Intraductal administration of transferrin receptor-targeted immunotoxin clears ductal carcinoma in situ in mouse models of breast cancer—a preclinical study

Abstract

The human transferrin receptor (TFR) is overexpressed in most breast cancers, including preneoplastic ductal carcinoma in situ (DCIS). HB21(Fv)-PE40 is a single-chain immunotoxin (IT) engineered by fusing the variable region of a monoclonal antibody (HB21) against a TFR with a 40 kDa fragment of Pseudomonas exotoxin (PE). In humans, the administration of other TFR-targeted immunotoxins intrathecally led to inflammation and vascular leakage. We proposed that for treatment of DCIS, intraductal (i.duc) injection of HB21(Fv)-PE40 could avoid systemic toxicity while retaining its potent antitumor effects on visible and occult tumors in the entire ductal tree. Pharmacokinetic studies in mice showed that, in contrast to intravenous injection, IT was undetectable by enzyme-linked immunosorbent assay in blood following i.duc injection of up to 3.0 μg HB21(Fv)-PE40. We demonstrated the antitumor efficacy of HB21(Fv)-PE40 in two mammary-in-duct (MIND) models, MCF7 and SUM225, grown in NOD/SCID/gamma mice. Tumors were undetectable by In Vivo Imaging System (IVIS) imaging in intraductally treated mice within 1 wk of initiation of the regimen (IT once weekly/3 wk, 1.5 μg/teat). MCF7 tumor–bearing mice remained tumor free for up to 60 d of observation with i.duc IT, whereas the HB21 antibody alone or intraperitoneal IT treatment had minimal/no antitumor effects. These and similar findings in the SUM225 MIND model were substantiated by analysis of mammary gland whole mounts, histology, and immunohistochemistry for the proteins Ki67, CD31, CD71 (TFR), and Ku80. This study provides a strong preclinical foundation for conducting feasibility and safety trials in patients with stage 0 breast cancer.

Repurposing an endogenous degradation domain for antibody-mediated disposal of cell-surface proteins

“…Thus, TfR overexpression has been exploited with a range of therapeutic approaches. For instance, an anti-TfR antibody has been used for tumour-directed delivery of immunotoxins (Wang et al, 2022 ), and TfR-directed chimeric antigen receptor (CAR) T-cells are effective in mouse models of T-cell acute lymphoblastic leukemia (Guo et al, 2021 ). To determine if PCTD could drive degradation of the reporter TfR construct, we purified from HEK-293F cells a chimeric protein composed of an anti-GFP nanobody (Rothbauer et al, 2008 ) (αGFP) fused to PCTD, termed PCTD-αGFP, and, as a control, we purified αGFP from E. coli .…”

Transferrin receptor in primary and metastatic breast cancer: Evaluation of expression and experimental modulation to improve molecular targeting

“…Previous studies on the use of transferrin conjugates in breast cancer considered primary breast tumors [ 8 , 24 ]. Here, we set out to study the distribution of transferrin conjugates to breast cancer metastases, utilizing commercially available fluorescent transferrin consisting of stable conjugates of recombinant transferrin to AlexaFluor-750 (AF750-Tf) or AlexaFluor-680 (AF680-Tf).…”

“…Transferrin was identified as a key factor for breast cancer cell survival about 40 years ago [19], and TfR was shown to be upregulated in breast cancer [6,16] relative to normal breast or fibromas [20,21]. Since then, preclinical models or early-stage clinical studies have addressed the potential of conjugating transferrin to chemotherapeutics [11,12], drug-carrying nanoparticles [9,[22][23][24], and optical [25][26][27] or radioactive [8,10] tracers to target breast cancer.…”

Engineering Biomimetic Protein Camouflage for Delivering Peptide/siRNA Nanocomplexes

“…Also, tumor cells often exhibit overexpressed protein receptors ( e.g. , GP60, , transferrin receptor, , and low-density lipoprotein receptors) that can be utilized for targeted delivery.…”

“…15 Besides, it would be beneficial to exploit proteins as targeting ligands for tumors, which are active sites for protein trafficking and catabolism. Also, tumor cells often exhibit overexpressed protein receptors (e.g., GP60, 16,17 transferrin receptor, 19,20 and low-density lipoprotein receptors 21 ) that can be utilized for targeted delivery.…”