20210921期 PD1 blockade enhances ICAM1-directed CAR T Efficacy

Clinical Cancer Research September 4, 2020

DOI: 10.1158/1078-0432.

Background:
  Thyroid cancer is a common malignancy with increasing incidence. As tumors quire increasing mutational burden, they under go progression from well-differentiated papillary thyroid cancer (WDPTC) to poorly differentiated thyroid cancer (PDTC) and finally anaplastic thyroid cancer (ATC). Patients with PDTC and ATC have disease-specific mortality rates that range between 40–100%, and these radioiodine-refractory tumors generally have a poor response to known systemic therapies. Monotherapies for PDTC/ATC targeting specific oncogenic molecules, such as BRAF and other kinase inhibitors, have also shown limited response inclinical trials. Recently, the FDA approved the combination treatment with inhibitors of BRAF and MEK for metastatic BRAF-mutant ATC. However, since the BRAF mutation (V600E) occurs in less than 30% of ATCs, the majority of ATC patients will not benefit from this combination therapy.
  Recently, blockades of the immunosuppressive interaction between programmed cell death protein 1 (PD1) and its ligand (PD-L1) have generated considerable interest within the solid tumor field due to durable response rates observed in melanoma and lung cancer. Monoclonal antibodies that target PD1 expressed by T cells improve the function of exhausted T cells by blocking PD1 binding to PD-L1 that is expressed in the tumor. Histopathologic reports show that PD-L1 and PD1 proteins are upregulated in PDTC and ATC tumors and tumor infiltrating lymphocytes, respectively, suggesting a potential opportunity for immune checkpoint therapy in advanced thyroid tumors. However, the initial reports of pembrolizumab (anti-PD1) monotherapy to treat advanced thyroid cancer have been disappointing, resulted in a response rate of less than 10% of the phase I clinical trials. Another clinical study using the anti-PD1 antibody, spartalizumab, reported a 17% overall response rate in ATC patients. Therefore, there is still a clear need to broaden treatment options.
  Previously, the authors group demonstrated that intercellular adhesion molecule 1 (ICAM1, CD54), a cell surface glycoprotein with cytokine-inducible expression and known roles related to T lymphocyte trafficking in the endothelium, displays high levels of membranous expression in aggressive PTC, PDTC, and ATC malignant cells. They have also shown that human T cells transduced with a lentiviral vector encoding a third-generation chimeric antigen receptor (CAR) construct targeting ICAM1 (either via a single-chain fragment variable (scFv) derived from the R6.5 monoclonal antibody or an affinity-tuned natural ligand inserted (I) domain derived from integrin called lymphocyte function-associated antigen (LFA)-1)) selectively eradicated ATC cells in an ICAM1-dependent fashion in a preclinical murine model. Furthermore, these ICAM1-targeting CAR T (ICAM1-CAR T) cells can be tracked in vivo either optically by incorporation of Renilla luciferase or via PET/CT by incorporation of a radioligand-sensitive receptor, somatostatin receptor 2 (SSTR2), to examine kinetics of T cell distribution in response to tumor recognition.

  Despite promising initial results of CAR T therapy in a number of solid tumors, its efficacy in general has been limited against solid tumors when compared to hematologic malignancies in part due to the highly immunosuppressive tumor microenvironment of solid tumors. Multiple specific tumor escape mechanisms have been proposed, including upregulation of PD1 expression on tumor-infiltrating CAR T cells. PD1 belongs to the immunoglobulin superfamily, and its expression on T cells is upregulated upon interaction with ligands PD-L1 and PD-L2 (12). The engagement of PD1 with PD-L1 inhibits T cell-mediated effector functions, conferring tumor protection against immune-mediated destruction.

 

Methods:

The author examined the expression of PD-L1 (a ligand of PD1) and intercellular adhesion molecule 1 (ICAM1) in thyroid tumors and ATC cell lines, and investigated the PD1 expression level in peripheral T cells of thyroid cancer patients. Next, they studied the tumor targeting efficacy and T cell dynamics of mono- and  combination treatments of ICAM1-targeting CART cells and anti-PD1 antibody in a xenograft model of ATC.

 
Results:
1. PD-L1 and ICAM1 expression correlate with PDTC/ATC histology and aggressive features.
  To determine association between ICAM1 and PD-L1 expression in thyroid tumors, they examined both PD-L1 and ICAM1 via IHC in patients with WDPTC (n = 19) or PDTC/ATC (n = 14) (Table 1). Given the small number of PDTC samples and similarly aggressive clinical behavior between PDTC and ATC, they grouped these tumor types together and compared them to WDPTC for the statistical analysis. They used a 1% cutoff for positivity of PD-L1, a criteria previously used for melanoma and head and neck squamous cell carcinoma. Compared to WDPTC, the PDTC or ATC tumors were more likely to be positive for PD-L1, and PD-L1 positive tumors were associated with circumferential membranous ICAM1 staining (Table 1& Fig. 1A). They previously reported that PDTC or ATC tumors were significantly correlated with the circumferential ICAM1 expression pattern on the tumors, whereas apical ICAM1 staining has been observed in WDPTC tumors. Additionally, patients with PD-L1 positive tumors were more likely to be presented with locally advanced tumors and distant metastases. No significant association was observed, however, between PD-L1 expression and BRAF^V600E mutational status. The diffuse and rampant  expression of both ICAM1 and PD-L1 on aggressive thyroid tumors suggests that targeting both molecules may boost efficacy toward PDTC and ATC.

   IFNγ has been described as a major soluble factor that induces ICAM1 expression in PDTC and ATC cell lines. PD-L1 expression is also regulated by the IFNγ signaling pathway through JAK1 and JAK2. They therefore investigated whether ICAM1 and PD-L1 expression is regulated by a common pathway in ATC. Treatment of multiple established ATC cell lines (8505C, FRO, KHM-5M) and ATC patient-derived cells (JV) with exogenous IFNγ in vitro resulted in a significant increase in ICAM1 expression (up to 9-fold in MFI) compared to untreated controls (Fig.1B-1C). In 8505C and FRO, the percentages of ICAM1-positive cells increased significantly after IFNγ treatment (Supplementary Fig. 1). Similarly, treatment with IFNγ significantly upregulated PD-L1 expression in the same ATC cells, albeit to a lesser extent than ICAM1 (up to 2-fold) except for JV cells where an 11-fold increase was observed. In JV cells, the frequency of PD-L1⁺ cells increased significantly relative to untreated cells (Supplementary Fig.1). Despite the quantitative differences in the level of IFNγ-induced ICAM1 and PD-L1 expression, the surface expression of both proteins was equally sensitive to co-treatment with the JAK2 inhibitor (JAK2i), AZD1480, which reduced expression back to basal levels (Fig. 1B-1C, Supplementary Fig. 1). In the absence of IFNγ stimulation, JAK2i did not alter either ICAM1 or PD-L1 expression, demonstrating that JAK2 is a crucial mediator of IFNγ triggered ICAM1 and PD-L1 expression but is not required for basal surface expression within unstimulated ATC cells. This finding is further corroborated by the induction patterns of mRNA encoding interferon regulatory factor 1 (IRF1), a critical transcription factor downstream of the JAK1/2 pathway within IFNγ treated ATC cells compared to unstimulated cells or co-treated with JAK2i (Fig. 1D). Therefore, an IFNγ signaling axis within ATC cells that requires JAK2 and drives IRF1 induction mediates expression of both ICAM1 and PD-L1. ICAM1 and PD-L1 are at least in part regulated by a common pathway in ATC, which is supported by the significant correlation between expression of both these proteins in de-differentiated thyroid cancer cells (Fig.1).

2. PD1 has increased expression on CD8+ PBMCs in patients with PDTC/ATC.

  Given the high expression of PD-L1 in PDTC/ATC tumors, they investigated whether increased PD-L1 expression in PDTC/ATC tumors is correlated with the level of PD1 expression in circulating T cells. Peripheral bloods isolated from patients (WDPTC, n = 8; PDTC/ATC, n = 7) and healthy donors (n = 5) were analyzed by flow cytometry for CD3, CD4, CD8, and PD1-positive T cells (Fig.2A). The prevalence of PD1+ cells amongst circulating CD8+T cells was low in both healthy volunteers and all patients with WDPTC (medianPD1+/CD8+ T cells 7%, interquartile range (IQR) 5% - 15%) compared to significantly elevated expression in patients with PDTC/ATC tumors (median PD1+/CD8+ T cells 54%, IQR 11% - 96%) (Fig. 2B). All patients with>40% PD1 expression on peripheral blood CD8+ T cells presented with lung metastasis at the time of diagnosis or recurrence. However, the percentage of circulating PD1+/CD8+ T cells from patient’s blood did not correlate with PD-L1 positivity in tumor (Fig. 2C), suggesting that the expression of PD1 in circulating T cells was more reflective of the degree of tumor aggressiveness rather than in situ PD-L1 expression in the tumor.

3. Co-administration of anti-PD1 antibody with ICAM1-targeted CAR T cells variably enhances in vitro cytotoxicity against ATC cell lines

  Next, they investigated the potential for checkpoint-mediated inhibition of ICAM1 targeting-CAR T therapy based on three key findings: (1) significant enrichment of PD-L1 on de-differentiated thyroid tumors; (2) the high level of PD1 expression in the peripheral blood of advanced thyroid cancer patients; (3) the common upstream signaling pathway of ICAM1 and PD-L1 induction in these tumors. To investigate this, they used primary CD3+ T cells derived from the peripheral blood of patients with thyroid tumors (ATC, PDTC, WDPTC) with varying degrees of differentiation and commercially available healthy donor leukopaks to generate CAR T cells. T cells were transduced with one of two ICAM1-directed CAR T constructs (F292A-CAR T (labeled as mAS) or R6.5-CAR T (RR), which were previously shown to enable efficient T cell-mediated lysis of ICAM1-expressing ATC cell lines. The established ATC cell line 8505C (intermediate ICAM1/high PD-L1), ATC patient-derived cell line JV (high ICAM1/intermediate PD-L1), and control cell lines, 293T (low ICAM1/lowPD-L1) and HeLa (high ICAM1/intermediate PD-L1) were chosen to represent various expression profiles of PD-L1 and ICAM1 (Fig. 3A). ATC cells had significantly lower surface expression of PD-L2 (PDCD1LG2), another ligand of PD1, compared to PD-L1 (Fig. 3A). The cytotoxic properties of ICAM1-CAR T cell batches generated with T cells from WDTC, PDPTC, or ATC patients as a representative of thyroid cancer with gradual loss of differentiation status were examined (Fig. 3B & Supplementary Table1). The baseline efficacy of ICAM1-CAR T cells was demonstrated by the lysis of approximately 50% of the 8505C cell line after co-culture at a 2.5:1 effector:target (E:T) ratio. The time equired to achieve 50% lysis varied between 18 to 48 hours depending on the donor blood used for CAR T manufacturing. To evaluate the effect of neutralizing PD1-signalling, the ability of ICAM-1 CAR T cells to lyse ATC cell lines in combination with anti-PD1 antibody treatment in vitro was compared to lysis mediated by ICAM-1 CAR T cells alone (Fig. 3C). Compared to ICAM1-CAR T alone, the cytotoxicity of ICAM1-CAR T cells generated with ATC and WDPTC patients was significantly enhanced when anti-PD1 antibody was supplemented to ATC target cell lines. Overall, two out of four patient donor ICAM1-CAR T cell batches showed significant improvement in their ability to eradicate co-cultured target ATC cells when anti-PD1 antibody was supplemented to the media (Supplementary Table 1). Addition of anti-PD1 antibody to non-transduced T (NT) cells did not affect cell behavior in the same settings. However, donor T cell PD1 expression level or donor thyroid tumor histopathological type (e.g. WDPTC vs. PDPTC and ATC) did not correlate with in vitro responders to anti-PD1 treatment (Supplementary Table 1).

4. Anti-PD1 therapy in combination with ICAM1-CAR T treatment confers a survival advantage over ICAM1-CAR T alone in a preclinical murine model.

  A metastatic model of human ATC was created by systemic engraftment of the 8505C-fLuc/GFP cell line into NSG mice. These mice were later treated with ICAM1-CAR T cells manufactured from three commercial donor leukopaks derived from healthy donors (CAR T-1, CAR T-2, CAR T-3) with different percentages of PD1-expressing CD8+T cells but similar CAR T transduction frequencies (Fig. 4A and Supplementary Table1). The CAR T manufacturing preserved the ratio of CD4:CD8 in CAR T cells of each donor leukopak (Fig. 4A and Supplementary Fig. 2A). The CAR T transduction frequencies in live T cells were determined by measuring the expression of the myc peptide tag that is fused to CAR at the N-terminal. The expression of SSTR2, which is co-expressed by a ribosome skipping sequence P2A within the CAR expression cassette was also confirmed to validate T cell trafficking by PET/CT. In vitro E:T experiments using CAR T cell batches manufactured from each of the three donors demonstrated cytotoxicity against target ATC cells (Fig. 4B). However, enhanced killing with anti-PD1 antibody was observed with CAR T-1 only. Next, the three CAR T cell batches were tested for activity in vivo with or without PD1 blockade. Mice were administered with CAR T cells five days after xenograft with 8505C cells; PD1 cohort additionally received twice-weekly injections of 150 µg of anti-PD1 antibody for 8 weeks. Whole body luminescence imaging showed that all three donor CAR T cell batches reduced the tumor burden drastically in treated animals compared to mice left untreated or treated with NT cells (Fig. 4C). However, when the rates of in vivo tumor luminescence reduction mediated by each donor CAR batch were compared, a disparate tumorlytic response was observed. Donor CAR T-1 cells, which displayed the highest level of PD1 expression and were responsive to anti-PD1 treatment in vitro (Fig. 4A), also exhibited an enhanced tumor lytic response when anti-PD1 antibody was administered together with CAR T cells: the CAR T-1 and anti-PD1 antibody combination demonstrated significantly enhanced tumor burden reduction when compared to the effect of CAR T-1 alone at 3-4 weeks post-xenograft, (P< 0.01) (Fig. 4C-D & Supplementary Table 1). Interestingly, CAR T-1 retained the highest percentage of CD62LlowCD4+ T cell subset population compared to other donor CAR T cells (Supplementary Fig. 2B). In non-small lungcancer patients, the frequency of CD62LlowCD4+ T cells has been correlated with long-term responders to anti-PD1 therapy. Overall, all mice receiving ICAM1-CAR T cells and anti-PD1 antibody together showed a significant survival benefit compared to mice treated with ICAM1-CAR T only (Fig. 4E). The additive benefit of anti-PD1 therapy to CAR T cells was more apparent in an in vivo condition with a long-term follow-up period. The combined treatment of ICAM1-CAR T cells and anti-PD1 antibody also showed improvement in survival in xenografts with KHM-5M, another ATC line with ICAM1 expression (Figure 1B),compared to treatment with ICAM1-CAR T cells alone (Supplementary Figure 3).

5.  Anti-PD1 antibody reduces the peak expansion of ICAM1-CAR T cells and facilitates subsequent contraction at the tumor site.

  ICAM1-specific CAR T cells co-express SSTR2, which upon injection of a positron-emitting SSTR2-specific tracer, 18F-NOTA-Octreotide (NOTAOCT), enables whole-body tracking and localization of CAR T cells by PET/CT (Fig. 5A). Previously, they able to detect multiple phases of CAR T cell activity in correlation with tumor clearance, characterized by rapid expansion and contraction of T cells at metastatic thyroid tumor sites in vivo. In the current study, the peak or near-peak expansion of ICAM1-targeting CAR T cells were detected in the lungs three weeks after tumor injection (Fig. 5A). To assess whether CAR T cell temporal kinetics was altered by anti-PD1 antibody, they quantified uptake of NOTAOCT tracers in 8505C-xenografted mice that were treated with different treatments (Fig. 5B). This analysis revealed that anti-PD1 antibody significantly reduced CAR T cell expansion at its peak (week 3-4) and contraction phase (week 5 post-xenograft). Lower expansion of CAR T cells in mice co-treated with anti-PD1 antibody was likely due to a faster elimination of tumor cells, avoiding a prolonged expansion of T cells against delayed clearance of tumor (Fig. 5B and 4C).

6. Anti-PD1 antibody restricts the activity and proliferation of ICAM1-CAR T cells at tumor cells.

  To examine the effect of anti-PD1 antibody on CAR T cells at the cellular levels, they then analyzed consecutive lung sections from 8505C xenografted mice by IHC. The number of tumor-infiltrating T cells with respect to tumor cells for the different treatment groups was compared using digital image analysis (Fig. 5C). The 3-week post xenograft (which is about 2-week post-CAR T treatment) was chosen as an approximate time point for maximal tumor lysis in both the CAR T alone and CAR T/anti-PD1 treatment groups. Lung tissue sections from 8505c-xenografted mice treated with CAR T cells demonstrated a marked reductionin tumor burden and increase in a number of T cell clusters (Fig. 5C-E). Dual treatment with anti-PD1 antibody and ICAM1-CAR T-1 cells revealed a further reduction in the number of tumor cells (Fig. 5E). Of the lung tumor sections that stained positive for tumor cells, specific co-localization of CD3+T cells and GFP+ tumor cells were observed both for CAR T with or without PD1. Close examination, however, revealed that PD1 treatment led to an increased number of T cells restricted to at and in the vicinity of the remaining GFP+ tumor cells compared to CAR T distribution without PD1 (Fig. 5E). This result may indicate that anti-PD1 antibody treatment curtailed excessive expansion of CAR T cells by augmenting cytotoxicity and proliferation capacity of CAR T cells limited to interaction with tumor cells. In comparison, xenografted mice treated with NT cells displayed the highest tumor burden as shown by the abundance of GFP+ tumor cells remaining in the lung (Fig. 5C-E). The additional administration of anti-PD1 antibody tomice treated with NT cells significantly elevated the density of T cells in the lung; however, T cell expansion was found to be non-specific to T cell interaction with tumor cells (Fig. 5D).

7. Anti-PD1 antibody relieves the inhibitory effects of PD1 induction in CAR T cells.

  As the addition of anti-PD1 antibody improves ICAM1-directed tumor lysis by CAR T in vitro and in vivo, they investigated whether this benefit is specific to the activity of the CAR T cells but not to non-tumor directed T cells. Compared to NT cells, PD1 expression was significantly induced in ICAM1-CAR T cells only when exposed to ICAM1-positive 8505C, JV, and HeLa cells compared to ICAM1-negative 293T cells (Fig. 6A-B). JAK2i abrogated PD1 induction in ICAM1-CAR T cells, implicating that PD1 is induced through the IFNγ-JAK2 signaling pathway. They further investigated PD1 expression in xenografts treated with ICAM1-CAR T-1 cells. In border to enumerate the density of tumor-infiltrating T cells, we analyzed the same high density tumor areas for PD1 and CD3 in the consecutive slides of the lung sections. At 3-week post-xenograft (2-week post-CAR T injection), the number of PD1+ T cells was significantly increased in the CAR T cohort relative to NT treatment group (Fig. 6C). The fraction of PD1+T cells was approximately 17% of CD3+ T cells, whereas 1% of NT cells were PD1+ (Treatment cohorts with anti-PD1 antibody were not analyzed for PD1+ due to epitope overlap or steric hindrance between the treatment antibody and IHC antibody. In contrast, they found that the PD-L1+cell density was significantly reduced in CAR T-treated mice compared to NT-treated mice (Fig. 6D). The majority of the remaining GFP+ tumor cells in CAR T-treatment group were also PD-L1+, whereas combination treatment with CAR T cells and anti-PD1 antibody eliminated most PD-L1 high cluster of tumor cells (Fig. 6D).