Rationale for combining tyrosine kinase inhibitors and T cell redirecting antibodies to mitigate cytokine release syndrome (CRS)

Keywords:

• Chimeric Antigen Receptor-(CAR-) T cells
• CD3 bispecific antibody
• Cytokine Release Syndrome (CRS)
• cytokines
• tyrosine kinase inhibitors

1. Introduction

T cell engagers are a promising class of cancer immunotherapies. By bridging T cells to tumor cells, they enable the formation of a cytotoxic synapse, where the release of cytotoxic granules mediates tumor cell killing. T cell engagers have shown remarkable clinical efficacy, particularly in the treatment of leukemia and lymphoma. As of today, four T cell bispecific antibodies; blinatumomab (CD19xCD3), tebentafusp (gp100-HLA-A*02xCD3), mosunetuzumab (CD20xCD3), and teclistamab (BCMAxCD3) have been approved. We have previously described 2 + 1 T cell bispecific antibodies (TCBs), e.g. cibisatamab (CEA-TCB), glofitamab (CD20-TCB), or WT1-TCB [1–3]. The mode of action of these therapies consists in redirecting T cell cytotoxicity toward tumor cells, facilitating the formation of an immunological synapse where the release of perforin and granzymes induces tumor cell killing [1–3]. At the same time, the on-target activity of T cell engagers triggers T cell activation and proliferation together with the release of cytokines and chemokines [4,5]. In some cases, this inflammatory response can cause a cytokine release syndrome (CRS) manifesting mild to very severe symptoms ranging from fever, hypotension, respiratory deficiency, and multi-organ failure [4]. Given the profound clinical implications of CRS, the development of mitigation strategies that can reduce cytokine release while retaining the anti-tumor activity is crucial to improve patient safety and treatment outcomes. Recent work deciphering the events involved in the inflammatory response shows that T cell-derived cytokines and chemokines can rapidly activate surrounding myeloid cells, which greatly amplify the cascade [5]. Considering the upstream contribution of T-cells in the cytokine release cascade, one approach to prevent CRS comprises targeting kinase signaling pathways involved in cytokine release downstream of TCR activation (Figure 1). Here, we will discuss the effects of different FDA-approved tyrosine kinase inhibitors on T cell functionality and cytokine release and provide a rationale for their use as mitigation strategies for CRS. This approach may be of particular interest for cancer indications where the respective tyrosine kinase inhibitors have anti-tumor activity.

Figure 1. a. Mechanisms of cytokine release following T cell bispecific antibody treatment. On-target T cell engagement leads to their activation and initiates cytokine release. This further activates neutrophils, monocytes and T cells, which amplify the cytokine release signal. b. JAK, mTOR, and BCR-Abl/ Src-Lck inhibitors target kinase-signaling pathways involved downstream of TCR activation and interfere with T cell cytokine release and/or T cell-mediated cytotoxicity. BCR-Abl/Src-Lck, mTOR and JAK inhibitors are also pharmacologically active in various cancer indications, making them attractive combination partners for T cell engaging therapies targeting the same cancer cells. Created with Biorender.com.

2. Expert opinion

Following a screening of 52 FDA approved kinase inhibitors for their impact on T cell proliferation and cytokine release after CD3 stimulation, we selected the dual Bcr-Abl/Src (dasatinib, ponatinib, and bosutinib), JAK (ruxolitinib, baricitinib, tofacitinib, and fedratinib), and mTOR inhibitors (sirolimus, temsirolimus, and everolimus) as potential drugs to fine-tune TCB-induced T cell activation, proliferation, and cytokine release and further evaluated their effects in T cell cytotoxicity assays using CEA-TCB, CD19-TCB, or WT1-TCB [6,7] (Figure 1).

Among the Bcr-Abl/Src inhibitors, we found that pharmacologically active doses of dasatinib most efficiently and reversibly switched-off cytotoxicity and cytokine release from PBMCs that were pre-stimulated with WT1-TCB (HLA-A2 TCR-like TCB) or CEA-TCB by inhibition of Src and Lck kinases, in line with findings from Leonard et al. [7,8]. In vivo, dasatinib was able to prevent CD19-TCB-mediated B cell depletion and cytokine release for up to 48 h in humanized NSG mice. Based on these findings, dasatinib was developed into a potential safety switch e.g. to be applied if there is a risk for on-target off-tumor activity, or in rare cases of life-threatening CRS. In this case, transient administration of dasatinib should start as early as possible after the first signs of CRS or on-target off-tumor activity to rapidly resolve symptoms. Besides, short administration of dasatinib around the first infusion with T cell engager therapies could also prevent the cytokine peaks and reduce the risk of CRS, while only transiently blocking activity.

In contrast to the Bcr-Abl/Src inhibitor dasatinib, mTOR and JAK inhibitors suppressed cytokine release while retaining T cell-mediated tumor cell cytotoxicity in the T cell dependent cytotoxicity assay, similarly to dexamethasone [6]. Therefore, they may be more attractive for the prophylactic mitigation of cytokine release with minimal effect on anti-tumor efficacy. The side-by-side in vitro comparison of JAK and mTOR inhibitors further revealed that the blockade of the mTOR pathway more extensively reduced cytokine release with lower impact on tumor cell killing than observed with the JAK1/2 inhibitor ruxolitinib, which retained IL-2 and GM-CSF release, and, to some extent, TNF-α release [6]. In the clinic, the prophylactic use of ruxolitinib in flotetuzumab-treated patients modulated the cytokine profiles, which was not sufficient to prevent CRS clinical signs [9]. Given the partial effect of JAK1/2 inhibition in reducing cytokine release, the combination of JAK1/2 inhibitors and other mitigating agents, including glucocorticoids and adalimumab (TNF-α blockade) for the mitigation of CRS could be explored. The side-by-side in vitro and in vivo comparison of anti-TNF-α, anti-IL-6R, dexamethasone, JAK, Src, and mTOR inhibitors reveals that mTOR inhibitors most efficiently reduce cytokine release while retaining anti-tumor activity mediated by T cell bispecific antibodies [10]. Considering the pharmacological anti-tumor activity of mTOR inhibitors in various solid cancers including breast, prostate, lung, liver, or renal cell carcinomas, a combination with a solid-tumor targeted T cell bispecific antibody may be of particular interest to prevent CRS risk and remains to be clinically explored (Table 1) [11,12].

Table 1. Rationale for potential combination dual BCR-Abl/Src and mTOR inhibitors with T cell engagers for enhanced safety profile and efficacy.

Combinations	Indications	Rationale	References
BCR-Abl/Src inhibition (dasatinib) + blinatumomab (CD19xCD3 T cell engager)	Acute Lymphoblastic Leukemia (ALL)	In the NCT02744768 trial, dasatinib eliminates ALL cells reducing tumor load before treatment with blinatumomab. When combined with blinatumomab, dasatinib rapidly switches off T cells for short period of time following stimulation with blinatumomab, resulting in lower cytokine release and prolonged efficacy	6^[Citation14]
mTOR inhibition (sirolimus, temsirolimus or everolimus) + T cell engagers directed against solid tumor	breast cancer, lung cancer, gastric carcinoma, colorectal cancer, prostate cancer, head and neck cancer, gynecologic cancer, glioblastoma, renal cell carcinoma	Preclinical studies show that mTOR inhibitors are pharmacologically active in various solid tumor indications. When combined with a T cell engager targeting the same indication, mTOR inhibition could improve efficacy and at the same time prevent cytokine release.	6^[Citation6,Citation11,Citation12]
mTOR inhibition (sirolimus, temsirolimus or everolimus) + T cell engagers directed against lymphoma	Lymphoma	Preclinical studies show that mTOR inhibitors are pharmacologically active in lymphoma indications. When combined with a T cell engager targeting the same indication, mTOR inhibition could improve efficacy and at the same time prevent cytokine release.	6^[Citation6,Citation11,Citation12]

An important feature of kinase inhibitors is their potential antitumor activity, which makes them ideal combination partners with T cell engaging therapies in the same cancer indications (Table 1) [13]. For indications where they are pharmacologically active, TKIs could be used as single-agent therapy to de-bulk tumor load, before being combined with a T cell bispecific antibody to safely eliminate the remaining tumor cells, and at the same time lower the CRS risk. A clinical example is the combination of dasatinib with blinatumomab to eradicate the residual tumor cells in patients with Philadelphia positive acute lymphoblastic leukemia (Ph+ ALL), who were previously treated with dasatinib to de-bulk the tumor load [14]. This schedule resulted in a safe and efficacious administration of blinatumomab [14]. Our preclinical findings suggest that rapid in vivo ON/OFF switches decreased the risk of CRS while retaining lasting blinatumomab efficacy [7]. As these mitigation therapies, even if applied acutely and not applied chronically, add extra cost to treatment and come with an increased risk of infection for the patients, the identification of predictive biomarkers of CRS would also be highly informative to stratify patient at risk beforehand. Another important feature of kinase inhibitors is their potential to reprogram T cell fate toward better effector phenotype. One example is the work from Zieger et al. demonstrating that on/off switches induced by dasatinib prolong T cell effector functions by preventing rapid T cell exhaustion, preserving long-term blinatumomab efficacy [15].

3. Conclusion

CRS remains dose-limiting toxicities associated with treatment with T cell engagers, accentuating the need to define precise interventions that retain or even enhance efficacy while efficiently reducing cytokine peaks. Reviewing the effects of Src-Lck, mTOR, and JAK inhibitors on cytokine release induced by T cell redirecting therapies sheds new light on the mitigation of CRS in the context of T cell bispecific antibodies. By interfering with signaling pathways downstream of TCR activation, these molecules can prevent T cell-derived cytokine release, which is the initial trigger of the inflammatory response that may cause CRS [5] (Figure 1). When compared side-by-side to tocilizumab and adalimumab, they also show superior reduction of cytokine release, which is comparable to dexamethasone, and at the same time, retained efficacy (with the exception of the Src-Lck inhibitor) [6]. For this reason, TKIs appear more attractive than tocilizumab and adalimumab to prevent the cytokine release cascade. In contrast to antibody-based approaches, small molecules TKIs are short-lived, enabling precise interventions for the mitigation of CRS. We would recommend giving TKIs before and shortly after treatment with T cell engagers to prevent the early inflammatory events, which may initiate the cascade of cytokine release, and ultimately cause CRS.

The dual Bcr-Abl/Src inhibitor dasatinib switches off both cytokine release and cytotoxicity following stimulation with T cell engager [7]. Consequently, dasatinib could be transiently used as a safety switch in rarer cases of acute toxicity, including high-grade CRS or the risk of on-target off-tumor activity [7]. In vivo, the reversible inhibitory properties and the combination of short PK/PD properties translates into rapid on/off switches [7]. Therefore, combining dasatinib with T cell redirecting antibodies for the duration of treatment could also prolong T cell effector functions by preventing exhaustion induced by the rapid on-target activation and, at the same time, reduce toxic peaks of cytokine release [15].

In contrast, JAK and mTOR inhibitors were shown to reduce cytokine release while retaining T cell bispecific antibody-mediated cytotoxicity [6]. mTOR inhibitors appear to be more potent than JAK inhibitors and dexamethasone in reducing cytokine release while minimally affecting tumor cell killing [6]. Therefore, they represent an attractive approach for the mitigation of CRS, in particular for indications where they are pharmacologically active [12]. JAK and mTOR inhibitors could be given transiently around the first treatment cycle to prevent the peak of cytokine release, or as a de-bulking therapy before treatment with T cell engagers to eliminate residual tumor cells. Yet this remains to be clinically proven. Another aspect to further explore for successful combinations would be their potential to reprogram T cells toward more functional phenotypes following TCR activation after treatment with T cell bispecific antibodies.

Declaration of interest

G Leclercq, M Bacac, and C Klein declare patent applications with Roche. All authors are employees of Roche. All the authors declare ownership of Roche stock. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Additional information

Funding

This paper was not funded.