Stability and activity of MCSP-specific chimeric antigen receptors (CARs) depend on the scFv antigen-binding domain and the protein backbone.

Chimeric antigen receptor (CAR)-modified T cells emerged as effective tools in the immunotherapy of cancer but can produce severe on-target off-tissue toxicities. This risk can conceivably be overcome, at least partially, by transient transfection. The design of CARs, however, has so far not been optimized for use in non-permanent T cell modification. Here we compared the performance of T cells modified with three different first- and second-generation CARs, each specific for MCSP (HMW-MAA) which is commonly expressed by melanoma cells. Upon RNA transfer, the expression of all receptors was limited in time. The second-generation CARs, which combined CD28-CD3ζ signaling, were expressed at higher levels and more prolonged than first-generation CARs with CD3ζ only. The CD28 domain increased the cytokine production, but had only an indirect effect on the lytic capacity, by prolonging the CAR expression. Especially for the second-generation CARs, the scFv clearly impacted the level and duration of CAR expression and the T cell performance. Thus, we identified a CAR high in both expression and anti-tumor cell reactivity. T cells transfected with this CAR increased the mean survival time of mice after challenge with melanoma cells. To facilitate clinical application, this CAR was used to redirect T cells from late-stage melanoma patients by RNA transfection. These T cells mediated effective antigen-specific tumor cell lysis and release of pro-inflammatory cytokines, even after cryoconservation of the transfected T cells. Taken together, the analysis identified a CAR with superior anti-melanoma performance after RNA transfer which is a promising candidate for clinical exploration.

Effect of adjuvant endocrine therapy on hormonal levels in premenopausal women with breast cancer: the ProBONE II study.

Endocrine therapy (ET) is a key treatment modality in hormone receptor positive (HR+) early breast cancer (BC) patients. Although the anticancer activity of adjuvant ET + zoledronic acid (ZOL) has been investigated, the potential effects of ET ± ZOL on endocrine hormones in premenopausal women with HR+ early BC are not well understood. ProBONE II was prospective, double-blind, randomized controlled trial. Premenopausal patients with histologically confirmed invasive BC with no evidence of metastases and a T score >-2.5 received ET ± ZOL 4 mg every 3 months for 2 years. Serum levels of estradiol (E2), follicle-stimulating hormone, anti-muellerian hormone (AMH), inhibins A and B, sex hormone-binding globulin, parathyroid hormone, total testosterone, and vitamin D were evaluated at baseline and at every scheduled visit. Of 71 women enrolled, 70 were evaluable (n = 34, ZOL; n = 36, placebo). No statistically significant differences were observed in hormone levels, except E2 and AMH, which showed minor differences. These included decreases in serum E2 levels, which reached a nadir after 3 and 9 months in placebo and ZOL groups, respectively, and decrease in serum AMH levels throughout the study with ZOL, but remained constant with placebo after 6 months. Adverse events in ZOL-treated group were influenza-like illness (32.4 %), bone pain (32.4 %), chills (20.6 %), and nausea (23.5 %). ET ± ZOL was well tolerated. This study showed no influence of ZOL on hormonal level changes that accompany ET, supporting inclusion of ZOL in adjuvant therapy for premenopausal women with HR + BC.

Targeting of DEC-205 on human dendritic cells results in efficient MHC class II-restricted antigen presentation.

The use of dendritic cells (DCs) in therapeutic cancer vaccination requires their loading with tumor-specific antigen(s). DEC-205, a phagocytosis receptor mediating antigen uptake, is associated with CD8(+) T-cell responses in mice. Here we fused an anti-DEC-205scFv to an HLA-DP4-restricted epitope from the tumor antigen MAGE-A3, and examined the suitability and efficacy of DEC-205 to deliver a helper epitope to human monocyte-derived DCs (moDCs). The construct specifically bound DEC-205 on human moDCs without negative impact on DC phenotype and function. We measured antigen presentation with specific autologous CD4(+) T cells, generated by TCR-RNA transfection. DEC-205 targeting resulted in significant major histocompatibility complex class II-restricted antigen presentation, and was superior to loading DCs by electroporation of mRNA encoding endosome-targeted MAGE-A3-DCLAMP or by direct peptide pulsing. Anti-DEC-205scFv-MAGE-A3 was presented 100 times more efficiently than the control constructs. DC maturation before or during incubation with anti-DEC-205scFv-MAGE-A3 reduced the interleukin-10/interleukin-2 ratio. Moreover, we successfully applied the DEC-205 targeting strategy to moDCs from malignant melanoma patients. Again, DEC-205-targeted mature DCs (mDCs) presented the antigen more efficiently than peptide-pulsed DCs and maintained their stimulatory capacity after cryoconservation. Thus, DEC-205 targeting represents a feasible and effective method to deliver helper epitopes to DCs in anticancer vaccine strategies, which may also be suitable for DC targeting in vivo.

Positive effects of zoledronate on skeletal-related events in patients with renal cell cancer and bone metastases.

INTRODUCTION: Approximately 30% of patients with renal cell cancer (RCC) develop bone metastasis causing skeletal-related events (SRE): pathologic fracture, spinal cord compression, surgery to bone and radiotherapy. Zoledronic acid demonstrated significant clinical benefit in RCC patients in a retrospective analysis. Primary objective of this prospective study was the proportion of patients experiencing ≥ 1 SRE during 12 months of zoledronic acid treatment and to verify the retrospective data. MATERIALS AND METHODS: Fifty patients with histologically confirmed RCC and evidence of ≥ 1 cancer-related bone lesion and ≤ 3 prior bisphosphonate applications were enrolled in 19 German centers between 2004 and 2007. The patients received 4 mg zoledronic acid every 3 weeks for 12 months followed by a follow up period for overall survival of 12 months. Bone lesions were diagnosed by bone scan or MRI-quickscan. Greater and equal to 1 lesion had to be confirmed by x-ray, CT or MRI scan. Additional bone scans were performed after completion of study treatment and if clinically indicated. In case of suspicion or evidence of a SRE it had to be confirmed radiologically. RESULTS: In total, 49 of the 50 enrolled patients were treated. Only 11 of them (22.4%) experienced any SRE until month 12. Patients with > 6 lesions and higher baseline MSKCC (Memorial Sloan-Kettering Cancer Center) score had a higher risk for SREs. Zoledronic acid was generally well tolerated and its known safety profile was affirmed. CONCLUSIONS: This prospective study confirms the results of prior data about the efficacy of zoledronic acid in patients with metastatic (m)RCC, supporting its beneficial use in these patients.

Shortened up-dosing with sublingual immunotherapy drops containing tree allergens is well tolerated and elicits dose-dependent clinical effects during the first pollen season.

BACKGROUND: This study compared a rapid home-based up-dosing schedule for sublingual immunotherapy (SLIT) drops containing tree pollen allergens with two previously established schedules. Furthermore, the clinical effect of the SLIT was investigated with respect to patients' first pollen season under treatment. METHODS: In this open-label, prospective, patient-preference, non-interventional study, local and systemic reactions were compared between three up-dosing groups using a SLIT formulation containing birch, alder, and hazel pollen extracts (ORALVAC® Compact Bäume). Clinical improvement after patients' first season under treatment was analysed using symptom scores, ARIA classification, symptom control, and the use of symptomatic medication and was compared with data from the previous, pre-treatment pollen season. As the real-life study design allowed no placebo group, the late-treated patients (co-seasonal) served as a control, and crowd-sourced symptom data from persons with hay fever were used from a free web-based online diary. RESULTS: In 33 study centres in Germany and Austria, 164 patients were included. The treatment was well tolerated, without difference between the groups during the up-dosing phase. At the end of the assessment, 96.1% rated the tolerability of the treatment as good or very good. Local reactions were mostly mild in severity and no serious adverse events occurred. Symptom scores decreased from the 2016 pollen season to the 2017 pollen season. As for the ARIA classification, 79.0% of patients had persistent, moderate-to-severe rhinitis before treatment, but only 18.6% had the same classification after treatment. In all, 62.4% of patients achieved symptom control, and 34.3% of patients required no symptomatic medication after treatment. The rhinoconjunctivitis score was 34.4% lower for pre-seasonal treatment initiation than for the control group. Crowd-sourced symptom load indices showed that the 2016 season caused slightly more symptoms; however, it is assumed that this difference of 0.3-0.5 (score range 0-10) was of less clinical relevance. CONCLUSION: The treatment administered using the rapid home-based up-dosing schedule was safe and well tolerated. Symptom relief and reduction in medication use were observed during the first pollen season with SLIT. TRIAL REGISTRATION NUMBER: NCT03097432 (clinicaltrials.gov).

A single chain immunotoxin, targeting the melanoma-associated chondroitin sulfate proteoglycan, is a potent inducer of apoptosis in cultured human melanoma cells.

A recombinant immunotoxin was constructed by fusing a single chain fragment variable antibody fragment, specific for the melanoma-associated chondroitin sulfate proteoglycan (MCSP), to a truncated variant of Pseudomonas exotoxin A (ETA'), carrying a C-terminal KDEL-peptide for improved retrograde intracellular transport. The resulting immunotoxin MCSP-ETA' was periplasmatically expressed in Escherichia coli and purified under native conditions by affinity chromatography resulting in a yield of approximately 30 mug/l bacterial culture. This immunotoxin induced antigen-specific apoptosis in the cultured human melanoma-derived cell lines A2058 and A375M, and treatment with a single dose of the agent eliminated up to 80% of these cells within 72 h. The dose needed for half-maximum killing (EC50) was approximately 1 nmol/l for both cell lines. MCSP-ETA' also displayed cytotoxic activity against cultured primary melanoma cells from patients with advanced disease (pathologic stages IIIC and IV), with net cell death reaching up to 70% within 96 h after treatment with a single dose of 14 nmol/l. MCSP-ETA' induced cell death synergistically with cyclosporin A, both in established human melanoma cell lines and cultured primary melanoma cells. The distinctive antigen-restricted induction of apoptosis and the synergy with cyclosporin A justify further evaluation of this novel agent with regard to its potential application for the treatment of malignant melanoma.

The ZOTECT study: Effect of zoledronic acid on bone metabolism in patients with bone metastases from prostate or breast cancer.

PURPOSE: The ZOTECT study assesses the effect of zoledronic acid (ZOL) on bone-marker levels and potential correlations with disease outcomes in bisphosphonate-naive patients. METHODS: This prospective, single-arm, open-label study in bisphosphonate-naive (≥6 months) patients with bone metastases from prostate cancer (PC; n=301) or breast cancer (BC; n=99) enrolled at 98 German sites (May 2006 to July 2008) investigated the effect of ZOL (4 mg intravenously every 4 weeks×4 months, with a final follow-up at 12 months) on bone-marker levels. Secondary assessments: skeletal-related event (SRE) rate, pain, quality of life (QoL), and prostate-specific antigen levels. Endpoints were assessed using summary statistics by visit/tumor type and Kaplan-Meier analyses. RESULTS: ZOL treatment significantly decreased bone-marker levels (amino-terminal propeptide of type I collagen [P1NP], C-terminal cross-linking telopeptide of type I collagen [CTX]; P<0.0001), and this decrease was maintained through the final 1-year follow-up visit. Baseline P1NP and CTX levels correlated with extent of bone disease (P<0.0001, each) and on-treatment decreases in marker levels. Skeletal disease burden and bone-marker levels were similar between PC and BC patients, and ZOL did not significantly influence osteoprotegerin/receptor activator of nuclear factor-κB ligand levels. Only 13 SREs occurred in 11 patients, supporting the known ZOL-mediated reduction in SREs. On-treatment bone-marker level changes did not correlate with SRE rate, pain scores, or QoL. Generally, ZOL was well tolerated and adverse events were consistent with its known safety profile. CONCLUSIONS: This study confirms that ZOL therapy significantly reduces bone turnover (measured as P1NP and CTX levels) in patients with bone metastases from PC or BC.

A fast and robust method to clone and functionally validate T-cell receptors.

Sequencing, cloning and functional testing of T-cell-receptor (TCR) alpha- and beta-chains from T-cell clones is often required in immunotherapy and in immunological research. However, the determination of the TCR chains by a simple PCR is not possible, since, in contrast to the 3' constant domain and untranslated region (UTR), no conserved sequences are present in the 5' region. Furthermore, subsequent functional testing of cloned TCRs requires laborious cell culture experiments, often involving primary human material and time-consuming viral transduction strategies. Here we present a universal PCR-based protocol, adapted from the capswitch technology, that allows for amplification of the TCR alpha- and beta-chain mRNAs without knowledge of the TCR variable domain subtype by attaching a designed sequence to the mRNA's 5' end. Two different MelanA/HLA-A2-specific and one HIVgag/HLA-A2-specific TCR were cloned that way, and were functionally tested by a newly developed easy, fast, and low-cost method: we electroporated Jurkat T cells simultaneously with TCR-encoding RNA and an NFAT-reporter construct, and measured the activation status of the cells upon specific stimulation. The results of this assay correlated with the cytokine release, functional avidity, proliferative activity, and the ability to recognize MelanA/HLA-A2-presenting tumor cells of bulk T cells electroporated with RNA encoding the same TCR. Together these two protocols represent a rapid and low-cost tool for the identification and functional testing of TCRs of T-cell clones, which can then be applied in immunotherapy or immunological research.