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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 explicit identification of CD8 + T cell subpopulation is important for deciphering the role of CD8 + T cells for protecting our body against invading pathogens and cancer. Our generated monoclonal antibody (mAb), named FE-1H10, recognized two novel subpopulations of peripheral blood CD8 + T cells, FE-1H10 + and FE-1H10 - CD8 + T cells. The molecule recognized by mAb FE-1H10 (FE-1H10 molecules) had a higher distribution on effector memory CD8 + T cell subsets. The functions of FE-1H10 - and FE-1H10 + CD8 + T cells were investigated. T cell proliferation assays revealed that FE-1H10 - CD8 + T cells exhibited a higher proliferation rate than FE-1H10 + CD8 + T cells, whereas FE-1H10 + CD8 + T cells produced higher levels of IFN-γ and TNF-α than FE-1H10 - CD8 + T cells. In T cell cytotoxicity assays, FE-1H10 + CD8 + T cells were able to kill target cells better than FE-1H10 - CD8 + T cells. RNA-sequencing analysis confirmed that these subpopulations were distinct: FE-1H10 + CD8 + T cells have higher expression of genes involved in effector functions (IFNG, TNF, GZMB, PRF1, GNLY, FASL, CX3CR1) while FE-1H10 - CD8 + T cells have greater expression of genes related to memory CD8 + T cell populations (CCR7, SELL, TCF7, CD40LG). The results suggested that mAb FE-1H10 identifies two novel distinctive CD8 + T cell subpopulations. The FE-1H10 + CD8 + T cells carried a superior functionality in response to tumour cells. The uncover of these novel CD8 + T cell subpopulations may be the basis knowledge of an optional immunotherapy for the selection of potential CD8 + T cells in cancer treatment., (© 2024 John Wiley & Sons Ltd.)

CD99 was demonstrated to be a potential target for antibody therapy on T-acute lymphoblastic leukemia (T-ALL). The ligation of CD99 by certain monoclonal antibodies (mAbs) induced T-ALL apoptosis. However, the molecular basis contributing to the apoptosis of T-ALL upon anti-CD99 mAb engagement remains elusive. In this study, using our generated anti-CD99 mAb clone MT99/3 (mAb MT99/3), mAb MT99/3 engagement strongly induced apoptosis of T-ALL cell lines, but not in non-malignant peripheral blood cells. By transcriptome analysis, upon mAb MT99/3 ligation, 13 apoptosis-related genes, including FOS, TNF, FASLG, BCL2A1, JUNB, SOCS1, IL27RA, PTPN6, PDGFA, NR4A1, SGK1, LPAR5 and LTB, were significantly upregulated. The epitope of CD99 recognized by mAb MT99/3 was then identified as the VDGENDDPRPP at residues 60-70 of CD99, which has never been reported. To the best of our knowledge, this is the first transcriptome data conducted in T-ALL with anti-CD99 mAb engagement. These findings provide new insights into CD99 implicated in the apoptosis of T-ALL. The identification of a new epitope and apoptosis-related genes that relate to the induction of apoptosis by mAb MT99/3 may serve as a new therapeutic target for T-ALL. The anti-CD99 mAb clone MT99/3 might be a candidate for further development of a therapeutic antibody for T-ALL therapy.

Background: Cannabinoid receptor 2 (CB2) of the cannabinoid system is predominantly expressed on immune cells and involved in a diverse range of immune functions. However, the role of CB2 in immunoregulation is still controversial. One of the challenges in the detailed characterization and functional study of CB2 is the lack of CB2-specific monoclonal antibodies (mAbs)., Objective: We aimed to produce mAbs against a native form of human CB2 using human CB2 expressing mouse myeloma cells as immunogens., Methods: Non-viral vector expression system was used to generate stable human CB2-expressing mouse myeloma cells and were utilized as an immunogen for mouse immunization. Hybridoma technique was employed in the production of mAbs. The produced mAbs were verified by flow cytometry and western blotting., Results: Using a non-viral vector expression system, myeloma clones, which stable expressed human CB2, were generated and used as immunogen for antibody production. Following mouse immunization process, the anti-CB2 polyclonal antibodies were induced. By hybridoma technique, a mAb against CB2 could be generated. This mAb reacted to CB2-expressing THP-1 cells, but not to non-CB2-expressing SH-SY5Y cells. By western blotting, the generated anti-CB2 mAb reacted with a 42 kDa protein presented in lysates of CB2-expressing THP-1 cells, but not with non-CB2-expressing SH-SY5Y cell lysates., Conclusion: A new approach using human CB2 expressing myeloma cells as immunogen for production of anti-CB2 mAb was developed. The generated anti-human CB2 mAb is regarded as a valuable tool for CB2 characterization. Moreover, the developed technique can be applied to produce other antibodies of interest.

Monoclonal antibodies (mAbs) are an effective drug for targeted immunotherapy in several cancer types. However, so far, no antibody has been successfully developed for certain types of cancer, including T-cell acute lymphoblastic leukemia (T-ALL). T-ALL is an aggressive hematologic malignancy. T-ALL patients who are treated with chemotherapeutic drugs frequently relapse and become drug resistant. Therefore, antibody-based therapy is promising for T-ALL treatment. To successfully develop an antibody-based therapy for T-ALL, antibodies that induce death in malignant T cells but not in nonmalignant T cells are required to avoid the induction of secondary T-cell immunodeficiency. In this review, CD99 tumor associated antigen, which is highly expressed on malignant T cells and lowly expressed on nonmalignant T cells, is proposed to be a potential target for antibody therapy of T-ALL. Since certain clones of anti-CD99 mAbs induce apoptosis only in malignant T cells, these anti-CD99 mAbs might be a promising antibody drug for the treatment of T-ALL with high efficiency and low adverse effects. Moreover, over the past 25 years, many clones of anti-CD99 mAbs have been studied for their direct effects on T-ALL. These outcomes are gathered here., Competing Interests: The authors declare that they have no conflicts of interest., (© The Author(s) 2024.)

Aim: The present study aims to generate chimeric mouse single-chain variable fragment (scFv) and immunoglobulin G1 (IgG1) crystallizable fragment (Fc) antibody against disialoganglioside (GD2) for the treatment of neuroblastoma (NB). The generated scFv-IgG Fc antibody, lacking first constant domain of heavy chain (CH1), is of a smaller size than the natural antibody and has anti-tumor activity., Methods: Vector for scFv-IgG Fc antibody was constructed and scFv-IgG Fc antibody was expressed in human embryonic kidney 293T (HEK293T) cell line. Purification of scFv-IgG Fc antibody from the culture supernatant of transfected HEK293T cells was performed by Protein G affinity chromatography. The structure and binding activity of scFv-IgG Fc antibody were verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blotting (WB), and immunofluorescence techniques. Anti-tumor activities by antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) were determined., Results: Using plasmid fusion-human IgG1-Fc2 tag vector (pFUSE-hIgG1-Fc2), a plasmid vector encoding chimeric mouse scFv and hIgG1 Fc antibody against GD2 was successfully constructed. This vector was transfected into human HEK293T cells to produce scFv-IgG Fc antibody. The transfected HEK293T cells could produce chimeric scFv-IgG Fc antibody against GD2, which lacks the IgG heavy chain CH1 domain but carries CH2 and CH3 domains. The chimeric antibodies could be purified from the culture supernatant of the transfected HEK293T culture in the presence of zeocin drug. The produced GD2 scFv-IgG Fc antibodies, which are smaller in size than the intact antibody, could trigger the killing of GD2 expressed NB cell line SH-SY5Y by ADCC and ADCP mechanisms., Conclusions: The results indicate that chimeric scFv-hIgG Fc antibody, lacking heavy chain CH1 domain, could mediate antibody induced anti-tumor activities. The small size of this type of chimeric antibody may be employed as anti-GD2 antibody for NB therapy., Competing Interests: The authors declare that they have no conflicts of interest., (© The Author(s) 2023.)