Nuchjira Takheaw

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.

Fine particulate matter (PM2.5) is an air pollutant that significantly impacts environmental and health outcomes. In Chiang Mai province, Thailand, residents face recurring PM2.5 pollution from November to May each year, primarily due to open biomass burning and transboundary emissions. The long-term health impacts of PM2.5 in Chiang Mai remain inadequately documented. This study aims to investigate leukocyte distribution and IgE level in response to long-term PM2.5 exposure. Participants residing in San Patong district, Chiang Mai, were recruited. Leukocyte distribution was investigated by the complete blood count and flow cytometry. IgE levels were measured by sandwich enzyme-linked immunosorbent assay. Long-term PM2.5 exposure is associated with significant changes in leukocyte distribution, including WBC count, neutrophil, monocyte, and basophil absolute count, while lymphocyte and eosinophil counts were not significantly affected. In the present study, flow cytometric immunophenotyping was performed during the late PM2.5 season to evaluate the distributions of major leukocyte populations and the expression of activation and inhibitory markers on lymphocyte subsets. Interestingly, IgE levels showed a decreasing trend. These findings highlight the immunological effects of repeated PM2.5 exposure and underscore the need for further research to clarify its mechanisms and health implications.

The immune system comprises a complex network of cells that continuously change during activation, infection, and the maintenance of balance. Immunophenotyping offers valuable insights into the regulation of immune responses. We systematically characterized the expression profile of CD99 ligands across distinct immune cell subsets using both conventional and high-dimensional flow cytometry. CD99 ligands were detected on NK cells and monocytes under both resting and IL-2-activated conditions, with non-classical monocytes and CD56 Dim NK cells exhibiting the highest expression levels. Notably, ligand expression in these subsets was further enhanced following IL-2 activation. In contrast, T lymphocytes (CD3⁺) displayed low basal levels of CD99 ligand expression, which increased modestly upon activation. Cellular activation was accompanied by an expansion of specific immune phenotypes characterized by elevated CD99 ligand expression alongside the upregulation of activation markers such as CD69 and CD137. Collectively, these findings suggest that the expression of the CD99 ligands may serve as an indicator of immune activation and demonstrate subset-specific regulation, particularly in response to IL-2 stimulation. These findings have revealed the distinct expression patterns of CD99 ligands, emphasizing their crucial role in modulating immune responses.

Recombinant antibody production in mammalian cells is essential for therapeutic, diagnostic, and research applications. Single‐expression plasmid systems that co‐express both heavy and light chains under balanced promoters offer advantages in improving expression consistency. However, the relationship between intracellular antibody expression levels and secretion efficiency following clonal selection remains unclear. In this study, a single‐expression plasmid encoding a chimeric anti‐CD99 antibody (ChAbMT99/1) was constructed and used to establish stably expressing HEK293T mini‐pool populations through zeocin selection. Intracellular ChAbMT99/1 expression levels in five selected mini‐pools were assessed using intracellular immunofluorescence staining and flow cytometry. Antibody secretion into the culture supernatants was quantified by indirect enzyme‐linked immunosorbent assay (ELISA) and further validated using indirect immunofluorescence assay and flow cytometry with native CD99‐expressing cells. The results revealed a strong positive correlation between intracellular antibody expression and secretion levels. Mini‐pools with higher intracellular fluorescence intensities produced greater antibody yields, indicating that intracellular antibody expression levels, within the context of single‐plasmid expression systems, can reliably predict secretion efficiency. This finding facilitates the efficient selection of high‐producing mini‐pools for large‐scale recombinant antibody production.

Monoclonal antibodies (mAbs) have emerged as targeted immunotherapies with clinical effectiveness and low adverse effects for various cancers. However, antibody drugs for treating aggressive T cell malignancies, T lymphoblastic leukemia/lymphoma (T-ALL/T-LBL), are still limited. Therefore, a potential mAb for treating T-ALL/T-LBL with minimal toxicity to normal cells needs to be developed. We have previously demonstrated that our in-house produced mouse anti-human CD99 mAb MT99/3 and its humanized version, HuMT99/3, which recognize a newly identified epitope of CD99 can induce apoptosis of T-ALL/T-LBL cells without affecting non-malignant peripheral blood cells. Nevertheless, the immune effector functions activated by HuMT99/3 against T-ALL/T-LBL cells remain unexplored. In this study, we evaluated the anticancer activities of HuMT99/3 against T-ALL/T-LBL cells via immune effector functions. T-ALL/T-LBL cell lines were used as target cells, including Jurkat E6.1, MOLT-4, and SUP-T1. The results demonstrated that HuMT99/3 could mediate potent antibody-dependent cellular cytotoxicity (ADCC) activity to kill all cell lines by activating the Fc receptor CD16 on effector cells. HuMT99/3 significantly enhanced the phagocytosis of monocytes on all three malignant T cell lines through antibody-dependent cellular phagocytosis (ADCP) activity. In addition, HuMT99/3 could activate complement to destroy T-ALL cell lines through complement-dependent cytotoxicity (CDC) activity, without affecting the T-LBL cell line and normal PBMCs. Furthermore, the mAb MT99/3 significantly inhibited tumor growth in a T-ALL xenograft model. These findings provide valuable insights into the development of monoclonal antibodies targeting CD99 as promising therapeutics for T-ALL/T-LBL treatment with minimal toxicity to normal peripheral blood cells.

Laopajon W, et al. Infect Chemother. 2025 Apr;57:e25. https://doi.org/10.3947/ic.2024.0145

BioPhi-guided humanization was utilized to enhance the humanness of a humanized single-chain variable fragment targeting CD99, leading to the development of two variants: HuScFvMT99/3^BP and HuScFvMT99/3^HY. The HuScFvMT99/3^BP variant incorporated framework region modifications, leading to modest improvements in humanness, particularly in the VH domain, although the VL domain remained suboptimal. To address this limitation, HuScFvMT99/3^HY was designed by combining the VL domain of wild-type with the VH domain of HuScFvMT99/3^BP. Molecular dynamics simulations employing AlphaFold2, AlphaFold3, and HADDOCK were performed to evaluate the HuScFv-CD99 peptide complexes. AF2-based simulations demonstrated enhanced binding free energy (ΔG_binding) for both variants compared to HuScFvMT99/3^WT. However, ΔG_binding values obtained from AF3 and HD simulations were inconsistent, with HuScFvMT99/3^BP exhibiting the weakest binding affinity. While ΔG_binding patterns derived from AlphaFold3 and HADDOCK simulations aligned, amino acid decomposition analysis revealed variations in the interaction coordinates of the predicted complexes. Root-mean-square deviation analysis indicated improved structural stability for HuScFvMT99/3^BP (0.975 Å) and HuScFvMT99/3^HY (1.075 Å) relative to HuScFvMT99/3^WT (1.225 Å). Biolayer interferometry further confirmed that HuScFvMT99/3^WT exhibited the highest binding affinity (K_D = 1.35 × 10⁻⁷ M) compared to HuScFvMT99/3^BP (K_D = 2.64 × 10⁻⁷ M) and HuScFvMT99/3^HY (K_D = 3.95 × 10⁻⁷ M). Supporting evidence was provided by ELISA and flow cytometry experiments. PITHA analysis revealed a high immunogenicity risk for all variants, despite HuScFvMT99/3^HY displaying improved humanness, a larger complementarity-determining region (CDR) cavity, and a more hydrophobic CDR-H3 loop. These findings highlight the delicate balance between enhancing humanness and preserving the structural and functional integrity critical for therapeutic antibody development.

CD99, a type I transmembrane protein, emerges as a promising therapeutic target due to its heightened expression in T cell acute lymphoblastic leukemia (T-ALL). This characteristic renders it a potential marker for minimal residual disease detection and an appealing target for antibody-based treatments. Previous studies have revealed that a mouse monoclonal antibody, mAb MT99/3, selectively binds to CD99, triggering apoptosis in T-ALL/T-LBL cells while preserving the integrity of healthy cells. By targeting CD99, mAb MT99/3 suppresses antigen presentation and disrupts T cell functions, offering promise for addressing hyperresponsive T cell conditions. To facilitate clinical translation, we developed a humanized ScFv variant of mAb MT99/3, termed HuScFvMT99/3 in “ScFvkh” design. Structural analysis confirms its resemblance to the original antibody, and the immunoreactivity of HuScFvMT99/3 against CD99 is preserved. The fully humanized version of antibody HuMT99/3 was further engineered, exhibiting similar binding affinity at the 10⁻¹⁰ M level and specificity to the CD99 epitope without antigenic shift. HuMT99/3 demonstrates remarkable selectivity, recognizing both malignant and normal T cells but inducing apoptosis only in T-ALL/T-LBL cells, highlighting its potential for safe and targeted therapy.

The diagnosis of mycobacterial infections, including both the Mycobacterium tuberculosis complex (MTBC) and non-tuberculous mycobacteria (NTM), poses a significant global medical challenge. This study proposes a novel approach using immunochromatographic (IC) strip tests for the simultaneous detection of MTBC and NTM. Traditional methods for identifying mycobacteria, such as culture techniques, are hindered by delays in distinguishing between MTBC and NTM, which can affect patient care and disease control. Molecular methods, while sensitive, are resource-intensive and unable to differentiate between live and dead bacteria. In this research, we developed unique monoclonal antibodies (mAbs) against Ag85B, a mycobacterial secretory protein, and successfully implemented IC strip tests named 8B and 9B. These strips demonstrated high concordance rates with conventional methods for detecting MTBC, with positivity rates of 93.9% and 85.9%, respectively. For NTM detection, the IC strip tests achieved a 63.2% detection rate compared to culture methods, considering variations in growth rates among different NTM species. Furthermore, this study highlights a significant finding regarding the potential of MPT64 and Ag85B proteins as markers for MTBC detection. In conclusion, our breakthrough method enables rapid and accurate detection of both MTBC and NTM bacteria within the BACTEC MGIT system. This approach represents a valuable tool in clinical settings for distinguishing between MTBC and NTM infections, thereby enhancing the management and control of mycobacterial diseases. Key points: • Panel of mAbs for differentiating MTB versus NTM • IC strips for diagnosing MTBC and NTM after the BACTEC MGIT • Combined detection of MTP64 and Ag85B enhances diagnostic accuracy