Auswahl der wissenschaftlichen Literatur zum Thema „Small molecule-radio conjugates“

Abstract Imaging procedures based on small molecule-radio conjugates targeting Fibroblast Activation Protein (FAP) are taking the spotlight in clinical practice for the diagnosis of a wide variety of cancer lesions. However, therapeutic applications of FAP-targeting radioligand therapeutics are limited by their short residence time in tumor lesions. We have recently described the discovery of OncoFAP, a novel ultra-high affinity ligand of FAP which has been already validated for PET imaging applications in more than twenty patients with solid tumors. In this abstract, we describe the development and in vivo characterization of BiOncoFAP, a dimeric FAP targeting small molecule based on OncoFAP. In particular, OncoFAP and BiOncoFAP displayed a superimposable affinity for recombinant human FAP (KD = 970 pM and 993 pM, respectively, measured by Fluorescence Polarization). Moreover, the two ligands exhibited a clear selectivity for the target, without significantly interacting with a wide panel of non-target proteins. BiOncoFAP was coupled to a DOTAGA chelator, suitable for radiolabeling with [177Lu]Lu for therapeutic applications. Cold [natLu]Lu-BiOncoFAP-DOTAGA showed an excellent stability in both mouse and human serum, with half-life longer than 5 days, supporting in vivo investigations. In a head-to-head in vivo biodistribution comparative study against [177Lu]Lu-OncoFAP-DOTAGA, [177Lu]Lu-BiOncoFAP-DOTAGA exhibited a more stable and prolonged residence time in FAP-positive tumors implanted in immunodeficient mice (~20% ID/g and ~200-to-1 tumor-to-blood ratio, 24 h after systemic administration). Notably, [177Lu]Lu-BiOncoFAP-DOTAGA did not significantly accumulate in healthy organs, thus showing an outstanding tumor-to-organ ratio (e.g., 12-to-1 tumor-to-kidney and 34-to-1 tumor-to-liver ratio, at the 24 h time point). These findings heighten BiOncoFAP as promising candidate for the development of anti-cancer radioligand therapeutics towards FAP-expressing tumor lesions. Citation Format: Samuele Cazzamalli, Andrea Galbiati, Aureliano Zana, Matilde Bocci, Jacopo Millul, Jacqueline Mock, Abdullah Elsayed, Dario Neri. A novel dimeric small molecule-radio conjugate targeting fibroblast activation protein with high and prolonged tumor uptake [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB527.

A novel competitive binding assay for protein kinase inhibitors has been developed for high-throughput screening (HTS). Unlike functional kinase assays, which are based on detection of substrate phosphorylation by the enzyme, this novel method directly measures the binding potency of compounds to the kinase ATP binding site through competition with a conjugated binding probe. The binding interaction is coupled to a signal amplification system based on complementation of β-galactosidase enzyme fragments, a homogeneous, nonisotopic assay technology platform developed by DiscoveRx Corp. In the present study, staurosporine, a potent, nonselective kinase inhibitor, was chemically conjugated to a small fragment of β-galactosidase (termed ED-SS). This was used as the binding probe to the kinase ATP binding pocket. The binding potencies of several inhibitors with diverse structures were assessed by displacement of ED-SS from the kinase. The assay format was specifically evaluated with GSK3α, an enzyme previously screened in a radio-active kinase assay (i.e., measurement of [33P]-γ-ATP incorporation into the kinase peptide substrate). Under optimized assay conditions, nonconjugated staurosporine inhibited ED-SS binding in a concentration-dependent manner with an apparent potency (IC50) of 11 nM, which was similar to the IC50 value determined in a radioactive assay. Furthermore, 9 kinase inhibitors with diverse structures, previously identified from chemical compound library screening, were screened using the competitive binding assay. The potencies in the binding assay were in very good agreement with those obtained previously in the isotopic functional activity assay. The binding assay was adapted for automated HTS using selected compound libraries in a 384-well microtiter plate format. The HTS assay was observed to be highly robust and reproducible (Z factors > 0.7) with high interassay precision ( R2 > 0.96). Interference of compounds with the β-galactosidase signal readout was negligible. In conclusion, the DiscoveRx competitive kinase binding assay, termed ED-NSIP™, provides a novel method for screening kinase inhibitors. The format is homogeneous, robust, and amenable to automation. Because there is no requirement for substrate-specific antibodies, the assay is particularly applicable to Ser/Thr kinase assay, in which difficulties in identifying a suitable substrate and antibody preclude development of nonisotopic assays. Although the nonselective kinase inhibitor, staurosporine, was used here, chemically conjugating the ED fragment to other small molecule enzyme inhibitors is also feasible, suggesting that the format is generally applicable to other enzyme systems.

Abstract Introduction: Malignant lymphomas are heterogeneous group of neoplasm comprising of proliferating lymphoid cells or their precursors. The two main types of lymphoma are Hodgkin Lymphoma and Non- Hodgkin Lymphoma. The current treatment modalities include chemotherapy; radiation therapy; passive immunotherapy; radio-immunotherapy and small-molecule inhibitors. Radiation therapy is the treatment of choice if disease is localized. Targeted therapy with antibody and small molecules may be more suitable for malignant lymphomas. Lymphoma cells are reported to over-express chemokine receptors that favors leukocyte infiltration, promotes tumorigenesis, angiogenesis, immune evasion, and metastasis. Human CXCL4 and CCL5 oligomerize with over 20 chemokine subfamilies to control leukocytes infiltration. CXCR4 antagonists disrupt the specific interaction of chemokines with CXCR4 and may elicit therapeutic potential. Chemokine receptor 4 (CXCR4) has been explored with radiolabelled CXCR4 antagonist (AMD3100) for imaging and future targeted radiation therapy for malignant lymphoma. Methods: Standardization of conjugation of AMD3100 with bifunctional chelating agents (DTPA, NOTA etc.). Optimization include radiolabelling of conjugated AMD3100 with 68Ga and 177Lu for imaging and therapeutic purpose respectively. The radionuclide and radiochemical purity of the product was determined. CXCR4 binding efficacy and toxicity studies were performed on CXCR4 expressing cancer cell-lines. The quality control of radiolabelled AMD3100 included, radiochemical purity, sterility, pyrogenicity, human serum and PBS stability. The biodistribution studies were performed in normal rats. After obtaining clearance from institutional ethics committee,68Ga-AMD3100-DTPA PET/CT was performed in lymphoma patients (n=2). The uptake was compared with 18FDG uptake for proof of concept. Results: DTPA conjugation of AMD3100 (1087 Da) and NOTA conjugation (1014 Da) was confirmed with MALDI-TOF. The radionuclide and radiochemical purity of 68Ga and 177Lu AMD3100 was >99%. Radio-ligand binding assay confirmed high specificity (Kd = 57.16 nM) of 177Lu-AMD3100-DTPA towards CXCR4 expressing cancer cells. Furthermore, the log absolute IC50 concentration of 177Lu-AMD3100-DTPA calculated for cytotoxicity assessment, via MTT assay, was 2.628 nM. Immunocytochemistry depicted positive nuclear staining of CXCR4 receptors in cancer cells. The synthesized radiopharmaceuticals were sterile and pyrogen-free. In-vivo physiological biodistribution of 68Ga-AMD3100-DTPA was found in liver, lung and spleen. The radiotracer showed faster renal clearance and low blood pool activity. In addition to physiological uptake, 68Ga-AMD3100-DTPA PET/CT showed similar lesions that were seen in 18FDG PET/CT of lymphoma patients. Conclusion: 68Ga/177Lu-AMD3100-DTPA demonstrated high target specificity towards CXCR4 expressing lymphoma cells. Cytotoxicity and patient study indicated its potential as theranostic agent. It can be used for the selection of patients, targeted therapy and response evaluation. Citation Format: Tamanna Lakhanpal, Jaya Shukla, Rajender Kumar, Harmanpreet Singh, Pankaj Malhotra, Alka Khadwal, Gaurav Prakash, Amanjit Bal, Yogesh Rathore, BR Mittal. Radiolabelled AMD3100 for the management of malignant lymphoma: Translational study [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A10.

Despite the different therapeutic options available, more than 10 million deaths every year are caused by cancer, thus representing one of the majors’ causes of death worldwide. Lung cancer (1.8 million), breast cancer (700’000), colorectal cancer (950’000), and liver cancer (830’000) represent half of the global cancer incidence. Common approaches for the treatment of cancer include surgery, radiotherapy (external beam radiation and Radioligand Therapy, RLT), chemotherapy and immunotherapy. While radiotherapy indistinctively targets all cells within the tumor environment, chemotherapeutic agents can interfere with the growth of rapidly dividing cancer cells. Both conventional therapeutic modalities lack selectivity, can affect healthy cells and elicit drug-resistance mechanisms that further reduce efficacy. To overcome the limitations of conventional treatments, pharmaceutical industries focused on the discovery of therapeutic agents which can be selectively active at the site of tumors. A strategy to generate targeted therapeutics consist in the use of active delivery vehicles of bioactive payloads including cytotoxic agents, radiations, and immunomodulatory agents. An increased number of products based on tumor targeting like monoclonal antibodies, peptides, and small molecules have been recently approved (e.g., ZynlontaTM, an Antibody-Drug Conjugate approved in 2021, and Sunitinib, a multikinase inhibitor approved in 2006), or are being studied in clinical trials. This thesis is focused on the design and development of Small Molecule-Drug Conjugates (SMDCs) and Small Molecule-Radio Conjugates (SMRCs) for the imaging and treatment of solid tumors. Some of the results presented in this thesis have been published in our paper “Immunotherapy with Immunocytokines and PD-1 blockade enhances the anticancer activity of Small Molecule-Drug Conjugates targeting Carbonic Anhydrase IX” (Molecular Cancer Therapeutics; 2021) and in our article “An Ultra-High Affinity Small Organic Ligand of Fibroblast Activation Protein for Tumor Targeting Applications” ( Proceedings of National Academy of Sciences; 2021).