BACKGROUND: The treatment of non-small cell lung cancer (NSCLC) patients is correlated with the efficacy of immune checkpoint blockade therapy (ICB) targeting programmed cell death ligand 1 (PD-L1) or its cognate receptor (PD-1) on cancer cells or infiltrating immune cells. Analysis of PD-L1/PD-1 expression in tumor tissue represents a crucial step before PD-L1/PD-1 blocker usage. METHODS: We used directed evolution of protein variants derived from a 13 kDa Myomedin loop-type combinatorial library with 12 randomized amino acid residues to select high-affinity binders of human PD-L1 (hPD-L1). After the ribosome display, individual clones were screened by ELISA. Detailed analysis of binding affinity and kinetics was performed using LigandTracer. The specificity of Myomedins was assessed using fluorescent microscopy on HEK293T-transfected cells and cultured cancer cells in vitro, formalin-fixed paraffin-embedded (FFPE) sections of human tonsils, and FFPE tumor samples of NSCLC patients. RESULTS: Seven identified PD-L1 binders, called MLE, showed positive staining for hPD-L1 on transfected HEK293T cells and cultured MCF-7 cells. MLE031, MLE105, MLE249, and MLE309 exhibited high affinity to both human and mouse PD-L1-transfected HEK293T cells measured with LigandTracer. The diagnostic potential of MLE variants was tested on human tonsillitis tissue and compared with diagnostic anti-PD-L1 antibody DAKO 28-8 and PD-L1 IHC 22C3 pharmDx antibody. MLE249 and MLE309 exhibited an excellent overlap with diagnostic DAKO 28-8 (Pearson ́s coefficient (r) = 0.836 and 0.731, respectively) on human tonsils on which MLE309 exhibited also excellent overlap with diagnostic 22C3 antibody (r = 0.876). Using three NSCLC tissues, MLE249 staining overlaps with 28-8 antibody (r = 0.455-0.883), and MLE309 exhibited overlap with 22C3 antibody (r = 0.534-0.619). Three MLE proteins fused with Fc fragments of rabbit IgG, MLE249-rFc, MLE309-rFc and MLE031-rFc, exhibited very good overlap with anti-PD-L1 antibody 28-8 on tonsil tissue (r = 0.691, 0.610, and 0.667, respectively). Finally, MLE249-rFc, MLE309-rFc and MLE031-rFc exhibited higher sensitivity in comparison to IHC 22C3 antibody using routine immunohistochemistry staining system Ventana, which is one of gold standards for PD-L1 diagnosis. CONCLUSIONS: We demonstrated the development of MLE Myomedins specifically recognizing hPD-L1 that may serve as a refinement tool for clinical PD-L1 detection.
The development of canine immunotolerant monoclonal antibodies can accelerate the invention of new medicines for both canine and human diseases. We develop a methodology to clone the naive, somatically mutated variable domain repertoire from canine B cell mRNA using 5'RACE PCR. A set of degenerate primers were then designed and used to clone variable domain genes into archival "holding" plasmid libraries. These archived variable domain genes were then combinatorially ligated to produce a scFv M13 phage library. Next-generation long-read and short-read DNA sequencing methodologies were developed to annotate features of the cloned library including CDR diversity and IGHV/IGKV/IGLV subfamily distribution. A synthetic immunoglobulin G was developed from this scFv library to the canine immune checkpoint receptor PD-1. This synthetic platform can be used to clone and annotate archived antibody variable domain genes for use in perpetuity in order to develop improved preclinical models for the treatment of complex human diseases.
- MeSH
- Programmed Cell Death 1 Receptor immunology MeSH
- Single-Chain Antibodies * immunology genetics MeSH
- Humans MeSH
- Antibodies, Monoclonal immunology genetics MeSH
- Neoplasms immunology therapy MeSH
- Peptide Library MeSH
- Dogs MeSH
- Recombinant Proteins immunology genetics MeSH
- Translational Research, Biomedical MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Dogs MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Developing methodological approaches for discovering novel pathways is a key challenge in the life science research. Biological pathways are regulated-in higher eukaryotes-by a vast diversity of linear peptide motifs that mediate combinatorial specificity in signal transduction pathways. The E3 ubiquitin ligase component (MDM2) is such a protein that interacts with target proteins containing linear motifs such as p53. Drug leads, such as Nutlin-3, that bind to the MDM2 hydrophobic pocket mimic p53 and can release p53 from MDM2 control and this can lead to cell death. However, these drug leads act allosterically, having agonist effects on MDM2's functions and there are other proteins whose steady state levels can be altered by Nutlin-3. As cell density can alter the proliferation state of cell populations, we examined the impact of Nutlin-3 on levels of newly synthesized proteins using pulse-SILAC mass spectrometry. The data demonstrate that at differing cell densities or population-wide proliferation rates, different newly synthesized proteins dominate the proteome landscape in a Nutlin-3 dependent manner. These data further confirm that the cell state in a population of cells can in turn impact on the MDM2 signalling landscape. This methodology forms a blueprint for biomarker discovery using clinical samples that can detect changes in the synthesis rate of proteins in cell populations treated with specific agents. Broader implications highlight tools that can be used to study allosteric regulation of protein-drug combinations.
- MeSH
- Imidazoles * pharmacology MeSH
- Humans MeSH
- Tumor Suppressor Protein p53 metabolism MeSH
- Piperazines * pharmacology MeSH
- Cell Proliferation drug effects MeSH
- Proteome * metabolism MeSH
- Proteomics methods MeSH
- Proto-Oncogene Proteins c-mdm2 metabolism MeSH
- Signal Transduction drug effects MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
PURPOSE: Patients with high-grade serous ovarian carcinoma (HGSOC) are virtually insensitive to immune checkpoint inhibitors (ICI) employed as standalone therapeutics, at least in part reflecting microenvironmental immunosuppression. Thus, conventional chemotherapeutics and targeted anticancer agents that not only mediate cytotoxic effects but also promote the recruitment of immune effector cells to the HGSOC microenvironment stand out as promising combinatorial partners for ICIs in this oncological indication. EXPERIMENTAL DESIGN: We harnessed a variety of transcriptomic, spatial, and functional assays to characterize the differential impact of neoadjuvant paclitaxel-carboplatin on the immunological configuration of paired primary and metastatic HGSOC biopsies as compared to neoadjuvant chemotherapy (NACT)-naïve HGSOC samples from five independent patient cohorts. RESULTS: We found NACT-driven endoplasmic reticulum stress and calreticulin exposure in metastatic HGSOC lesions culminates with the establishment of a dense immune infiltrate including follicular T cells (TFH cells), a prerequisite for mature tertiary lymphoid structure (TLS) formation. In this context, TLS maturation was associated with an increased intratumoral density of ICI-sensitive TCF1+PD1+ CD8+ T cells over their ICI-insensitive TIM-3+PD1+ counterparts. Consistent with this notion, chemotherapy coupled with a PD1-targeting ICI provided a significant survival benefit over either therapeutic approach in syngeneic models of HGSOC bearing high (but not low) tumor mutational burden. CONCLUSIONS: Altogether, our findings suggest that NACT promotes TLS formation and maturation in HGSOC lesions, de facto preserving an intratumoral ICI-sensitive T-cell phenotype. These observations emphasize the role of rational design, especially relative to the administration schedule, for clinical trials testing chemotherapy plus ICIs in patients with HGSOC. See related commentary by Bravo Melgar and Laoui, p. 10.
- MeSH
- CD8-Positive T-Lymphocytes * immunology drug effects MeSH
- Tertiary Lymphoid Structures * immunology pathology MeSH
- Hepatocyte Nuclear Factor 1-alpha * genetics metabolism MeSH
- Immune Checkpoint Inhibitors * therapeutic use pharmacology MeSH
- Carboplatin administration & dosage pharmacology therapeutic use MeSH
- Humans MeSH
- Tumor Microenvironment * immunology drug effects MeSH
- Ovarian Neoplasms * drug therapy immunology pathology MeSH
- Neoadjuvant Therapy methods MeSH
- Paclitaxel administration & dosage therapeutic use pharmacology MeSH
- Antineoplastic Combined Chemotherapy Protocols therapeutic use pharmacology MeSH
- Cystadenocarcinoma, Serous drug therapy pathology immunology MeSH
- Endoplasmic Reticulum Stress drug effects immunology MeSH
- Lymphocytes, Tumor-Infiltrating immunology drug effects metabolism MeSH
- Check Tag
- Humans MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
INTRODUCTION: Approximately one-third of all AML patients have a mutation in the Fms-like tyrosine kinase 3 (FLT3) gene, which is associated with a poor prognosis in these individuals. The 2017 approval of midostaurin, the first FLT3 inhibitor, spurred extensive development of more potent and selective inhibitors with an improved safety profile. AREAS COVERED: This review analyzes patent inventions for the treatment of AML using FLT3 inhibitors, covering developments from the earliest to the most recent, disclosed in 2024. Our search using the global Espacenet database identified numerous compounds with low nanomolar inhibitory concentrations against FLT3-ITD and FLT3-TKD mutants. These compounds have shown promise in preclinical studies. Co-inhibition strategies and combinatorial therapies to overcome resistance and enhance anti-leukemic efficacy are also discussed. EXPERT OPINION: Recent patents highlight advances in the field of FLT3 inhibitors with a focus on overcoming resistance, improving selectivity and potency. Future strategies may include third-generation inhibitors such as type III allosteric inhibitors, irreversible inhibitors, or PROTACs. Personalized medicine approaches utilizing genetic profiling to tailor therapies are emphasized. Exploration of novel combination regimens with emerging therapies like CAR T-cell therapy, immune checkpoint inhibitors, and small molecules targeting critical AML pathways is ongoing to further enhance anti-leukemic efficacy.
- MeSH
- Leukemia, Myeloid, Acute * drug therapy genetics MeSH
- Drug Resistance, Neoplasm * MeSH
- Precision Medicine MeSH
- Protein Kinase Inhibitors * pharmacology MeSH
- Humans MeSH
- Mutation * MeSH
- Patents as Topic * MeSH
- Prognosis MeSH
- Antineoplastic Agents * pharmacology MeSH
- Staurosporine analogs & derivatives pharmacology MeSH
- fms-Like Tyrosine Kinase 3 * antagonists & inhibitors genetics MeSH
- Drug Development MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
Bruton's tyrosine kinase (BTK) inhibitors have revolutionized the treatment of B-cell malignancies. They target BTK, a key effector in the B-cell receptor (BCR) signaling pathway, crucial for B-cell survival and proliferation. The first-in-class irreversible BTK inhibitor, ibrutinib, was approved for various B-cell malignancies but has limitations due to off-target effects. Second-generation inhibitors, such as acalabrutinib and zanubrutinib, offer improved selectivity and reduced side effects. However, resistance to BTK inhibitors, driven by BTK mutations, remains a challenge. Combinatorial therapies with PI3K inhibitors, immune checkpoint inhibitors, BH3 mimetics, and anti-CD20 antibodies show promise in overcoming resistance. Noncovalent BTK inhibitors and proteolysis-targeting chimeras (PROTACs) are emerging strategies with potential to combat resistance. Overall, advancements in BTK-targeted therapies provide hope for improved outcomes in patients with B-cell malignancies and a promising avenue to address drug resistance. Further research is needed to optimize combination therapies and identify optimal treatment regimens.
- MeSH
- Lymphoma, B-Cell drug therapy metabolism pathology MeSH
- Drug Resistance, Neoplasm * MeSH
- Protein Kinase Inhibitors * therapeutic use pharmacology MeSH
- Tyrosine Kinase Inhibitors MeSH
- Humans MeSH
- Piperidines * therapeutic use pharmacology MeSH
- Agammaglobulinaemia Tyrosine Kinase * antagonists & inhibitors MeSH
- Pyrazoles * therapeutic use pharmacology MeSH
- Pyrimidines * therapeutic use pharmacology MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
Arteriální hypertenze (AH) ve spojitosti s kardiometabolickými komorbiditami představuje terapeutickou výzvu. Následující text má připomenout a zdůraznit farmakologické strategie nezbytné pro léčbu pacientů s AH a přidruženými metabolickými poruchami. Článek podává přehled současných doporučení a léčebných postupů a zdůrazňuje význam individualizovaného přístupu při řešení komplexních komorbidních stavů, jako je diabetes mellitus (DM), dyslipidemie a obezita. Pojednává mimo jiné o úloze inhibitorů RAAS, BKK, β-blokátorů a o úpravách životního stylu při dosahování optimální kontroly krevního tlaku a zlepšování kardiovaskulární morbidity a mortality. Dále se zabývá novými nadějnými léčebnými postupy a zdůrazňuje přínos fixních kombinací u farmakologické léčby.
Arterial hypertension (AH) in association with cardiometabolic comorbidities represents a therapeutic challenge. The following text is intended to remind and highlight the pharmacological strategies necessary for the treatment of patients with AH and associated metabolic disorders. The article reviews current recommendations and therapies and highlights the importance of individualized medicine in addressing the complexity of comorbid conditions such as diabetes mellitus (DM), dyslipidemia, and obesity. It discusses the role of RAAS inhibitors, CCBs, β-blockers, and lifestyle modifications, among others, in achieving optimal blood pressure control and improving cardiovascular morbidity and mortality. It also discusses promising new therapies and highlights the benefits of combinatorial pharmacological treatment.
- MeSH
- Diabetes Mellitus drug therapy MeSH
- Dyslipidemias drug therapy MeSH
- Drug Combinations MeSH
- Weight Loss MeSH
- Hypertension * diagnosis drug therapy classification MeSH
- Hypolipidemic Agents pharmacology therapeutic use MeSH
- Comorbidity * MeSH
- Humans MeSH
- Obesity diet therapy drug therapy MeSH
- Motor Activity MeSH
- Risk Factors MeSH
- Age Factors MeSH
- Check Tag
- Humans MeSH
- Publication type
- Review MeSH
BACKGROUND: Programmed cell death 1 (PD-1) belongs to immune checkpoint proteins ensuring negative regulation of the immune response. In non-small cell lung cancer (NSCLC), the sensitivity to treatment with anti-PD-1 therapeutics, and its efficacy, mostly correlated with the increase of tumor infiltrating PD-1+ lymphocytes. Due to solid tumor heterogeneity of PD-1+ populations, novel low molecular weight anti-PD-1 high-affinity diagnostic probes can increase the reliability of expression profiling of PD-1+ tumor infiltrating lymphocytes (TILs) in tumor tissue biopsies and in vivo mapping efficiency using immune-PET imaging. METHODS: We designed a 13 kDa β-sheet Myomedin scaffold combinatorial library by randomization of 12 mutable residues, and in combination with ribosome display, we identified anti-PD-1 Myomedin variants (MBA ligands) that specifically bound to human and murine PD-1-transfected HEK293T cells and human SUP-T1 cells spontaneously overexpressing cell surface PD-1. RESULTS: Binding affinity to cell-surface expressed human and murine PD-1 on transfected HEK293T cells was measured by fluorescence with LigandTracer and resulted in the selection of most promising variants MBA066 (hPD-1 KD = 6.9 nM; mPD-1 KD = 40.5 nM), MBA197 (hPD-1 KD = 29.7 nM; mPD-1 KD = 21.4 nM) and MBA414 (hPD-1 KD = 8.6 nM; mPD-1 KD = 2.4 nM). The potential of MBA proteins for imaging of PD-1+ populations in vivo was demonstrated using deferoxamine-conjugated MBA labeled with 68Galium isotope. Radiochemical purity of 68Ga-MBA proteins reached values 94.7-99.3% and in vitro stability in human serum after 120 min was in the range 94.6-98.2%. The distribution of 68Ga-MBA proteins in mice was monitored using whole-body positron emission tomography combined with computerized tomography (PET/CT) imaging up to 90 min post-injection and post mortem examined in 12 mouse organs. The specificity of MBA proteins was proven by co-staining frozen sections of human tonsils and NSCLC tissue biopsies with anti-PD-1 antibody, and demonstrated their potential for mapping PD-1+ populations in solid tumors. CONCLUSIONS: Using directed evolution, we developed a unique set of small binding proteins that can improve PD-1 diagnostics in vitro as well as in vivo using PET/CT imaging.
- MeSH
- Programmed Cell Death 1 Receptor * metabolism MeSH
- HEK293 Cells MeSH
- Humans MeSH
- Mice MeSH
- Cell Line, Tumor MeSH
- Lung Neoplasms diagnostic imaging pathology metabolism genetics MeSH
- Carcinoma, Non-Small-Cell Lung diagnostic imaging pathology metabolism MeSH
- Positron-Emission Tomography * methods MeSH
- Protein Engineering * MeSH
- Amino Acid Sequence MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
BACKGROUND: Metastatic esophageal carcinoma (EC) has a poor prognosis and only limited treatment options. While immune checkpoint inhibitors (ICIs) have improved the treatment of a broad spectrum of cancers, patients with EC mostly fail to respond to this treatment. For that reason, it is crucial to understand the immune phenotype of each cancer patient and moreover, to understand how different therapies modulate the cancer microenvironment and sensitize the tumors to the treatment with ICIs. SUMMARY: We have conducted a systematic review of the literature to evaluate the potential of ICI therapy in combination with chemotherapy, radiotherapy, and/or biologic therapy in EC patients. In our review, we have discussed the effects of diverse treatment approaches on the tumor microenvironment of EC. In addition, we have reviewed the current phase II and III clinical trials in EC patients to provide a rationale for immunotherapy application in combination settings with chemotherapy, radiotherapy, and/or biologic therapy. KEY MESSAGES: A great effort is already underway in clinical trials evaluating the combinatorial administration of ICIs and other treatment modalities in metastatic EC patients. PD-L1 expression status was shown to be higher in the squamous cell carcinoma (SCC) as compared to adenocarcinoma. Thus, ICIs plus chemotherapy are being discussed as a particularly feasible option for patients with SCC. Radiation was shown to induce the expression of immune checkpoint molecules and to promote the priming and activation of cytotoxic T cells which provides a rationale for ICI administration in a combination with radiotherapy. The combination of ICIs with biologic therapy was shown to be safe; however, the impact on the clinical outcomes of EC patients varied among studies.
- MeSH
- Immunotherapy MeSH
- Carcinoma * MeSH
- Humans MeSH
- Tumor Microenvironment MeSH
- Esophageal Neoplasms * drug therapy MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
Multiple myeloma (MM) has witnessed improved patient outcomes through advancements in therapeutic approaches. Notably, allogeneic stem cell transplantation, proteasome inhibitors, immunomodulatory drugs, and monoclonal antibodies have contributed to enhanced quality of life. Recently, a promising avenue has emerged with chimeric antigen receptor (CAR) T cells targeting B-cell maturation antigen (BCMA), expressed widely on MM cells. To mitigate risks associated with allogenic T cells, we investigated the potential of BCMA CAR expression in natural killer cells (NKs), known for potent cytotoxicity and minimal side effects. Using the NK-92 cell line, we co-expressed BCMA CAR and soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL) employing the piggyBac transposon system. Engineered NK cells (CAR-NK-92-TRAIL) demonstrated robust cytotoxicity against a panel of MM cell lines and primary patient samples, outperforming unmodified NK-92 cells with a mean difference in viability of 45.1% (±26.1%, depending on the target cell line). Combination therapy was explored with the proteasome inhibitor bortezomib (BZ) and γ-secretase inhibitors (GSIs), leading to a significant synergistic effect in combination with CAR-NK-92-TRAIL cells. This synergy was evident in cytotoxicity assays where a notable decrease in MM cell viability was observed in combinatorial therapy compared to single treatment. In summary, our study demonstrates the therapeutic potential of the CAR-NK-92-TRAIL cells for the treatment of MM. The synergistic impact of combining these engineered NK cells with BZ and GSI supports further development of allogeneic CAR-based products for effective MM therapy.
