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

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.

Trial registration: ClinicalTrials.gov NCT02499770.

Keywords: clinical trials; drug evaluation; investigational; phase II as topic; preclinical; therapies.

Figure 1

Addition of trilaciclib to oxaliplatin and αPD-L1 combination therapy, and other multiple chemotherapy and ICI combinations, led to enhanced antitumor activity and durability of response in MC38 and CT26 tumor models. (A) Three dosing schedules were initially tested in the MC38 model. The induction plus maintenance schedule was carried forward in all other experiments in both the MC38 and CT26 models with the substitution of 5-FU for oxaliplatin, or anti-PD-1 for anti-PD-L1 as indicated. (B–D) Tumor growth and overall survival of MC38 and CT26 mice treated with chemotherapy/ICI ±trilaciclib combination therapy (n=10–15 per treatment group). *P≤0.05. 5-FU, 5-fluorouracil; αPD-L1, anti-programmed death-ligand-1; I, induction; ICI, immune checkpoint inhibitor; IM, induction plus maintenance; M, maintenance; OP, oxaliplatin plus anti-programmed death-ligand-1; TOP, trilaciclib plus oxaliplatin and anti-programmed death-ligand-1.

Figure 2

Addition of trilaciclib to OP treatment combination resulted in transient proliferation arrest followed by a faster recovery of CD8⁺ and CD4⁺ T cells compared with Tregs. (A) Baseline percent proliferation status of immune cell populations in spleen and tumors in MC38 tumor-bearing mice (n=16 biological replicates), and proliferation of intratumor lymphoid and myeloid immune cell types at (B) 6–24 hours (n=4 biological replicates) and (C) days 2, 4 and 7 (n=4 or 5 biological replicates) after trilaciclib treatment. Percent proliferation was defined as proportion of EdU⁺ cells, and relative proliferation was defined as (% EdU⁺ cells in trilaciclib-treated samples)/(% EdU⁺ cells in vehicle-treated samples) x 100. Each biological replicate consists of a pool of 3 animals. (D) Proliferation of CD8⁺ T cells in coculture with trilaciclib-treated Tregs (n=3 independent experiments, each performed with three biological replicates per culture condition). Data represent mean±SD. *p<0.05; **p<0.01. EdU, 5-ethynyl-2ʼ-deoxyuridine; gMDSC, granulocytic myeloid-derived suppressor cell; mMDSC, monocytic myeloid-derived suppressor cell; NK, natural killer; OP, oxaliplatin plus anti-programmed death-ligand-1; TOP, trilaciclib plus oxaliplatin and anti-programmed death-ligand-1; Treg, T-regulatory cell.

Figure 3

Transient G1 arrest led to changes in the proportion of intratumor T-cell subsets favoring effector T-cell function. (A) Flow cytometric analysis of intratumor CD8⁺ T cells, CD4⁺ T cells, myeloid cell types (macrophages, mMDSC and gMDSC populations) and Tregs; (B) proportion of Treg in total tumor or spleen CD4⁺ T cells and (C) ratio of CD8⁺ T cells to Tregs (% CD8⁺ T cells/% Tregs) in the CD45⁺ population or spleen (n=5–8 tumors analyzed per treatment group and time point); and (D) proportion of activated (% CD69⁺) cells in CD8⁺ or CD4⁺ T cells. *P<0.05. gMDSC, granulocytic myeloid-derived suppressor cell; mMDSC, monocytic myeloid-derived suppressor cell; OP, oxaliplatin plus anti-programmed death-ligand-1; TOP, trilaciclib plus oxaliplatin and anti-programmed death-ligand-1; Treg, T-regulatory cell.

Figure 4

Trilaciclib in combination with chemotherapy and ICI enhanced the gene signature of cytotoxic antitumor response. (A) Between-group comparison of the geometric mean of log2-normalized transcript levels of genes upregulated in either OP or TOP versus vehicle associated with each KEGG pathway and (B) associated heat maps showing mean log2 transcript levels of each gene in the pathway; and (C) T-cell repertoire analyses of MC38 tumors from OP-treated or TOP-treated mice (n=5 per treatment group) on day 17, presented as either clonality score and proportion of T cells in tumor (T-cell fraction; left panel), or median clonality score and TIL fraction (right panel). Error bars denote SE, and dotted lines represent the median value for TIL fraction and clonality score. *P<0.05. For the heat map analyses, p<0.01 for comparison of the difference in gene expression levels in TOP versus OP and trilaciclib versus vehicle groups on day 17, for all three KEGG pathways. Tumor clonality was significantly higher for comparison of TOP versus vehicle (p=0.024), but not OP versus vehicle, while the TIL fraction was significantly elevated in both OP and TOP versus vehicle comparisons (p=0.006). ICI, immune checkpoint inhibitor; IFN, interferon; KEGG, Kyoto Encyclopedia of Genes and Genomes; NK, natural killer; OP, oxaliplatin plus anti-programmed death-ligand-1; TCR, T-cell receptor; TIL, tumor-infiltrating lymphocyte; TOP, trilaciclib plus oxaliplatin and anti-programmed death-ligand-1; Treg, T-regulatory cell.

Figure 5

Addition of trilaciclib to E/P preserved and enhanced lymphocyte counts and function in patients with SCLC. (A) Mean absolute cell count (cells/µL) for B-cell and T-cell subsets and activated CD8⁺ T cells (CD3⁺CD8⁺CD38⁺HLA-DR⁺), and ratio of absolute whole blood cell counts for CD8⁺ T cells to Tregs (CD3⁺CD4⁺CD25⁺CD127^low), at the indicated time points. Error bars represent 95% CIs. (B) Per-patient T-cell repertoire analysis comparing mean ratio of clonality versus pretreatment level (C1D1) and number of expanded T-cell clones at the indicated time points. Bars represent median and IQR. (C) Proportion of patients with high (≥median) number of expanded T-cell clones at C3D1 (≥15 clones) and C5D1 (≥17 clones). (D) Progression-free and overall survival analysis of all patients with low (<median) and high (≥median) numbers of expanded T-cell clones on C3D1 and C5D1. *P=0.0098. C1D1, cycle 1 day 1; C3D1, cycle 3 day 1; C5D1, cycle 5 day 1; D, day; E/P, etoposide and carboplatin; T/E/P, trilaciclib prior to etoposide and carboplatin; PTV, post-treatment visit; SCLC, small cell lung cancer; Treg, T-regulatory cell.