The majority of clinically diagnosed cutaneous T‐cell lymphomas (CTCL) highly express the cell‐surface markers CC chemokine receptor 4 (CCR4) and/or CD25. Recently, we have developed diphtheria toxin‐based recombinant Ontak®‐like human IL2 fusion toxin (IL2 fusion toxin) and anti‐human CCR4 immunotoxin (CCR4 IT). In this study, we first compared the efficacy of the CCR4 IT vs IL2 fusion toxin for targeting human CD25⁺CCR4⁺ CTCL. We demonstrated that CCR4 IT was more effective than IL2 fusion toxin. We further constructed an IL2‐CCR4 bispecific IT. The bispecific IT was significantly more effective than either IL2 fusion toxin or CCR4 IT alone. The bispecific IT is a promising novel targeted therapeutic drug candidate for the treatment of refractory and recurrent human CD25⁺ and/or CCR4⁺ CTCL.

Cutaneous T‐cell lymphoma (CTCL) encompasses two main subtypes: mycosis fungoides and Sezary syndrome. Global response rates for the systemic treatment of mycosis fungoides and Sezary syndrome are approximately 30%, and none of these treatments are thought to be curative. C–C chemokine receptor type 4 (CCR4) and CD25 are encouraging targets for the treatment of CTCL and are individually targeted by mogamulizumab and denileukin diftitox, respectively. We developed a novel CCR4‐IL2 bispecific immunotoxin (CCR4‐IL2 IT) targeting both CCR4 and CD25. CCR4‐IL2 IT demonstrated superior efficacy against CCR4⁺CD25⁺CD30⁺ CTCL in an immunodeficient NSG mouse tumor model. Investigative New Drug‐enabling studies of CCR4–IL2 IT are ongoing, including Good Manufacturing Practice production and toxicology studies. In this study, we compared the in vivo efficacy of CCR4‐IL2 IT versus the US Food and Drug Administration–approved drug, brentuximab, using an immunodeficient mouse CTCL model. We demonstrated that CCR4–IL2 IT was significantly more effective in prolonging survival than brentuximab, and combination treatment of CCR4–IL2 IT and brentuximab was more effective than brentuximab or CCR4–IL2 IT alone in an immunodeficient NSG mouse CTCL model. Thus, CCR4–IL2 IT is a promising novel therapeutic drug candidate for CTCL treatment.

The thrombopoietin:cMPL signaling axis is a critical regulator of early hematopoiesis. However, the utility of cMPL as a standalone marker for identifying long-term repopulating hematopoietic stem cells (LT-HSCs) within the adult human CD34+ hematopoietic stem and progenitor cell (HSPC) population has not been validated. In this study, we established high cMPL surface expression as a defining feature of human LT-HSCs. Targeting the cMPL receptor facilitated the separation of human LT-HSCs from mature progenitors, a delineation not achievable with c-KIT (CD117). Leveraging this finding, we explored the therapeutic potential of cMPL as a novel target for pretransplant conditioning regimens. We developed a cMPL-targeting immunotoxin and demonstrated its ability to preferentially deplete host cMPLhigh LT-HSCs in murine xenograft models. Evaluation in rhesus macaques confirmed these findings and highlighted a favorable safety profile with rapid systemic clearance within 24 hours of administration. Proof of concept experiments validated the immunotoxin as a novel conditioning agent, enabling donor HSPC engraftment without the use of chemotherapy or irradiation. These findings advance our understanding of the molecular determinants of human hematopoiesis and underscore the potential of cMPL-targeting preparative regimens to improve therapeutic transplantation outcomes.

Epidermal growth factor receptor (EGFR) is often overexpressed in head and neck squamous cell carcinoma (HNSCC) and represents a top candidate for targeted HNSCC therapy. However, the clinical effectiveness of current Food and Drug Administration (FDA)‐approved drugs targeting EGFR is moderate, and the overall survival rate for HNSCC patients remains low. Therefore, more effective treatments are urgently needed. In this study, we generated a novel diphtheria toxin‐based bivalent human epidermal growth factor fusion toxin (bi‐EGF‐IT) to treat EGFR‐expressing HNSCC. Bi‐EGF‐IT was tested for in vitro binding affinity, cytotoxicity, and specificity using 14 human EGFR‐expressing HNSCC cell lines and three human EGFR‐negative cancer cell lines. Bi‐EGF‐IT had increased binding affinity for EGFR‐expressing HNSCC compared with the monovalent version (mono‐EGF‐IT), and both versions specifically depleted EGFR‐positive HNSCC, but not EGFR‐negative cell lines, in vitro. Bi‐EGF‐IT exhibited a comparable potency to that of the FDA‐approved EGFR inhibitor, erlotinib, for inhibiting HNSCC tumor growth in vivo using both subcutaneous and orthotopic HNSCC xenograft mouse models. When tested in an experimental metastasis model, survival was significantly longer in the bi‐EGF‐IT treatment group than the erlotinib treatment group, with a significantly reduced number of metastases compared with mono‐EGF‐IT. In addition, in vivo off‐target toxicities were significantly reduced in the bi‐EGF‐IT treatment group compared with the mono‐EGF‐IT group. These results demonstrate that bi‐EGF‐IT is more effective and markedly less toxic at inhibiting primary HNSCC tumor growth and metastasis than mono‐EGF‐IT and erlotinib. Thus, the novel bi‐EGF‐IT is a promising drug candidate for further development.

CD47 is overexpressed on the surface of many types of cancer cells, including T-cell acute lymphoblastic leukemia (T-ALL) cells. In this study, we have developed a diphtheria toxin (DT)–based bivalent anti-human CD47 immunotoxin (bi-CD47-IT) for the targeted therapy of CD47+ cancers using a unique DT-resistant yeast Pichia pastoris expression system. Bi-CD47-IT demonstrated compelling in vivo efficacy in multiple T-ALL cell line–derived xenograft (CDX) and patient-derived xenograft (PDX) mouse models. Bi-CD47-IT significantly prolonged the median survival of the tumor-bearing mice and highly effectively depleted the T-ALL blast cells in the peripheral blood, spleen, liver, bone marrow, brain, and spinal cord in the T-ALL CDX and PDX mouse models. Bi-CD47-IT cured 60% of tumor-bearing mice in a T-ALL Molt-4 CDX mouse model. Because CD47 is also expressed on normal tissues, including red blood cells and lymphocytes, specificity is a concern. We thus analyzed the in vitro binding avidity and hemagglutination of bi-CD47-IT in human red blood cells, finding no binding or hemagglutination. We further performed a toxicity study of bi-CD47-IT in humanized mice, which showed that bi-CD47-IT transiently depleted the human lymphocytes for ∼4 weeks after the 10-day treatment. No clinical adverse events were observed. As a result, bi-CD47-IT appears to possess the “optimal” binding avidity, with effective binding to human CD47+ T-ALL tumor cells, no binding to human red blood cells, and weak binding to human lymphocytes. We believe that bi-CD47-IT is a promising and safe therapeutic drug candidate for the targeted therapy of CD47+ cancers.