May 2026· Turkish journal of biology = Turk biyoloji dergisi· Vol 50, pp. 306 - 320· 0 citations· 36 references
Medicine
TL;DR
Only one monoclonal antibody was identified as a potential therapeutic drug candidate thanks to its high capacity for checkpoint blockade and affinity, as well as its unique sequence specificity, comparable to those of anti-PD-L1 antibodies currently used in clinical practice.
Abstract
Background/aim Targeted therapies with monoclonal antibodies provide cancer patients with better prognosis and disease-free survival. The blockade of immune checkpoints, including programmed cell death protein-1 (PD-1) and its ligand PD-L1, with monoclonal antibodies may boost immune responses against tumors and is regarded as an effective strategy in cancer immunotherapy. We describe the generation of anti-PD-L1 monoclonal antibodies with high affinity and specificity, and we assess their potential for therapeutic use in cancer. Materials and methods Hybridomas were selected for PD-L1 specificity and cross-reactivity with other immune checkpoint proteins and PD-L1 orthologs using indirect ELISA. Immunofluorescence and Western blotting assays were conducted for further characterization of the antibodies. The affinities of the antibodies for PD-L1 were determined using surface plasmon resonance. Receptor blocking activities were examined through competitive ELISA and cell-based luciferase reporter assays. Sequences of variable regions of the selected antibodies were determined by Sanger sequencing and subjected to BLAST analysis. Results A total of 25 PD-L1-specific monoclonal antibodies were generated. While most clones reacted with PD-L1 from cynomolgus monkeys, none of the antibodies displayed cross-reactivity with other checkpoint proteins. Immunofluorescence assays showed that the selected clones stained PD-L1-expressing cell membranes specifically, but not those of PD-L1-negative cells. Western blotting revealed that most of the clones recognized both glycosylated and nonglycosylated PD-L1, and a few reacted with the glycosylated form only. Only two clones with subnanomolar affinity for human PD-L1 were effective at blocking PD-1/PD-L1 and CD80/PD-L1 interactions. Sequence analysis of their variable regions revealed their unique specificity. Conclusion Of the 25 monoclonal antibodies produced in this study, only one was identified as a potential therapeutic drug candidate thanks to its high capacity for checkpoint blockade and affinity, as well as its unique sequence specificity. These properties are comparable to those of anti-PD-L1 antibodies currently used in clinical practice.
The potential of bioengineered PD-L1 siRNA entities for NSCLC immunotherapy is demonstrated and their immunotherapeutic efficacy against non-small cell lung cancer (NSCLC) in vitro is defined.
N. Batra, Mei-Juan Tu, Su Guan et al.· Non-Coding RNA· 0 citations
Background/Objectives: Therapeutic antibodies have become an important modality for cancer treatment; however, effective targeted antibody therapies for T-cell acute lymphoblastic leukemia (T-ALL) remain limited. CD99 is a promising therapeutic target due to its high expression in leukemic T cells and its capacity to transmit apoptotic signals. However, the efficacy of antibody-mediated apoptosis may depend on the CD99 region recognized by the antibody. This study compared the in vitro apoptotic activity of two chimeric anti-CD99 antibodies, ChAbMT99/1 and ChAbMT99/3, which recognize distinct regions of CD99 in T-ALL models. Methods: ChAbMT99/1 and ChAbMT99/3 were generated as human IgG1 antibodies. Antibody reactivity and binding site specificity were characterized using peptide-based ELISA. Binding to native CD99, apoptosis induction in two-dimensional (2D) Jurkat E6.1 and MOLT-4 suspension cultures and three-dimensional (3D) Jurkat E6.1-derived spheroid models, and cytotoxic effects on peripheral blood mononuclear cells (PBMCs) were assessed by flow cytometry. In vitro hematologic effects were assessed using hemagglutination and platelet aggregation assays. Results: ChAbMT99/1 and ChAbMT99/3 specifically recognized peptides corresponding to distinct CD99 regions, with peptide-binding EC50 values of 0.813 and 0.819 μg/mL, respectively. Both antibodies exhibited binding reactivity to native CD99 on the tested cells. Functionally, both antibodies induced Annexin V/7-AAD-defined cell death in both 2D and 3D T-ALL models compared with controls in a crosslinking-dependent manner. In contrast, both antibodies induced low levels of cell death (<10%) in bulk PBMCs, with no visible hemagglutination or platelet aggregation observed in samples from five selected donors under the stated assay conditions. Conclusions: Both chimeric anti-CD99 antibodies demonstrated in vitro apoptotic activity against T-ALL models despite recognizing distinct regions of CD99. These findings provide a rationale for further investigation of their other mechanisms of action to support the future development of CD99-targeted antibody therapy for T-ALL.
CP-1 exhibits balanced, dual-nanomolar inhibitory activity against PD-L1 and CD73 and displays potent immunomodulatory effects at the cellular level and serves as a promising lead candidate for developing novel bifunctional agents to advance tumor immunotherapy.
Jing-Jing Du, Sen Wu, Shiyun Cheng et al.· Frontiers in Immunology· 0 citations
The immune system plays crucial role to fight against cancer. Still, cancer cells have evolved mechanisms to evade immune responses, especially through immune checkpoint proteins such as cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), and programmed death-ligand 1 (PD-L1). These checkpoints inhibit T-cell activation and are involved in immune tolerance in the tumor microenvironment. Checkpoint inhibitor-based immunotherapy, such as anti-CTLA-4 therapy, has transformed cancer care, but there are still unresolved issues concerning its safety and effectiveness, as well as immune-related adverse events (irAEs). To overcome these drawbacks, we considered using small-molecule CTLA-4 inhibitors instead of antibodies. To screen a library of 10 million compounds to identify small molecules that could bind to a specific site on the CTLA-4 protein and interfere with its interaction with CD80, we applied artificial intelligence (AI) in this research. This screening identified promising compounds, which were subsequently evaluated in vitro using AlphaLISA and luciferase reporter assays. These findings showed that the D11 and A9 compounds were capable of blocking the interaction between CTLA-4 and CD80 and had a desirable biosafety profile relative to conventional antibody-based treatments such as ipilimumab. Moreover, D11 and A9 were found to be effective at inhibiting tumor growth and prolonging survival in mouse models expressing humanised CTLA-4, highlighting their ability to overcome the limitations of anti-CTLA-4 antibodies. Also, we discovered in our research that the combination of D11 and anti-PD-1 therapy increased the therapeutic efficacy and did not lead to severe adverse reactions. These data suggest that small molecules can be a new and more efficient alternative to existing immune checkpoint inhibitors, with greater specificity and fewer adverse events.
Chong Yu, Xiaohua Zhou, Wenqiang Chen et al.· International Immunopharmaco...· 0 citations
