Introduction: Immunotherapy is recommended for treating advanced colon cancer with high microsatellite instability. PD-L1 and infiltrating CD8+ T cells serve as biomarkers for tumoral response but their quantification by immunochemistry has major limitations as the tumoral microenvironment is dynamic and heterogeneous. Whole-body immunoPET imaging could be a powerful tool to assess the immune status of tumors throughout the treatment course.

The research of biomarkers is particularly relevant in predicting response to therapeutic combinations to improve the efficiency of immunotherapies. The combination of JQ1, an inhibitor of the BRD4 protein, and anti-PD-L1 immunotherapies has a synergic therapeutic potential for treating several cancer types [1]. The BRD4 protein belongs to the BET (bromodomain and extra-terminal domain) family and allows the transcription of genes coding for oncogenic proteins, notably the CD274 gene coding for PD-L1. Additivity as an anti-PD-L1 therapeutic strategy ties in with the critical challenge of target occupancy. This issue is of particular interest for immune checkpoint inhibitors, given their variable intratumoral pharmacokinetics/pharmacodynamics within and among individuals [2].

In this study, we investigated dual PD-L1 and CD8⁺ T cell immunoPET imaging to predict the response to the therapeutic combination of an anti-PD-L1 antibody and JQ1 in a syngeneic model of colon cancer.

Methods: The anti-PD-L1 C4 minibody and the anti-mCD8α minibody (variable region of the clone YTQS169.4) were radiolabeled with fluorine-18 site-specifically by enzymatic conjugation according to the method described by Tran et al. [3]. 11 days after subcutaneous implantation of the syngeneic colon cancer MC38 cells (1.10⁶ cells per flank of C57Bl/6 mice, n=30); PD-L1 tumoral expression and CD8⁺ T cell infiltration were imaged with C4 minibody-LAP-[¹⁸F]FPyOcta (¹⁸F-LAP-miniC4, n=19) and the anti-mCD8α-LAP-[¹⁸F]FPyOcta (¹⁸F-LAP-miniαCD8, n=16) 4h post-injection. The mice were randomized into 4 groups (n=7 per group) and treated daily with intra-peritoneal injection of JQ1 at 50 mg/kg or its vehicle for 13 days and with three injections of 400 µg of avelumab or its isotype. After treatment, PD-L1 tumoral expression and CD8⁺ T cell infiltration were re-assessed by PET imaging. Tumor volumes were assessed every two days until reaching humane endpoints.

Results: JQ1 efficiently reduced PD-L1 extracellular expression of MC38 cells in vitro and in vivo, confirming the relevance of this tumor model in investigating the therapeutic combination of avelumab and JQ1. Avelumab and JQ1 treatments alone or in combination led to significant tumor growth inhibition (TGI) and increased survival of mice compared to the control group. However, no synergic effect of the combination was observed (TGI_Avelumab  = 59±36%, n=12 versus TGI_Avelumab+JQ1 =62±34%, n=10). Imaging of CD8⁺ T cell infiltration with ¹⁸F-LAP-miniαCD8 was not predictive of the response to the treatment and the radiolabeled minibody’s tumoral uptake was not increased after treatment.

The C4 antibody, at tracer concentration, cannot displace avelumab bound to PD-L1. It suggests that the ¹⁸F-LAP-miniC4 can be used to quantify unoccupied-PD-L1 levels in tumors during treatment. Imaging the unoccupied-PD-L1 with the¹⁸F-LAP-miniC4 post-treatment was predictive of the TGI observed in mice treated with avelumab alone or in combination (R²=0.55, p= 0.001). A maximal tumor uptake inferior to 2.1 %ID/cc is associated with better survival of mice (38 days versus 26 days, p= 0.041).

Conclusion: The research of biomarkers is particularly relevant in predicting response to therapeutic combinations to improve the efficiency of immunotherapies. ImmunoPET imaging of the unoccupied fraction of PD-L1 with the C4 Minibody-LAP-[¹⁸F]FPyOcta predicts response to the therapeutic combination of immune checkpoint inhibitor and the targeted therapy JQ1. The treatment efficacy does not seem to be correlated with CD8⁺ T cell tumor infiltration. This study highlights the relevance of imaging biomarkers throughout the treatment course to improve the efficiency of immunotherapies.

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Image/Figure Caption:

ImmunoPET imaging of the unoccupied fraction of PD-L1 predicts response to the therapeutic combination of immune checkpoint inhibitor and the targeted therapy JQ1. (A) Timeline of the study. PD-L1 imaging was performed with C4 Minibody-LAP-[¹⁸F]FPyOcta, and CD8 imaging was performed with an anti-mCD8α Minibody-LAP-[¹⁸F]FPyOcta at 4h post-injection. Animals were treated with Avelumab or its isotype intravenously three times (400µg per dose) and JQ1 or its vehicle intraperitoneally every day (50 mg/kg). (B) Competition assay between C4 and Avelumab on high-PD-L1 expressing cells B16F10. (C) Representative C4 Minibody-LAP-[¹⁸F]FPyOcta PET images at 4h post-injection of mice from each treatment group. White arrows indicate tumors. (D) Tumor average uptake of the C4 Minibody-LAP-[¹⁸F]FPyOcta before and after treatment. (E) Correlation between maximal tumor uptake of C4 Minibody-LAP-[¹⁸F]FPyOcta and tumor growth inhibition TGI for mice receiving Avelumab +/- JQ1. (F) Survival curves of mice treated with Avelumab +/- JQ1, the median value of tumor maximal uptake was used as a threshold to stratify the mice.

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