Intraductal administration of a Transferrin Receptor-directed immunotoxin eliminates ductal carcinoma in situ in preclinical mammary in-duct (MIND) models of breast cancer

Abstract Background: Increased detection of ductal carcinoma in situ (DCIS) has been a consequence of mammographic screening. Currently there is a reappraisal of the necessity of aggressive treatment, with their attendant toxicities for a preneoplastic lesion with 15-20% chance of recurrence or progression. The recombinant immunotoxin, TFR(fv)PE40, binds to the abundantly expressed transferrin receptors on cancer cells, is internalized, processed and trafficked to the cytosol. The released Pseudomonas exotoxin (PE) catalyzes the transfer of ADP-ribose from NAD to elongation factor 2 (EF2), inactivates EF2, and leads to an arrest in protein translation, and cell death by apoptosis. However, TFR(fv)PE40 is toxic to liver cells. We hypothesized that, when administered via the opening at the mammary teat, the immunotoxin will destroy preneoplasias in the entire ductal tree. Because of the closed nature of the ductal system, the toxic effects of the TFR(fv)PE40 will be confined to the tumor cells alone, and systemic effects will be minimal or absent. The intraductal approach, we proposed, will provide a direct, safe and effective treatment for DCIS. Methods: We tested this concept in two models of DCIS using two luc-tagged cell lines-SUM225-luc, derived from a human DCIS, and in MCF-7-luc. In both cell lines, the IC50 of the toxin was in the nM range. DCIS develop within one week in MIND models following intraductal (i.duc) injection of tumor cells (right and left 4th mammary gland) of ex-breeder NSG mice. DCIS progressed to invasive cancer in 2-3 weeks. At day 7, mice were administered vehicle, TFR mAb alone, or the HB21(fv)PE40 conjugate at two doses (0.15 or 1.5 ug per teat x 2) by the i.duc route, 3 times at weekly intervals, and observed for 1-3 months. Mice treated with the TFR-Ab showed no significant reduction in tumor size. Treated with HB21(fv)PE40 i.duc, by IVIS imaging, tumors were undetectable in both models (P<0.0001) within two weeks. No recurrence was observed during 3 months of followup. Mammary whole mount analysis and histopathology showed that i.duc toxin rendered more than 90% of the mice cancer-free. Immunohistochemical analysis of tumor sections for huTFR (CD71), Ki67, and CD31 supported the loss of TFR-expressing and proliferating cancer cells, and reduction of blood vessels. Pharmacokinetics studies showed that toxin was detectable by TFR-ELISA in blood 5, 30 and 60 min after i.p injection, but was undetectable in the blood of i.duc-treated mice. Conclusions: Overall, we have shown that i.duc HB21(fv)PE40 has remarkable antitumor activity in cell culture with an IC50 in the nM range, and in two MIND models of DCIS. Blood levels of the toxin after i.duc injection were undetectable, suggesting that the toxin remains inside the ductal system during its 30 min half-life, and its effect occurs during this time. This preclinical study provides a strong basis for conducting human trials. The toxicity of HB21(Fv)PE40 by the i. duc route of drug administration could be determined in dose escalation studies in patients with DCIS and early breast cancer who will undergo mastectomy as part of their treatment regimen, using a previously successful study design (PMID:2230751). This design will allow detailed examination of the local and systemic effects of HB21(fv)PE40 administered i.duc and reintroduce a very potent agent into DCIS treatment, and in the future, cancer prevention in women at high risk of developing breast cancer. Citation Format: Saraswati Sukumar, Guannan Wang, Alok Kumar, Wanjun Ding, Preethi Korangath, Priya Pai, Kathleen Gabrielson, Ira Pastan. Intraductal administration of a Transferrin Receptor-directed immunotoxin eliminates ductal carcinoma in situ in preclinical mammary in-duct (MIND) models of breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-08-03.