Off-tumor IDO1 target engagements determine the cancer-immune set point and predict the immunotherapeutic efficacy

Background Indoleamine-2,3-dioxygenase 1 (IDO1) has been intensively pursued as a therapeutic target to reverse the immunosuppressive cancer-immune milieu and promote tumor elimination. However, recent failures of phase III clinical trials with IDO1 inhibitors involved in cancer immunotherapies highlight the urgent need to develop appropriate methods for tracking IDO1 when the cancer-immune milieu is therapeutically modified. Methods We utilized a small-molecule radiotracer, 11C-l-1MTrp, to quantitatively and longitudinally visualize whole-body IDO1 dynamics. Specifically, we first assessed 11C-l-1MTrp in mice-bearing contralateral human tumors with distinct IDO1 expression patterns. Then, we applied 11C-l-1MTrp to longitudinally monitor whole-body IDO1 variations in immunocompetent melanoma-bearing mice treated with 1-methyl-l-tryptophan plus either chemotherapeutic drugs or antibodies targeting programmedcell death 1 and cytotoxic T-lymphocyte-associated protein 4. Results 11C-l-1MTrp positron emission tomography (PET) imaging accurately delineated IDO1 expression in xenograft mouse models. Moreover, we were able to visualize dynamic IDO1 regulation in the mesenteric lymph nodes (MLNs), an off-tumor IDO1 target, where the percentage uptake of 11C-l-1MTrp accurately annotated the therapeutic efficacy of multiple combination immunotherapies in preclinical models. Remarkably, 11C-l-1MTrp signal intensity in the MLNs was inversely related to the specific growth rates of treated tumors, suggesting that IDO1 expression in the MLNs can serve as a new biomarker of the cancer-immune set point. Conclusions PET imaging of IDO1 with 11C-l-1MTrp is a robust method to assess the therapeutic efficacy of multiple combinatorial immunotherapies, improving our understanding of the merit and challenges of IDO1 regimens. Further validation of this animal data in humans is ongoing. We envision that our results will provide a potential precision medicine paradigm for noninvasive visualizing each patient’s individual response in combinatorial cancer immunotherapy, and tailoring optimal personalized combination strategies.


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was thoroughly mixed into the daily feed ration. Mice ate 3.5-3.7 g/day, similar to the dietary consumption without the drug. L-1MTrp was administered in the feed for ten days, starting on day 7 after tumor implantation, with freshly prepared L-1MTrp feed given every other day. CPA (150 mg/kg per injection) or PTX (13.3 mg/kg per injection) was administered intravenously on days 7, 10, 13, and 16. The mice in the vehicle group received a corresponding dose of vehicle and placebo powder diet without the active ingredient. To examine the character of 11 C-L-1MTrp PET/CT imaging in the different treatment outcomes among the three treatment strategies, we selected these 23 day-mice after implantation as the imaging subjects, who were quiescent after approximately 1 week following all treatment stimulations. For determining the interrelationship between the dynamic IDO1 expression in the MLNs and the cancer-immune set point in individuals, a longitudinal 11 C-L-1MTrp PET/CT imaging and distribution studies were performed on days 0, 7, 10, 13, 16, 23, 30, and 40 after tumor inoculation throughout the entire treatment process in mice treated with L-1MTrp + CPA. Mice were humanely sacrificed using isoflurane anesthesia when they were moribund or at the experimental endpoint of 40 days after tumor implantation.
In the anti-PD-1 and anti-CTLA4 dual-blockade experiment, male C57BL/6J mice were inoculated subcutaneously with 5 × 10 4 B16F10 cells into the flank in a total volume of 0.1 mL serum-free medium. On days 7, 10, and 13 after tumor inoculation, 10 mg/kg anti-PD-1 plus 5 mg/kg anti-CTLA4 therapeutic antibodies were mixed in a single injection and administered via BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) intraperitoneal injection combined with intravenous injection of 150 mg/kg CPA. To generalize 11 C-L-1MTrp PET imaging for monitoring the cancer-immune set point and antitumor response in mice receiving different immunotherapies, on day 13 and 25 after tumor transplantation, all mice received a triple-treatment regimen of anti-PD-1 + anti-CTLA4 + CPA were used for imaging study after 11 C-L-1MTrp injection. At the end of the study, the mice were segregated into two groups termed group a (Tumor volume more than 0.5 cm 3 represents the poor responder) and group b (Tumor volume less than 0.5 cm 3 represents the good responder) on day 32 after tumor inoculation.

Tumor response measurement
Tumor response was monitored every 2-3 days by measuring tumor volume (V) using calipers. The formula V = (length × width 2 ) × 0.5 was used to estimate tumor volume, and results are presented in cm 3 . To objectively describe the real-time antitumor response during the posttreatment followup period, the volumetric growth rate of each tumor per day was quantified by the SGR, the percentage volume change per day in measurement intervals, using the formula SGR = ln (V1 / V0) / (t1 -t0) x 100%, where V0 is the tumor volume at the start of measurement (t0), and V1 is the volume at the end of this period (t1).
Blocking experiments were performed by intravenous co-injection an excess of unlabeled IDO1 inhibitor INCB024360 (10 mg/kg) or L-1MTrp (50 mg/kg) with the radiotracer. All list-mode acquisition data were sorted into three-dimensional sinograms, which were then Fourier-rebinned into two-dimensional sonograms, and corrected for scanner dead time, randoms, and decay of the injected radiotracer. Dynamic images were reconstructed with filtered back-projection using Pearson's correlation analysis was used to estimate the relationship between radioactivity and the SGR of tumors by using the percentage uptake in the MLNs as a dependent variable and the tumor SRG as an independent variable indicator.

Supplementary Figures
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Supplementary Tables
Supplementary Table 1. Biodistribution characterization of 11 C-L-1MTrp uptake in immunodeficient mice bearing human tumors. Ex vivo biodistribution data for 11 C-L-1MTrp uptake in immunodeficient BALB/c nude mice bearing s.c. NCI-H69 and MDA-MB231 tumors; data were collected at 5, 15, 30, 60, and 90 min after radioinjection. Data are expressed as the mean %ID/g tissue ± standard error of the mean (s.e.m.; n = 3).

Supplementary Table 2. Biodistribution characterization of 11 C-L-1MTrp uptake in B16F10
tumor-bearing immunocompetent mice treated with IDO1 blockade-containing combinatorial immunotherapies or monotherapies. Ex vivo biodistribution data for 11 C-L-1MTrp uptake collected at 60 min after 11 C-L-1MTrp injection in six cohorts: vehicle, L-1MTrp, CPA, PTX, L-1MTrp + PTX, and L-1MTrp + CPA. A competition study was performed in L-1MTrp + CPA treatment mice by co-injection the "cold" IDO1 inhibitor INCB024360 (10 mg/kg) with 11 C-L-1MTrp. Data are expressed as the mean %ID/g tissue ± standard error of the mean (s.e.m.; n = 4-6).   To gauge the tumor elimination efficacy of an immunotherapy, an imaging toolbox has been developed. 3-11 13 14 16-18 However, these imaging tools cannot specifically address the immunoediting response against tumor cells because their target molecules, such as CD3, CD4, CD8, and CD20, 11 19-21 are expressed by both resting and active immune cells, including anti-inflammatory immune cells 8 . Molecules expressed by activated immune cells have also been pursued as imaging targets, for instance, OX40 (CD134), inducible T-cell costimulatory (ICOS), IFN-γ and granzyme B. 10 15 16 18 However, these targets are usually transiently expressed, diffusible, and located primarily in the extracellular matrix. Moreover, it is also worth noting that all the abovementioned methods narrowly focus on tumor sites, which are still too fragmented to produce an understanding of the immunoediting process in a living body. Continuous crosstalk is known to occur between tumors and the host immune system, which shapes antitumor immune responses and determines the efficacy of immunotherapy; hence, methods for imaging offtumor biomarkers on a whole-body scale could provide a more complete picture of the cancer-immune interaction occurring during immunotherapeutic intervention.