Ram Kumar Selvaraju

Supplemental Table 1: Patient data from ATC IHC samples

Supplemental Figure 2: Representative HPLC-SEC chromatograms of UU-40 and DOTA-UU-40 and SPR sensorgrams of CD44v6-binding. A. HPLC evaluation of samples UU-40 and DOTA-UU-40 stored at -20C or -80C for 23 days. B. SPR sensorgrams of UU-40 (top row) and DOTA-UU-40 (bottom row) to CD44iso4-Fc (naturally occurring v6-positive CD44, Fc-fused) and human v6-peptide after storage at either 4C, -20C or 80C for 23 days. Affinity to the Fc-fused CD44v6 iso4-protein should be regarded as apparent affinity due to influence from avidity during measurements.

Supplemental Figure 3: CD44v6 antigen expression levels of BHT-101 and ACT-1. A. In vitro specificity of 125I- and 177Lu-UU-40 with/without 100-fold molar excess (block) of unlabeled UU-40 on BHT-101 cells. B. In vitro specificity of 125I- and 177Lu-UU-40 with/without 100-fold molar excess (block) of unlabeled UU-40 on ACT-1 cells. C. IHC staining of CD44v6 in BHT-101 xenografts. D. IHC staining of CD44v6 in ACT-1 xenografts. Unpaired t-tests assessed the level of significance between blocked and unblocked wells of both radioconjugates on both cell lines. P < 0.0001 (****), p < 0.001 (***).

Supplemental Figure 1: Representative images of IHC-stainings for CD44v6 from tumors from ATC patients. Images were obtained by high resolution microscope using 400x magnification. No image-adjustments have been made. Case no T1-T39 are cross-linked with data found in Supplemental Table 1.

Supplemental Figure 4: Biodistribution of BHT-101 therapy mice. Left: Biodistribution of animals carrying BHT-101 xenografts treated with 177Lu-UU-40 (6.5 MBq) dissected on the day of termination due to reaching TBL. Right: Biodistribution of animals carrying BHT-101 xenografts treated with isotype control antibody, 177Lu-B1-8 (7 MBq), dissected on the day of termination due to reaching TBL.

Supplemental Figure 3: CD44v6 antigen expression levels of BHT-101 and ACT-1. A. In vitro specificity of 125I- and 177Lu-UU-40 with/without 100-fold molar excess (block) of unlabeled UU-40 on BHT-101 cells. B. In vitro specificity of 125I- and 177Lu-UU-40 with/without 100-fold molar excess (block) of unlabeled UU-40 on ACT-1 cells. C. IHC staining of CD44v6 in BHT-101 xenografts. D. IHC staining of CD44v6 in ACT-1 xenografts. Unpaired t-tests assessed the level of significance between blocked and unblocked wells of both radioconjugates on both cell lines. P < 0.0001 (****), p < 0.001 (***).

Supplemental Figure 4: Biodistribution of BHT-101 therapy mice. Left: Biodistribution of animals carrying BHT-101 xenografts treated with 177Lu-UU-40 (6.5 MBq) dissected on the day of termination due to reaching TBL. Right: Biodistribution of animals carrying BHT-101 xenografts treated with isotype control antibody, 177Lu-B1-8 (7 MBq), dissected on the day of termination due to reaching TBL.

Anaplastic thyroid cancer (ATC) is a rare but severe form of thyroid cancer responsible for approximately 50% of thyroid cancer deaths. Consequently, the identification of innovative therapies remains crucial for the effective treatment of ATC. Molecular radiotherapy is a rapidly growing field within oncology, and the cell surface antigen CD44v6, which is overexpressed in several cancers, is a plausible target for molecular radiotherapy of ATC. IHC of 39 patient samples with ATC was evaluated for CD44v6 expression. Biodistribution and dosimetry of iodine-125 (125I)–/lutetium-177 (177Lu)–labeled UU-40, a CD44v6-specific antibody, followed by in vivo efficacy in two ATC xenograft models with varying target expression levels (ACT-1 and BHT-101), accompanied by single-photon emission computed tomography (SPECT) imaging, evaluated radiolabeled UU-40 for therapeutic efficiency in ATC xenografts. The IHC revealed CD44v6 immunoreactivity in 46% of patient samples with ATC. The biodistribution favored 177Lu-labeled UU-40 over the 125I-labeled antibody and confirmed the in vivo specificity of both radioconjugates. The in vivo efficacy and accompanied SPECT imaging of a moderate CD44v6-expressing xenograft model (BHT-101) verified the tumor specificity, as well as the target-specific effect of 177Lu-labeled UU-40 on tumor growth and survival. A 100% complete response rate was demonstrated as a result of therapy using a single dose of 16 MBq 177Lu-labeled UU-40 in a high CD44v6-expressing xenograft model (ACT-1), and SPECT imaging revealed excellent tumor uptake of the radioconjugate at 14 days after injection. This study verifies the expression of CD44v6 in ATC and strengthens the superiority and promise of 177Lu-labeled UU-40 over 131I-labeled UU-40 for antibody-based molecular radiotherapy of CD44v6-positive ATC.

Supplemental Figure 1: Representative images of IHC-stainings for CD44v6 from tumors from ATC patients. Images were obtained by high resolution microscope using 400x magnification. No image-adjustments have been made. Case no T1-T39 are cross-linked with data found in Supplemental Table 1.

Supplemental Table 1: Patient data from ATC IHC samples