Targeted radionuclide therapy against GARP expressing T regulatory cells after tumour priming with external beam radiotherapy in a murine syngeneic model

Abstract

Purpose: Radiation therapy (RT) exerts its anti-tumour efficacy by inducing direct damage to cancer cells but also through modification of the tumour microenvironment (TME) by inducing immunogenic antitumor response. Conversely, RT also promotes an immunosuppressive TME notably through the recruitment of regulatory T cells (Tregs). Glycoprotein A repetitions predominant (GARP), a transmembrane protein highly expressed by activated Tregs, plays a key role in the activation of TGF-β and thus promotes the immunosuppressive action of Tregs. The development of a theranostic approach targeting GARP combining imaging and targeted radionuclide therapy (TRT) was carried out.

Methods: A preclinical model of 4T1 triple negative breast tumour-bearing BALB/c mice was used to show that GARP expression is increased after external beam radiation in the TME of our cancer model. We generated a theranostic probe through the bioconjugation of the chelating agent DOTAGA onto an anti-GARP monoclonal antibody. The bioconjugation with DOTAGA allows the radiolabelling of the DOTAGA-GARP conjugate with both Indium-111 for SPECT imaging and Lutetium-177 for TRT purposes.

Results: We demonstrate that GARP expression is increased following RT in vivo and can be specifically detected and quantified using in vivo SPECT imaging with [¹¹¹In]In-DOTAGA-GARP. In addition, ¹⁷⁷Lu-DOTAGA-GARP limits tumour growth in our cancer model.

Conclusion: This theranostic strategy may allow for the personalization of cancer treatments by early detection of activated Tregs infiltration following RT and identification of patients likely to respond to Tregs-targeted therapy via TRT.

Keywords: External beam radiotherapy; GARP; SPECT imaging; T regulatory cells; Targeted radionuclide therapy.

Fig. 1

GARP expression with or without external beam radiation therapy in 4T1 tumour model. A. In vitro detection of membrane GARP expression in 4T1 cells with or without external beam radiation therapy at the dose of 8 Gy (48h post irradiation) by flow cytometry. Results are presented as the median with the interquartile range, p=0.0002. B. Representative immunostaining and quantification of GARP (red) in 4T1 tumour from mice 7 days after 8 Gy irradiation (n=6) or not (n=7). Results are presented as the median with the interquartile range, p=0.0082. C. Schematic representation of the experimental design: ex vivo detection of GARP expression in 4T1 tumours by flow cytometry. 14 days after 4T1 cells injection mice were divided into two groups: control or 8 Gy irradiation. 7 days after irradiation, flow cytometry was performed on dissociated tumours. 1. Percentage of GARP + cells in alive cells in tumours (p=0.0093). 2. Percentage of CD3+CD4+CD25+FoxP3 + cells expressing GARP in the tumour (p=0.018). 3. Percentage of GARP + cells within CD3+CD4+CD25+FoxP3+ regulatory T cells (p=0.0067).

Fig. 2

Bioconjugation of DOTAGA on anti-GARP antibody: binding assays. A/B. Saturation curve of the recombinant GARP protein with the unmodified antibody GARP; the DOTAGA-GARP 10eq (Kd = 153 nM); 20eq (Kd = 226 nM) and 30eq (Kd = 189 nM). C. Radioligand saturation binding to 4T1 cells of [¹¹¹In]In-DOTAGA-GARP in the absence (total binding) or presence (non-specific binding) of excess (10 μM) unlabelled DOTAGA-GARP antibody. Specific binding was calculated by subtraction of non-specific binding from total binding.

Fig. 3

External beam radiation therapy increases [¹¹¹In]In-GARP-DOTAGA uptake in vivo by SPECT/CT imaging. A. 14 days after 4T1 triple negative murine breast cancer, mice were assigned in 2 groups: external beam radiation at the dose of 8 Gy or control without irradiation. 7 days post irradiation, they underwent SPECT/CT imaging at 24h and 48h post-injection with [¹¹¹In]In-GARP-DOTAGA. Mice were sacrificed after the last imaging and gamma counting was performed ex vivo on tumours. B. Representative SPECT/CT images of 4T1 tumour-bearing mice irradiated (n = 17) or not (n = 18) at 24h and 48h post-injection of 15MBq/100 μL of [¹¹¹In]In-DOTAGA-GARP. Tumours are highlighted with circles. C. The scatter dot plot represents the percentage of the injected dose of [¹¹¹In]In-DOTAGA-GARP per gram of tumour (%ID/g) of 4T1 tumour-bearing mice irradiated (n = 17) or not (n = 18) at 24h and 48h post-injection of 15 MBq/100 μL of [¹¹¹In]In-DOTAGA-GARP. Results are presented as the median with the interquartile range, (∗∗∗∗p < 0.0001 ∗∗∗p = 0.0001). D. The scatter dot plot represents the percentage of injected dose of ¹¹¹In-DOTAGA-GARP per gram of tumour of 4T1 tumour-bearing mice irradiated (n = 11) or not (n = 12) at 48h post-injection of 15 MBq/100 μL of [¹¹¹In]In-GARP-DOTAGA measured by gamma counting. Results are presented as the median with the interquartile range, (∗∗p = 0.0056). E. Representative SPECT/CT images of 4T1 tumour-bearing mice irradiated (8Gy) at 24h and 48h post-injection of 15MBq/100 μL of [¹¹¹In]In-DOTAGA-GARP (n = 6) or [¹¹¹In]In-DOTAGA-GARP + 100x unlabelled DOTAGA-GARP (blocking, n = 3). The scatter dot plot represents the percentage of the injected dose of [¹¹¹In]In-DOTAGA-GARP per gram of tumour (%ID/g) of 4T1 tumour-bearing mice irradiated (8Gy) at 24h and 48h post-injection of 15 MBq/100 μL of [¹¹¹In]In-DOTAGA-GARP or blocking. Results are presented as the median with the interquartile range, (∗p < 0.05). F. The scatter dot plot represents the percentage of the injected dose of [¹¹¹In]In-DOTAGA-GARP per gram of tumour (%ID/g) of 4T1 tumour-bearing mice irradiated (8Gy) at 48h post-injection of 15 MBq/100 μL of [¹¹¹In]In-DOTAGA-GARP or blocking measured by gamma-counting. Results are presented as the median with the interquartile range, (∗p < 0.05).

Fig. 4

¹⁷⁷Lu-DOTAGA-GARP limits tumour growth. A. Schematic representation of the experimental design: 4T1 tumour-bearing mice were randomized into 5 groups of 12 mice at 14 days post-implantation, mice in each group are subdivided in two groups receiving (IR n = 6) or not (NT n = 6) external radiation therapy at the dose of 8Gy 7 days post-irradiation, mice received: one i.v. dose of 25 μg/3,7 MBq/100 μL of [¹⁷⁷Lu]Lu-DOTAGA-GARP per mouse (G1 = 6 NT and G2 = 6 IR 8Gy), one i.v. dose of 25 μg/11,7 MBq/100 μL of [¹⁷⁷Lu]Lu-DOTAGA-GARP per mouse (G3 = 6 NT and G4 = 6 IR 8Gy), one i.v. dose of 25 μg/11,7 MBq/100 μL of [¹⁷⁷Lu]Lu-DOTAGA-irrelevant antibody per mouse (G5 = 3 NT and G6 = 3 IR 8Gy), one i.v. dose of 25 μg/100 μL of non-radiolabelled DOTAGA-GARP per mouse (G7 = 6 NT and G8 = 6 IR 8Gy), one i.v. dose of 100 μL PBS per mouse(G9 = 6 NT and G10 = 6 IR 8Gy). B/C/D/E. 4T1 delta tumour growth (mm³) measured 3 times per week according to treatment group previously described. Results are presented as the median with the interquartile range. F/G/H. Total white blood cells (10³/mm³), lymphocytes (10³/mm³) and platelets (10³/mm³) measured 2 times per week according to treatment group previously described. Results are presented as the median with the interquartile rang. I. Kaplan-Meier survival plot for 4T1 tumour-bearing mice treated with treatment previously described.