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Although Chimeric Antigen Receptor (CAR) T-cells have shown high efficacy in hematologic malignancies, they can cause severe to life-threatening side effects. To address these safety concerns, we have developed adaptor CAR platforms, like the UniCAR system. The redirection of UniCAR T-cells to target cells relies on a Target Module (TM), containing the E5B9 epitope and a tumor-specific binding moiety. Appropriate UniCAR-T activation thus involves two interactions: between the TM and the CAR T-cell, and the TM and the target cell. Here, we investigate if and how alterations of the amino acid sequence of the E5B9 UniCAR epitope impact the interaction between TMs and the UniCAR.We identify the new epitope E5B9L, for which the monoclonal antibody 5B9 has the greatest affinity. We then integrate the E5B9L peptide in previously established TMs directed to Fibroblast Activation Protein (FAP) and assess if such changes in the UniCAR epitope of the TMs affect UniCAR T-cell potency. Binding properties of the newly generated anti-FAP-E5B9L TMs to UniCAR and their ability to redirect UniCAR T-cells were compared side-by-side with the ones of anti-FAP-E5B9 TMs. Despite a substantial variation in the affinity of the different TMs to the UniCAR, no significant differences were observed in the cytotoxic and cytokine-release profiles of the redirected T-cells. Overall, our work indicates that increasing affinity of the UniCAR to the TM does not play a crucial role in such adaptor CAR system, as it does not significantly impact the potency of the UniCAR T-cells.

T cells can be armed with chimeric antigen receptors (CARs) to target tumor cells, a strategy which has proven successful against some hematological cancers. To overcome some of the dangerous side-effects, a modular system termed RevCAR has been developed. Its components are the RevCAR T cells, which alone cannot bind to any targets, and a bispecific targeting molecule (RevTM). Since the presence of the RevTM is necessary for the system to be functional, it can be used as a safety switch. In addition, different RevTMs can be used with the same T cells to target different antigens, achieving more flexibility. In order to achieve a successful treatment of solid tumors, however, the immune suppression of the tumor microenvironment needs to be overcome. To do this, novel RevTMs were designed which target immune checkpoint molecules such as PD-L1, a pathway which is often exploited by cancer cells to block the immune response. We have successfully shown that, in the presence of these new RevTMs, RevCAR T cells are able to specifically target and kill a wide range of PD-L1-expressing cells, both in monolayer and 3D models. They also release pro-inflammatory cytokines and express activation markers after the co-culture. Furthermore, a combinatorial targeting approach of both a TAA and an immune checkpoint has been proven. Together, these results demonstrate a promising new strategy to apply the RevCAR platform to solid tumors.

Discussed are two picolinate appended bispidine ligands (3,7-diazabicyclo[3.3.1]nonane derivatives) in comparison with an earlier described bis-pyridine derivative, which are all known to strongly bind CuII. The radiopharmacological characterization of the two isomeric bispidine complexes includes quantitative labeling with 64CuII at ambient conditions with high radiochemical purities and yields (molar activities > 200 MBq/nmol). Challenge experiments in presence of EDTA, cyclam, human serum and SOD demonstrate high stability and inertness of the 64Cu-bispidine complexes. Biodistribution studies performed in Wistar rats indicate a rapid renal elimination for both 64Cu-labeled chelates. The bispidine ligand with the picolinate group in N7 position was selected for further biological experiments, and its backbone was therefore substituted with a benzyl-NCS group at C9. Two tumor target modules (TMs), targeting prostate stem cell antigen (PSCA), overexpressed in prostate cancer, and the fibroblast activation protein (FAP) in fibrosarcoma, were selected for thiourea coupling with the NCS-functionalized ligand and lysine residues of TMs. Small animal PET experiments on tumor-bearing mice showed very good and specific accumulation of the 64Cu-labeled TMs in tumors.

Item does not contain fulltext, BACKGROUND: Linezolid is an effective, but toxic anti-tuberculosis drug that is currently recommended for the treatment of drug-resistant tuberculosis. Improved oxazolidinones should have a better safety profile, while preserving efficacy. Delpazolid is a novel oxazolidinone developed by LegoChem Biosciences Inc. that has been evaluated up to phase 2a clinical trials. Since oxazolidinone toxicity can occur late in treatment, LegoChem Biosciences Inc. and the PanACEA Consortium designed DECODE to be an innovative dose-ranging study with long-term follow-up for determining the exposure-response and exposure-toxicity relationship of delpazolid to support dose selection for later studies. Delpazolid is administered in combination with bedaquiline, delamanid and moxifloxacin. METHODS: Seventy-five participants with drug-sensitive, pulmonary tuberculosis will receive bedaquiline, delamanid and moxifloxacin, and will be randomized to delpazolid dosages of 0 mg, 400 mg, 800 mg, 1200 mg once daily, or 800 mg twice daily, for 16 weeks. The primary efficacy endpoint will be the rate of decline of bacterial load on treatment, measured by MGIT liquid culture time to detection from weekly sputum cultures. The primary safety endpoint will be the proportion of oxazolidinone class toxicities; neuropathy, myelosuppression, or tyramine pressor response. Participants who convert to negative liquid media culture by week 8 will stop treatment after the end of their 16-week course and will be observed for relapse until week 52. Participants who do not convert to negative culture will receive continuation phase treatment with rifampicin and isoniazid to complete a six-month treatment course. DISCUSSION: DECODE is an innovative dose-finding trial, designed to support exposure-response modelling for safe and effective dose selection. The trial design allows assessment of occurrence of late toxicities as observed with linezolid, which is necessary in clinical evaluation of novel oxazolidinone

06 augustus 2023, Contains fulltext : 295321.pdf (Publisher’s version ) (Open Access)

Engineered T cells that express chimeric antigen receptors (CARs) show a big potential in cancer immunological research. They are synthetic receptors that can target antigens independently from the MHC complex. However, most tumor associated antigens are not only expressed on the tumor site but also on healthy tissues. To switch off CAR T cells in case of on-target/off-tumor effects occur, an adaptor CAR platform, called the universal CAR (UniCAR), was developed. In contrast to conventional CAR systems this platform consists of the engineered T cells and a linker molecule, called target module (TM), that is both binding to the target antigen and recognized by the UniCAR T cell. With this approach there is the possibility to switch the system on and off when needed. Fibroblast activation protein (FAP) is a protein of high interest in cancer research as it is upregulated in most of epithelial cancers but shows a comparably low expression in healthy tissues. It is shown to be expressed on cancer associated fibroblasts and therefore plays an important role in the tumor microenvironment where it has pro-tumorigenic activity both in the FAP positive and surrounding cells. Given this, the goal of this work was to develop new TMs against FAP. To achieve such aim, we cloned two TM formats, either based on a single-chain variable fragment (scFv) or an IgG4 based backbone. The difference in the size of the backbones translates into different half-lifes and kinetics. These different TM formats can be used to customize UniCAR therapy according to the given circumstances. We were able to purify and perform various experiments with the mentioned TMs. We could show binding to the target cells using flow cytometry. We could also show that the TMs could be used to induce specific lysis in vitro on monolayer cells and spheroids. We were also able to show UniCAR T cell infiltration into the spheroids and their consequent activation. Moreover, in mouse models the TMs could be used for P

T-cells genetically modified to express chimeric antigen receptors (CARs) are playing a more and more important role in targeted cancer immunotherapy. However, these living drugs can also cause lifethreatening side effects. To overcome such limitations and improve the safety of CAR T-cell therapy, adaptor CAR platforms such as the universal CAR (UniCAR) have been developed. This platform consists of the UniCAR T-cell and a target module (TM) cross-linkage effector and tumor cells. Here, we have established a novel UniCAR system targeting Fibroblast Activation Protein (FAP) that is highly expressed in the tumor microenvironment of epithelial cancers and a marker for cancer-associated fibroblasts. For that, we constructed two novel FAP-directed TMs possessing different sizes and pharmacokinetic properties, in which one is based on a single-chain variable fragment (scFv), and the other is based on an IgG4 backbone. We have shown that both TMs were able to bind to FAP-expressing cells and redirect UniCAR T-cells in vitro to monolayer and spheroid target cells inducing effective killing. Furthermore, we could show infiltration and activation of T-cells in the spheroid setting. Using in vivo models, the TMs were proven to be suitable to be used for PET imaging showing FAP-specific accumulation at the tumor site. Moreover, the immunotherapeutic effect of UniCAR T-cells in combination with FAP TMs was demonstrated using mouse models. In conclusion, in this work, we could show that the two novel anti- FAP TMs prove to hold great theranostic potential for diagnostic imaging and immunotherapy.