Combination Bacillus Calmette–Guérin and indoleamine 2,3-dioxygenase 1 inhibitor therapy of murine orthotopic bladder cancer

Sze Han Lee1, Ratha Mahendran2, Sin Mun Tham2,3, Thomas Paulraj Thamboo4, Dorinda Yan Qin Kioh1, Lloyd Wei Tat Tang1, Edmund Chiong2,3, Kesavan Esuvaranathan2,3, Eric Chun Yong Chan1,*
1 Department of Pharmacy, National University of Singapore, Singapore 117543, Singapore; 2 Department of Surgery, National University of Singapore, Singapore 119074, Singapore; 3 Department of Urology, National University Hospital, Singapore 119074, Singapore; 4 Department of Pathology, National University Hospital, Singapore 119074, Singapore
*Corresponding author: Professor Eric Chun Yong Chan ([email protected])
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Article type : Research Communication
Article Category: Research Correspondence
Dear Editor,
Non-muscle invasive bladder cancer (NMIBC) can be managed initially with transurethral resection of a bladder tumor. In carcinoma in situ (CIS) or papillary high- grade NMIBC patients, further intravesical Bacillus Calmette–Guérin (BCG) is recommended for the prevention of recurrences. About 20% of patients suffer from BCG intolerance thereby limiting its use; up to 40% are BCG-refractory and face tumor recurrence despite initial BCG induction; finally there are BCG-resistant patients who respond to neither initial nor repeated BCG instillations [1]. Hence, improvements to the therapeutic outcomes of intravesical BCG are essential. One mechanism of action of BCG involves a shift in the urinary cytokine milieu from Th2-like (e.g. IL-4, IL-5, IL-10) to Th1-like (e.g. IL-2, IL-12, and interferon-γ) to achieve BCG response [2]. Indoleamine 2,3-dioxygenase 1 (IDO1) can attenuate Th1 response via metabolism of tryptophan (TRP) to kynurenine (KYN) and resultant activation of aryl hydrocarbon receptor (AhR). This in turn leads to IL-10 transcription, regulatory T cells activation and CD8+ T cells suppression which collectively create an immunological tumor milieu that is defective in tumor cells recognition and elimination [3]. Thus, the IDO1-KYN-AhR metabolic axis represents a viable therapeutic target for augmenting response towards BCG. We aimed to evaluate the therapeutic efficacy of combination immunotherapy with IDO1 inhibitor, linrodostat, and BCG using our murine MB49-PSA orthotopic bladder tumor model [4].
After tumor induction on Day 0, intravesical BCG (or saline) was instilled on Day 7, 14, 21, 28, while oral linrodostat (or 0.5% methylcellulose) was delivered daily via gavage from Day 7. For BCG, 1×106 cfu/100µL was administered intravesically into each mouse weekly, equivalent to one-third of the full human dose to recapitulate the mixed clinical response. Mesylate salt of linrodostat (Bristol-Myers Squibb, NY, United States) was freshly prepared on a weekly basis as a 10 mg/mL suspension in 0.5% methylcellulose. Mice were given a daily oral gavage of 0.1 mL per 20 g body weight (equivalent to 40.5 mg/kg of linrodostat). By quantitating normalized urinary human prostate-specific antigen (PSA) to creatinine (PSA/CR) as a surrogate marker of murine bladder tumor load, we measured the post-treatment responses of mice when administered saline, BCG alone and BCG combined with linrodostat. Plasma kynurenine (KYN) was measured to detect the systemic inhibition of IDO1 by linrodostat. Immunohistochemistry staining was performed to measure CD3-positive T cells and IDO1 expression in tumor.
The combination of linrodostat and BCG reduced PSA/CR, a surrogate of tumor load, to a greater extent as compared to BCG monotherapy although it was not statistically significant (Fig. 1A). For the responders of both treatment groups who demonstrated substantial reduction in tumor load, we observed the consistent inhibition IDO1 functional activity by linrodostat, as confirmed by the suppression of plasma KYN to levels comparable to non-tumor controls (Fig. 1B). Non-responders to BCG showed significantly higher PSA/CR than responders (p=0.0006); similarly, non-responders to combination therapy showed significantly higher PSA/CR than responders (p=0.0259) (Fig. 1B). Immunohistochemistry revealed similar extent of CD3-positive T cells infiltration into the tumor (Fig. 1C). Likewise, IDO1 expression was homogenous across the tumor (Fig. 1D), confirming that IDO1 was available as the therapeutic target for linrodostat. The inconsistent responses towards the therapies were not due to the poor recruitment of immune cells nor non-expression of IDO1 enzymes in the tumor.
We successfully recapitulated 50% response rate in BCG monotherapy which was comparable to the clinical failure rate of BCG therapy at 50% [5]. This intended variable response was at least partially associated with the sub-therapeutic dose of BCG adopted in this study. Additionally, a lack of immune reactivity towards BCG antigens might also impair an initial immune response in some mice.
Interestingly, combinatorial therapy using BCG and linrodostat increased the response rate marginally to 60% although the difference was not statistically significant compared to BCG monotherapy. The heterogeneity of MB49 cell line was recently characterized, showing adherent and non-adherent spheroidal MB49 cells in the same culture to possess different degree of metastatic potential [6]. The proliferation of non-IDO1 expressing sub-clones of MB49 cells is not likely due to similar extent of expression of IDO1 between responders and non-responders. However, the lack of key downstream immune cell activation and cytokines may account for the differential responses. Secondly, while the dosing regimen of linrodostat adopted in this study inhibited the systemic metabolism of TRP to KYN, it does not necessarily translate to the most optimal regimen to elicit the pharmacological effect.
Linrodostat was recently initiated in a Phase II clinical trial (NCT03519256) for the treatment of NMIBC [7], with patients randomized to 1 of 4 treatment arms with nivolumab (anti-PD-1) ± linrodostat ± BCG. Notably, the specific combination of linrodostat and BCG was not investigated. With an early hint of potential combinatorial efficacy of BCG and linrodostat, further studies are warranted to invesitigate the efficacy and safety of this combination in the treatment of bladder cancer.
Sze Han Lee, Ratha Mahendran, Sin Mun Tham, Thomas Paulraj Thamboo, Dorinda Yan Qin Kioh, Lloyd Wei Tat Tang, Edmund Chiong, Kesavan Esuvaranathan, Eric Chun Yong Chan

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Funding The project was funded by the National University Health System (NUHS) Innovation Grant (NUHSRO/2018/041/Innov/03). Linrodostat was kindly donated by the Bristol-Myers Squibb Company.

Compliance with ethical standards
Conflict of interest The authors declare no conflict of interest.

Figure Legend
Figure 1
(A) Therapeutic efficacies of Bacillus Calmette–Guérin (BCG) monotherapy or in combination with indoleamine 2,3-dioxygenase 1 inhibitor (linrodostat) against murine MB49-PSA orthotopic bladder cancer where urinary PSA/CR is a surrogate of the tumor load. NR and R refer to nonresponders and responders. A response to treatment is defined as a reduction of PSA/CR (tumor load) to below the lowest level observed for the saline-treated tumor mice. (B) Terminal (blood drawn at point of sacrifice) plasma KYN in combinatorial treatment arm of BCG and linrodostat is suppressed to levels similar to the non-tumor control, illustrating sufficient dose of linrodostat for systemic suppression of indoleamine 2,3-dioxygenase (IDO1). Conversely, the terminal plasma KYN levels in BCG monotherapy arm are comparable to the levels of the tumor control arm, illustrating there is no systemic suppression of IDO1 in the absence of linrodostat. CD3 (C) and IDO1 (D) immunohistochemistry of MB49-PSA bladder tumors, with a focus on the non-responders (NR) and responders (R) towards BCG monotherapy and combinatorial BCG and linrodostat therapy. Mice administered with monotherapy and combinatorial therapy were coded with a prefix of ‘2’ and ‘3’ respectively.