CDK4/6 inhibition enhances antitumor efficacy of chemotherapy and immune checkpoint inhibitor combinations in preclinical models and enhances T-cell activation in patients with SCLC receiving chemotherapy

Background Combination treatment with chemotherapy and immune checkpoint inhibitors (ICIs) has demonstrated meaningful clinical benefit to patients. However, chemotherapy-induced damage to the immune system can potentially diminish the efficacy of chemotherapy/ICI combinations. Trilaciclib, a highly potent, selective and reversible cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitor in development to preserve hematopoietic stem and progenitor cells and immune system function during chemotherapy, has demonstrated proof of concept in recent clinical trials. Furthermore, CDK4/6 inhibition has been shown to augment T-cell activation and antitumor immunity in preclinical settings. Therefore, addition of trilaciclib has the potential to further enhance the efficacy of chemotherapy and ICI combinations. Methods In murine syngeneic tumor models, a schedule of 3 weekly doses of trilaciclib was combined with chemotherapy/ICI regimens to assess the effect of transient CDK4/6 inhibition on antitumor response and intratumor T-cell proliferation and function. Peripheral T-cell status was also analyzed in patients with small cell lung cancer (SCLC) treated with chemotherapy with or without trilaciclib to gain insights into the effect of transient exposure of trilaciclib on T-cell activation. Results Preclinically, the addition of trilaciclib to chemotherapy/ICI regimens enhanced antitumor response and overall survival compared with chemotherapy and ICI combinations alone. This effect is associated with the modulation of the proliferation and composition of T-cell subsets in the tumor microenvironment and increased effector function. Transient exposure of trilaciclib in patients with SCLC during chemotherapy treatment both preserved and increased peripheral lymphocyte counts and enhanced T-cell activation, suggesting that trilaciclib not only preserved but also enhanced immune system function. Conclusions Transient CDK4/6 inhibition by trilaciclib was sufficient to enhance and prolong the duration of the antitumor response by chemotherapy/ICI combinations, suggesting a role for the transient cell cycle arrest of tumor immune infiltrates in remodeling the tumor microenvironment. These results provide a rationale for combining trilaciclib with chemotherapy/ICI regimens to improve antitumor efficacy in patients with cancer.

ABSTRACT Background Combination treatment with chemotherapy and immune checkpoint inhibitors (ICIs) has demonstrated meaningful clinical benefit to patients. However, chemotherapy-induced damage to the immune system can potentially diminish the efficacy of chemotherapy/ ICI combinations. Trilaciclib, a highly potent, selective and reversible cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitor in development to preserve hematopoietic stem and progenitor cells and immune system function during chemotherapy, has demonstrated proof of concept in recent clinical trials. Furthermore, CDK4/6 inhibition has been shown to augment T-cell activation and antitumor immunity in preclinical settings. Therefore, addition of trilaciclib has the potential to further enhance the efficacy of chemotherapy and ICI combinations. Methods In murine syngeneic tumor models, a schedule of 3 weekly doses of trilaciclib was combined with chemotherapy/ICI regimens to assess the effect of transient CDK4/6 inhibition on antitumor response and intratumor T-cell proliferation and function. Peripheral T-cell status was also analyzed in patients with small cell lung cancer (SCLC) treated with chemotherapy with or without trilaciclib to gain insights into the effect of transient exposure of trilaciclib on T-cell activation. Results Preclinically, the addition of trilaciclib to chemotherapy/ICI regimens enhanced antitumor response and overall survival compared with chemotherapy and ICI combinations alone. This effect is associated with the modulation of the proliferation and composition of T-cell subsets in the tumor microenvironment and increased effector function. Transient exposure of trilaciclib in patients with SCLC during chemotherapy treatment both preserved and increased peripheral lymphocyte counts and enhanced T-cell activation, suggesting that trilaciclib not only preserved but also enhanced immune system function.
Conclusions Transient CDK4/6 inhibition by trilaciclib was sufficient to enhance and prolong the duration of the antitumor response by chemotherapy/ICI combinations, suggesting a role for the transient cell cycle arrest of tumor immune infiltrates in remodeling the tumor microenvironment. These results provide a rationale for combining trilaciclib with chemotherapy/ICI regimens to improve antitumor efficacy in patients with cancer.
BACKGROUND Trilaciclib (G1T28) is a highly potent, selective and reversible cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitor that maintains G1 cell cycle arrest of cells that are dependent on CDK4/6 for regulation of the G1 to S transition. By transiently maintaining G1 arrest of proliferating hematopoietic stem and progenitor cells in the bone marrow during chemotherapy treatment, trilaciclib proactively protects them from chemotherapy-induced damage, leading to faster recovery of neutrophils, red blood cells (RBCs), lymphocytes and platelets after chemotherapy treatment. 1 2 In a phase II trial (NCT02499770) evaluating trilaciclib administered prior to etoposide and carboplatin (E/P) therapy in patients with newly diagnosed extensive-stage small cell lung cancer (SCLC), trilaciclib demonstrated myelopreservation across multiple hematopoietic lineages (including neutrophils, RBCs and lymphocytes), resulting in fewer supportive care interventions and dose reductions, an improved safety profile and no detriment to antitumor efficacy. 3 In addition to improving the safety of chemotherapy, trilaciclib improved overall survival (OS) among patients with metastatic triple-negative breast cancer (mTNBC) when added prior to gemcitabine and carboplatin. 4 Possible mechanisms of trilaciclib-mediated Open access enhanced antitumor efficacy include maintenance of chemotherapy dose intensity (ie, fewer dose reductions), protection of lymphocyte populations and increased immune activation. Trilaciclib and other CDK4/6 inhibitors have been shown to augment antitumor responses in preclinical settings 5 by enhancing T-cell activation through modulation of nuclear factor of activated T-cell activity, 6 as well as increasing antigen presentation through upregulation of major histocompatibility complex class I and II in CDK4/6-sensitive tumors and myeloid cells. 7 8 Additionally, CDK4/6 inhibition can upregulate and stabilize the protein expression of programmed death-ligand 1 (PD-L1) on tumor cells, leading to increased vulnerability of tumors to immune checkpoint inhibitor (ICI) treatment. 9 Furthermore, CDK4/6 inhibition reduces a T-cell exclusion and immune evasion gene signature that is predictive of resistance to ICI treatment. 10 These results suggest that trilaciclib has the potential to enhance the efficacy of chemotherapy, as well as chemotherapy and ICI combinations.
Chemotherapy and ICI combinations have shown superior benefits compared with chemotherapy or ICI monotherapy in various clinical settings, including non-SCLC, SCLC and TNBC. [11][12][13][14][15] The enhanced efficacy by chemotherapy and ICI combinations is likely attributed to various immunostimulatory properties by different classes of chemotherapeutic agents. [16][17][18][19] However, because chemotherapy indiscriminately kills proliferating cells, the full benefit of chemotherapy plus ICI combinations may not be realized due to the resulting myelosuppression and immunosuppression 20 21 that occurs when normal proliferating hematopoietic stem and progenitor cells and immune cells are exposed to chemotherapy. Therefore, addition of trilaciclib to chemotherapy and ICI combinations is a rational approach to maintain and/ or enhance immune system function to fully exploit the therapeutic potential of chemotherapy/ICI combination regimens and minimize toxicity. The goal of this study was to evaluate the ability of trilaciclib to enhance antitumor response when combined with chemotherapy plus ICI combinations.

In vivo tumor studies
Nine-week-old female C57BL/6 (C57BL/6NCrl) and BALB/c mice were implanted subcutaneously with 5×10 5 MC38 22 or CT26 American Type Culture Collection (ATCC) tumor cells, respectively (cell lines supplied by Charles River Laboratories). Two to 3 weeks after tumor injection and prior to treatment start (day 1 of the study), animals with individual tumor volumes from 80 to 120 mm 3 were sorted into the appropriate number of treatment groups, with group mean tumor volumes of 100 mm 3 .
Tumors were measured using calipers twice per week. Each animal was euthanized when its tumor volume reached the 1000 mm 3 end point or at last day of the study. A partial response (PR) indicated that the tumor volume was ≤50% of its day 1 volume for three consecutive measurements during the course of the study, and ≥13.5 mm 3 for at least one of these three measurements. A complete response (CR) indicated that the tumor volume was <13.5 mm 3 for three consecutive measurements during the course of the study. Animals were scored only once during the study for a PR or CR event, and only as CR if both PR and CR criteria were satisfied.

Whole tumor gene expression analysis
Total RNA was isolated from flash-frozen tumors, harvested on days 9 and 17, from vehicle-treated, trilaciclib-treated, OP-treated and TOP-treated MC38 mice following the IM treatment schedule indicated in figure 1A (n=5 per treatment group), using the AllPrep DNA/RNA/miRNA Universal Kit (Qiagen; catalog number 80224). Gene expression analysis was performed using the PanCancer Immune Profiling Panel (NanoString; catalog number XT-CSO-HIP1-12). Count data for panels were normalized to positive control probes for each sample and background corrected using NanoStringQCPro software (Bioconductor). The background-corrected counts were normalized using trimmed mean of M-value normalization and transformed with voom to log2 counts per million with associated precision weights. Significant genes with raw p-value <0.05 and fold change ≥1.3 from each comparison were analyzed for enrichment of Kyoto Encyclopedia of Genes and Genomes pathway membership using a hypergeometric test. Significant genes with each comparison were also analyzed for enrichment of Gene Ontology (GO) terms across all three GO ontologies using a hypergeometric test. For all analyzes, enrichment (p<0.05) was assessed separately for upregulated and downregulated genes. The conditional algorithm implemented in the GOstats R package (Bioconductor) was used to estimate if each term was statistically over-represented; this was performed separately for each ontology and the results merged. Gene expression analysis was performed at EA Genomics/Q2 Solutions (Morrisville, North Carolina, USA), and data analysis was performed at Fios Genomics (Edinburgh, UK).

Peripheral blood immunophenotyping in an SCLC clinical trial
This was a phase 1b (open-label, dose-finding) and phase II (randomized, double-blind, placebo-controlled) study of the safety, efficacy and pharmacokinetics of trilaciclib in combination with E/P therapy for treatment-naïve patients with extensive-stage SCLC ( ClinicalTrials. gov identifier NCT02499770). Patients received trilaciclib or placebo prior to E/P on days 1-3 of each cycle. 3 During part 2 of the study, whole blood was collected from those patients who received at least four cycles of chemotherapy for analysis of immune subsets by flow cytometry at Covance Central Laboratory Services (CLS). For patients treated with E/P and trilaciclib prior to E/P (T/E/P), samples were collected on day 1 of cycles 1 (C1D1; 30 and 28 samples, respectively), 3 (C3D1; 24 and 27 samples, respectively) and 5 (C5D1; 19 and 22 samples, respectively), and at the post-treatment visit (28 and 19 samples, respectively).
A linear mixed-effect model was fitted on the longitudinal data for absolute count of CD3 + total T cells, CD4 + T cells, CD8 + T cells and CD19 + B cells to assess the effect of treatment on lymphocyte levels over time (table 1). The dependent variable in the model was 'test result,' the absolute cell count (cells/µL). The data were restricted to patients who received ≥4 cycles of chemotherapy and considered a random effect, while time (weeks), treatment group (T/E/P or E/P) and their interaction were modeled as fixed effects. The analysis used an unstructured variance-covariance structure, according to the following model: Test Result ∼ Weeks + Treatment + Weeks : Treatment + PatientID (random effect).
From the model, the slope for each treatment group reflects the trend of change along the time for absolute cell count; hence, the slope difference between treatment groups can be used to evaluate the treatment effect of trilaciclib. The R package lme4 was used to fit a linear mixed-effect model, 23 and lmerTest was used to generate p-values for the fitted model. 24 All statistical analysis of Open access flow cytometry data was performed by Fios Genomics (Edinburgh, UK).

T-cell receptor β CDR3 sequencing
The T-cell receptor (TCR)β CDR3 regions were amplified and sequenced from purified genomic DNA in human peripheral blood mononuclear cells or murine MC38 tumors using the immunoSEQ Assay (Adaptive Biotechnologies, Seattle, Washington, USA). 25 26 The estimated proportion of T cells in peripheral blood mononuclear cells or tumors was calculated as previously described. 27 28 To derive the overall clonality score, Shannon entropy was calculated on the clonal abundance of all productive TCR sequences in the data set, and normalized to the range by dividing by the logarithm of the number of unique productive TCR sequences. The clonality score was determined as the inverted value of the normalized entropy value. Expanded T-cell clones (ie, those whose frequencies increased in post-treatment versus pretreatment [C1D1] samples, in a given patient), were computationally identified as previously described. 29 A binomial test was used to compute a p value for each clone across the two samples against the null hypothesis that the population abundance of the clone was identical in both samples, and corrected for multiple testing to control the false discovery rate using the Benjamini-Hochberg procedure, employing a significance threshold of adjusted p<0.01. Survival analysis was performed using R packages Survival and Survminer. Statistical analysis was performed using the Cox proportional hazard regression analysis.

RESULTS
Addition of trilaciclib to chemotherapy and ICI combinations enhances antitumor activity in preclinical cancer models with established tumors To evaluate the effect of trilaciclib on the antitumor immune response when combined with chemotherapy plus ICI, murine subcutaneous syngeneic colon tumor models MC38 and CT26 were used. The dose of trilaciclib was derived from preclinical data and pharmacokinetic/pharmacodynamic modeling of the biologically effective dose in humans. 1-3 30 31 Both tumor models were insensitive to trilaciclib treatment alone, as there was no significant change in tumor growth inhibition when tumor-bearing animals were treated with varying schedules of trilaciclib as a single agent. Neither continuous nor intermittent dosing schedules of trilaciclib plus anti-PD-L1 combination treatment improved tumor growth inhibition in either model (Lai et al. online supplemental figure S1).
TOP combinations substantially enhanced overall response rate and durability of the response compared with the OP groups with all three dosing schedules (table 2; figure 1B-D; online supplemental figure S1C). Specifically, TOP animals had significantly higher rates of CR and prolonged survival on day 100 (table 2). Using the IM dosing strategy, the addition of trilaciclib to OP plus anti-PD-1 or 5-FU plus anti-PD-L1 combinations consistently improved antitumor response and survival (figure 1C; table 2). Although the CT26 tumor model was less sensitive to OP treatment than the MC38 model, TOP-treated CT26 animals dosed with the IM schedule demonstrated higher tumor growth inhibition (figure 1D), more CRs and longer OS compared with OP treatment (table 2). Tumor growth curves for individual animals within each of the treatment groups are provided in online supplemental figure S1D-F. These results demonstrate that trilaciclib can significantly enhance antitumor response of various chemotherapy and ICI combinations in murine models with established tumors.

Intermittent dosing of trilaciclib modulates the proliferation kinetics of T-cell subsets in the tumor microenvironment
To determine the mechanism by which trilaciclib contributes to an enhanced antitumor response seen in preclinical models, in vivo EdU incorporation was performed in MC38 tumor-bearing mice to identify proliferating intratumor immune cell populations that are sensitive to CDK4/6 inhibition. In untreated tumor-bearing animals, intratumor T cells (CD8 + , CD4 + and Tregs), NK cells and mMDSCs were significantly more proliferative compared with their counterparts in the spleen (Lai et al. online supplemental figure S2; figure 2A). Conversely, only a  To further examine the effect of trilaciclib in modulating the proliferation of intratumor T cells when combined with chemotherapy and ICI, proliferation of T-cell subsets was analyzed at multiple time points after OP or TOP treatment in MC38 tumor-bearing mice (figure 2C). On day 2, 1 day after the first dose of combination treatment, proliferation of intratumor CD8 + T cells in TOP-treated animals decreased by 82.5%, while the proliferation status of intratumor CD8 + T cells in OP-treated animals remained unchanged compared with vehicle treatment ( figure 2C). However, the proliferation of intratumor CD8 + T cells in the TOP group was quickly restored, resulting in a 20% increase in proliferating CD8 + T cells compared with the OP group on days 4 and 7, 3 days after the first and second doses of anti-PD-L1, respectively (figure 2C). While the recovery of cell proliferation in CD4 + T cells was initially slower than that of CD8 + T cells, the proportion of proliferating CD4 + T cells was twofold higher in TOP-treated versus OP-treated animals by day 7 (figure 2C). In contrast, transient G1 arrest of Tregs resulted in much slower recovery of proliferation in this cell population. On day 7, the proportion of EdU + Tregs in TOP-treated animals was 46% lower than that of the OP group (figure 2C). To further understand the impact of each treatment on T-cell proliferation, rates of absolute proliferation of T-cell subsets were also analyzed (Lai et al. online supplemental figure S3). These results indicate that in the presence of chemotherapy and ICI combination treatment, trilaciclib transiently arrests all major intra-tumor T-cell subsets, with subsequent faster recovery and enhanced proliferation in CD8 + T and CD4 + T cells and slower recovery of Treg proliferation. The differences in proliferation and recovery result in an increased effector T cell to Treg ratio in the tumor microenvironment.
In addition to the slower recovery of Treg proliferation, we further investigated whether CDK4/6 inhibition in Tregs affected their immunosuppressive function (online supplemental methods). A dose-dependent inhibition of Treg function was observed after trilaciclib treatment in vitro, as evidenced by an increase in CD8 + T-cell proliferation in the presence of trilaciclib pretreated Tregs compared with untreated Tregs ( figure 2D). This result further indicates that trilaciclib can attenuate the proliferation and immunosuppressive properties of Tregs.

Transient G1 arrest alters the proportion of intratumor T-cell subsets favoring effector T-cell function
Because TOP treatment led to changes in the proliferation kinetics of T-cell subsets, we next investigated whether   immunosuppression. In the spleen, the proportion of Tregs in CD4 + T cells and ratio of splenic CD8 + T cells to Tregs were similarly altered in TOP-treated compared with OP-treated animals on day 5 ( figure 3B,C). Furthermore, both splenic CD8 + and CD4 + T cells had a higher proportion of cells expressing the activation marker CD69 Trilaciclib in combination with chemotherapy and ICI enhances the gene signature associated with a cytotoxic antitumor response To gain insight into whether modulation of T-cell subsets by trilaciclib enhanced effector T-cell function when combined with chemotherapy and ICI, whole tumor gene expression profile was compared between vehicle-treated, OP-treated and TOP-treated MC38 tumor-bearing mice on days 9 and 17, after two or three doses of chemotherapy, respectively. Compared with vehicle treatment, upregulated genes in both the OP and TOP groups were enriched for two immune-related pathways, including TCR signaling pathway and NK cell-mediated cytotoxicity on days 9 and 17 (online supplemental data file S1). While the expression level of most individual genes within the two enriched immune pathways was not significantly upregulated in the TOP versus OP groups, the mean expression level of genes within both pathways was significantly higher in tumors from TOP-treated versus OP-treated animals on day 17 ( figure 4A,B). Importantly, the same set of genes within each pathway was also more highly expressed in tumors from animals treated with trilaciclib alone versus vehicle ( figure 4A,B), further indicating the contribution of transient CDK4/6 inhibition by trilaciclib in augmenting antitumor gene expression signatures when combined with chemotherapy and ICIs. The IFN-γ pathway is an important component of cytotoxic antitumor response shown to predict clinical response to PD-1 blockade. 32 The GO term 'positive regulation of IFN-γ production' was enriched in tumors of TOP-treated but not OP-treated animals on day 17 (online supplemental data file S1). Accordingly, the average expression level of genes associated with this GO term was elevated in TOP-treated compared with OP-treated tumors on day 17 ( figure 4A,B). In agreement with enhanced antitumor immunity gene signatures, T-cell repertoire analysis indicated that the addition of trilaciclib to OP treatment resulted in a higher proportion of intratumor T cells and TCR clonality (figure 4C), suggesting a more robust clonal expansion of T cells within tumors. 33 Collectively, these results support the notion that transient CDK4/6 inhibition modulated the tumor microenvironment through changes in the proportion of T-cell subsets leading to enhanced effector T-cell function.
Addition of trilaciclib to chemotherapy preserves and enhances lymphocyte function in patients with SCLC To assess the myelopreservation and immunomodulatory properties of trilaciclib in combination with chemotherapy in patients with SCLC, peripheral blood immunophenotyping was performed in patients from the E/P and T/E/P groups at various time points. Although the CD19 + B-cell population was significantly depleted by E/P, CD19 + B-cell numbers in the T/E/P group remained unchanged ( figure 5A). Furthermore, while E/P treatment did not decrease the overall number of peripheral T cells, T/E/P treatment resulted in an increased number of total T cells (CD3 + ), CD4 + T cells and CD8 + T cells (figure 5A). Profile longitudinal analysis confirmed significantly decreased B-cell numbers in the E/P group but not the T/E/P group, and increased CD4 + and CD8 + T-cell numbers with T/E/P compared with E/P treatment over time (table 1).
Functional evaluation of the T-cell subpopulations demonstrated that the trilaciclib group had a higher proportion of activated or effector CD8 + T cells (figure 5A; Lai et al. online supplemental figure S5), as well as activated or effector CD4 + T cells (online supplemental figure S5B and C). On stimulation, a higher proportion of CD8 + T cells produced IFN-γ in the T/E/P group compared with the E/P group (online supplemental figure S5D). Furthermore, the ratio of both CD8 + T cells to Tregs and activated CD8 + T cells to Tregs increased with T/E/P compared with E/P (figure 5A; online supplemental figure S5E). These results suggest that the addition of trilaciclib to chemotherapy at least preserved, if not enhanced, T-cell function during E/P treatment.
While flow cytometry results demonstrated differential treatment effects on T-cell levels (figure 5A), the overall diversity of the TCR repertoire, measured by the clonality score, was not significantly altered by E/P versus T/E/P treatment (figure 5B). Patients receiving T/E/P treatment, however, exhibited an increased number of T-cell clones expanded when measured on C3D1 (after roughly 6 weeks of therapy (statistically significant)) and C5D1 (after roughly 15 weeks of therapy (not statistically significant)) when compared with patients treated with E/P (figure 5B). Increased clonal expansion in the T/E/P group was more pronounced at C3D1 (figure 5B), and a higher proportion of patients had above the median number of expanded clones with T/E/P compared with E/P (figure 5C). These data further support a role of trilaciclib in increasing the level of T-cell activation.
Regardless of treatment group, high levels of clonal expansion at C3D1 or C5D1 were associated with improved progression-free survival (PFS) and numerically longer median OS (figure 5D), suggesting a correlation between peripheral T-cell activation status and antitumor response. Comparison of PFS in patients with high clonal expansion in the T/E/P versus E/P groups indicated an additional improvement with trilaciclib (online supplemental figure S5F and G), pointing at a role for trilaciclib in mediating an enhanced antitumor response.

DISCUSSION
While the combination of chemotherapy and ICIs is efficacious, and several chemotherapy/ICI regimens have recently gained US Food and Drug Administration approval, combination therapy results in significant hematologic toxicity and fails to achieve adequate benefit Open access in many patients. Diminishing the myelosuppressive and immunosuppressive effects of chemotherapy has the potential to improve therapeutic benefit by simultaneously reducing toxicity and increasing efficacy. The latter may even offer the potential for improved outcomes of chemotherapy/ICI regimens in indications that do not currently benefit.
The addition of trilaciclib to multiple chemotherapy and ICI combinations in murine syngeneic tumor models enhanced antitumor response compared with chemotherapy and ICI combinations alone, in part by modulating the proliferation and composition of T-cell subsets in the tumor microenvironment. A schedule of 3 weekly doses of trilaciclib given 30 min before chemotherapy was sufficient to enhance and prolong the duration of the antitumor response. In contrast, efficacy of the anti-PD-L1 antibody in established tumors was not increased by continuous co-administration of trilaciclib in the  Open access absence of chemotherapy. We speculate that prolonged exposure to CDK4/6 inhibition can dampen the ability of tumor-specific T cells to exert their cytotoxic functions. It is also possible that chronic exposure to CDK4/6 inhibition could prevent the differentiation of effector T cells to memory T cells, as intratumor T cells are highly dependent on the CDK4/6 pathway for proliferation.
Given that T-cell proliferation is a critical component of an effective immune response and is highly sensitive to CDK4/6 inhibition, 6 it is counterintuitive that cell cycle arrest in T cells could positively influence an antitumor immune response. In this study, we characterized a cell cycle-dependent effect of trilaciclib in modulating the composition of intratumor T-cell subsets and provided a plausible explanation of why transient T-cell proliferation arrest could be beneficial to an ongoing immune response. Following transient G1 arrest, there was a faster restoration of proliferative capacity in CD4 + and CD8 + T cells compared with a significantly slower rate of recovery of Treg proliferation, corresponding to a decreased proportion of Tregs in the tumor microenvironment and enhanced effector T-cell function. Consistent with the ability of trilaciclib to enhance or preserve effector T-cell function during exposure to chemotherapy, we have previously shown ex vivo stimulation of splenic T cells from animals treated with trilaciclib plus 5-FU resulted in the increased production of IFN-γ and IL-2 compared with 5-FU treatment alone. 34 Further investigation is needed to determine whether trilaciclib-mediated G1 arrest results in Treg depletion or apoptosis, or if transient proliferative arrest leads to the differentiation of Tregs into effector CD4 + T cells. In the current study, intratumor T-cell populations were defined using CD4 and CD8 cell surface markers only. More detailed analysis of the effects of trilaciclib on T-cell subsets (eg, effector memory, central memory, activated effector T cells, exhausted T cells), through the incorporation of CD3 staining, as well as other cell surface markers specific to different subsets and activation/exhaustion status, may provide additional insights into how transient trilaciclib treatment might enhance antitumor response.
Consistent with increased effector T-cell function in the tumor microenvironment, we observed elevated proportions of activated CD4 + and CD8 + T cells and a high ratio of CD8 + T cells to Tregs in the spleens of tumor-bearing animals after treatment with TOP versus OP combinations. This suggests that the activation status and composition of T cells in the peripheral compartment may be indicative of changes within the tumor microenvironment. 35 36 In agreement with these preclinical observations, patients with SCLC who received trilaciclib with chemotherapy showed an increase in number of T cells and the proportion of activated CD8 + T cells in peripheral blood, and had increased T-cell clonal expansion relative to patients who did not receive trilaciclib with chemotherapy. The more pronounced T-cell clonal expansion at C3D1 in the trilaciclib group is consistent with more robust T-cell proliferation previously observed at earlier time points with immunotherapy. 35 37 While trilaciclib did not significantly improve PFS (hazard ratio 0.7; p=0.1695) or OS (hazard ratio 0.87; p=0.6107) when added to E/P, the study was not powered to show such an effect. 3 However, there are several notable observations providing indirect clinical evidence that trilaciclib may enhance immune-mediated antitumor response through mechanisms demonstrated in preclinical models. Because SCLC is predominately deficient in the retinoblastoma gene and protein function and therefore CDK4/6 independent, 38 the positive trend in PFS is not expected to be mediated by direct cell cycle effects on the tumor cells, but rather through effects on immune cells within the tumor microenvironment, as shown in the MC38 model. Peripheral blood immunophenotyping and clonal expansion provides support that trilaciclib can enhance immune function in patients. While tumor tissue was not available for evaluation in patients with SCLC, the congruence between trilaciclib's effects on immune activation within the tumor and periphery of preclinical models and the peripheral blood of patients supports the hypothesis that trilaciclib can enhance immune activation within the tumors of patients with SCLC. Finally, while patients with above the median clonal expansion had a longer PFS and trended toward longer OS regardless of treatment group, the observation that patients receiving trilaciclib with a high number of expanded clones may have additional PFS benefits may also reflect the effects of trilaciclib on the immune cells within the tumor microenvironment. 39 While these observations are thought provoking, tumor tissue and/or larger studies will be needed to confirm these hypotheses in patients.
The more robust immune status in T/E/P patients suggests that trilaciclib has the potential to enhance the efficacy of chemotherapy and ICI combinations in tumors that are sensitive to ICIs. While SCLC has a high degree of genomic instability and a smoking-associated mutational profile, the low expression of major histocompatibility complex class I and class II suggests that it will be recalcitrant to immuno-oncology therapeutic approaches, 40 which is supported by a number of underwhelming clinical trials. 41 Trilaciclib has been tested in combination with E/P/atezolizumab for the first-line treatment of patients with SCLC. 42 Similar to the results shown here, trilaciclib treatment enhanced T-cell immune response as measured by peripheral blood immunophenotyping and T-cell clonal expansion, but failed to significantly improve PFS or OS. In contrast, the addition of trilaciclib to chemotherapy improved OS in mTNBC, 4 a tumor type with an immune landscape more amenable to ICI therapy. 15 43 While preclinical and correlative data from patients with SCLC have demonstrated that trilaciclib can enhance T-cell activity, direct mechanistic evidence to explain the improved OS results in mTNBC is still needed. 4 Given the clinical proof-of-concept findings that trilaciclib can reduce chemotherapy-induced multi-lineage myelosuppression in patients with SCLC, 3 improve OS when added to chemotherapy in patients with mTNBC 4 Open access and enhance antitumor response and survival in preclinical models when added to chemotherapy and ICI combinations, further clinical exploration of trilaciclib plus chemotherapy or chemotherapy/ICI combinations in tumors that are sensitive to immunotherapies is warranted.