in both BALB/c and C57BL/6 mice to RP3/IEd- and RP3/CIITA-expressing cells. Also, we observed immunogenicity of RP3-derived phosphopeptides in ELISpot assays, supporting the hypothesis that the aberrant autophosphorylation of RP3 is a source of tumor-specific immunogenicity. Finally, utilizing a RP3-expressing vaccinia vector for immunization of C57BL/6 mice, we identified peptide sequences that appear to be immunogenic on the basis of unique conformation of the fusion protein that impacts antigen processing. Taken together these results could pave the way for better vaccine approaches without accompanying autoimmunity. Citation Format: Laura Sponton, Gabriela Cosma, Mark Mendonca, Mirella Giovarelli, Laurence Crane Eisenlohr. Identification of tumor-specific antigens associated with RET/PTC3 expression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1294. doi:10.1158/1538-7445.AM2015-1294