Immune checkpoint blockade and CAR-T cell therapy in hematologic malignancies

PD-L1 expression in murine leukemia cell line C1498 was upregulated in vivo, and blocking PD-L1/PD-1 pathway resulted in decreased AML burden and longer survival time [22]. In myelodysplastic syndromes (MDS) and AML patient samples, PD-L1 was detectable (> 2% PD-L1⁺ cells) in 100% of patients with common expression on non-tumor hematopoietic cells, while PD-L2 expression was largely absent [23]. PD-L1 expression on AML cells is significantly higher in the relapse setting than at the newly diagnosed stage [24, 25]. In BM aspirates from patients with TP53 mutation, PD-L1 positivity was more frequently noted [25]. Higher PD-L1 expression level was positively correlated with poor risk cytogenetic and molecular abnormalities [25, 26]. In a similar manner to solid tumor, interferon-γ (IFN-γ) induced PD-L1 expression on AML cells protected them from cytotoxic T cell lysis [27]. In BM aspirates from AML patients, T cell subsets, such as CD4⁺ effector T cells, CD8⁺ T cells, and Tregs, had significantly higher PD-1 expression in untreated and relapsed AML patients compared with healthy donors [28]. PD-1 expression on CD4⁺ and CD8⁺ T cells was upregulated at relapse after allogeneic stem cell transplantation (allo-SCT) [29]. In peripheral blood of patients with chronic myeloid leukemia (CML), PD-1 expression on CD8⁺ T cells was higher in comparison with healthy donors. In CML mouse model, PD-1 was highly expressed on CML-specific cytotoxic T cells, while PD-L1 expression was higher in blast crisis CML (bcCML) than chronic phase CML (cpCML), indicating that CML cells utilized PD-L1 to avoid immune surveillance. PD-1-deficient mice with bcCML survived significantly longer than wild type mice, suggesting that myeloid leukemia cells impaired host immune responses via PD-L1/PD-1 pathway [30]. Hence, the upregulation of PD-L1 on MDS/AML cells leads to immune escape and supports the potential benefit of using PDL-1/PD-1 inhibitors to treat MDS/AML.

Single agent nivolumab (humanized anti-PD-1 IgG4 mAb) as maintenance therapy demonstrated a complete remission (CR) rate of 71% in 14 transplant ineligible patients with high-risk features including adverse cytogenetics, treatment-related AML, and history of prior relapse (Table 1) [33]. Early results of pembrolizumab (humanized anti-PD-1 IgG4 mAb) plus cytarabine yielded CR rate of 35% and minimal residual disease (MRD)-negative remission in 56% of patients (Table 1) [34]. Idarubicin plus cytarabine and nivolumab in newly diagnosed AML reported complete remission or complete remission with incomplete count recovery (CR/CRi) in 34 of 42 patients and MRD-negative remission in 18 patients. Furthermore, the median relapse-free survival for the complete responders was 18.5 months. The risk of graft versus host disease (GvHD) was not significantly elevated in the 18 patients who proceeded to allo-SCT. Interestingly, baseline BM analysis of those who achieved CR/CRi had a higher frequency of CD3⁺ T cell infiltrate as compared to non-responders who had higher number of CD4⁺ effector T cells co-expressing markers of an exhausted phenotype (Table 1) [35, 36]. While the use of nivolumab and ipilimumab (humanized anti-CTLA-4 IgG1 mAb) in the post allo-SCT relapse setting in hematologic malignancies has demonstrated potent anti-tumor effects, significant immune-related adverse events (irAE) have also been reported (Table 1) [31, 49, 50]. Ipilimumab use with various hematologic malignancies resulted in significant irAE including 1 death, GvHD leading to drug discontinuation in 4 patients, pneumonitis and colitis [31]. In addition, fatal acute respiratory distress syndrome (ARDS), antiphospholipid syndrome, fatal acute grade 3 GvHD, and worsening of chronic GvHD were reported with nivolumab use in two other clinical trials [49, 50]. These data highlight a need for caution of T cell-mediated GvHD when treating patients with ICB after allo-SCT. Mechanistically, one study has demonstrated that persistent expression of PD-L1 by parenchymal cells reduces the proliferation of donor-derived CD8⁺ T cells in GvHD target tissues, leading to amelioration of GvHD in a mouse model [51]. Another group has showed similar result that elevated levels of PD-L1 from organ-specific microenvironments (e.g., lymph nodes) dampen cytotoxic T lymphocyte (CTL)-mediated GvHD after allo-SCT [52]. Reduced CTL activity in lymph nodes, however, also contributed to local tumor escape, which could be reversed by anti-PD-1 blockade [52]. It would be important to balance the possible risk of exacerbating GvHD and achieving maximum tumor killing.

PD-L1/PD-L2 expression is increased on HL cell lines and malignant Reed Sternberg (RS) in classical HL (cHL), due to upregulation and amplification of 9p24.1 JAK and MEK/ERK signaling [53, 54]. Although cHL does not have a high mutational burden, a necessary biomarker predicting responses to ICB, high frequency of PD-L1/PD-L2/PD-1/JAK2 genetic alterations in RS cells and high proportion of PD-1⁺ TILs determine sensitivity to PD-L1/PD-1 inhibitors [55, 56]. Receptor PD-1 was markedly increased on TILs as well as peripheral T cells of HL patients [55, 57]. Functionally, mAb targeting PD-L1 was able to inhibit tyrosine phosphorylation of SHP-2 and restore the production of IFN-γ by tumor-infiltrating T cells [57]. Within the tumor microenvironment (TME) of cHL, PD-1 and PD-L1 were elevated on natural killer (NK) cells and tumor-associated macrophages (TAMs), respectively. As expected, PD-1 inhibition reactivated both T and NK cells by blocking interactions between PD-1⁺ T/NK cells and PD-[39]L1⁺ malignant B cells/TAMs [58]. In addition, expanded numbers of CD4⁺PD-1⁻ Th1-polarized Tregs and PD-1⁺ differentiated T effectors were observed within the TME of cHL, where these cells might utilize PD-L1/PD-1 pathway to exert complementary mechanisms to suppress host anti-tumor immune responses [59].

Clinically, both pembrolizumab and nivolumab showed favorable responses and acceptable safety profile in patients with cHL that has relapsed or progressed after autologous stem cell transplantation (auto-SCT) and brentuximab vedotin (BV), leading to their approval in 2016 by US FDA. The phase I clinical trials, KEYNOTE-013 with pembrolizumab and CheckMate 039 with nivolumab, produced overall response rates (ORRs) of 65% (CR 21%) and 87% (CR 17%) in relapsed and refractory (RR) HL, respectively (Table 1) [37, 38, 43]. CheckMate-205, the phase II multi-cohort study of 243 patients with BV naïve-cohort A, BV after auto-SCT cohort B, and BV before and after auto-SCT cohort C, demonstrated ORR of 69% and a median duration of response (DOR) of 16.6 months (Table 1) [41]. Correlative studies of 45 available tumor samples showed concordant alteration of the PD-L1 and PD-L2 loci in the RS cells. Fluorescence in situ hybridization of the RS cells showed 26 cases with copy gain of PD-L1/PD-L2, 12 cases with PD-L1/PD-L2 amplification, and 7 cases with polysomy 9. Furthermore, complete responders had higher PD-L1 than non-responders [42]. Similarly, KEYNOTE-087, the multi-cohort phase II trial with pembrolizumab monotherapy in RR HL patients who progressed after auto-SCT and subsequent BV therapy (cohort 1), salvage chemotherapy and BV (cohort 2), or auto-SCT but no BV (cohort 3), demonstrated ORR of 72% and CR rate of 28% with a median DOR of 11.1 months (Table 1) [45, 46]. Combination therapy of ipilimumab plus nivolumab has also shown efficacy with ORR of 74% in HL (CheckMate 039, Table 1) [40].

Nivolumab plus BV produced ORR of 82% and CR rate of 61% as the first-line salvage therapy (Table 1) [47]. ECOG-ACRIN E4412 study of nivolumab, ipilimumab, and BV demonstrated ORR of 82% (18/22), with a CR rate of 68% (15/22) (Table 1) [48]. Nivolumab followed by treatment with adriamycin, bleomycin, vinblastine, and dacarbazine (ABVD) for patients at high risk of relapse (NCT03033914) and pembrolizumab for patients unsuitable for ABVD (PLIMATH NCT03331731) are being explored in the first-line setting for HL. Pembrolizumab (NCT02684292) and nivolumab (CheckMate-812 NCT03138499) with or without BV are being evaluated in phase III clinical trials in the relapsed setting as well (Table 1).

In contrast to HL, PD-L1 expression in NHL is markedly heterogeneous. Of two distinct clinical subtypes of DLBCL, PD-L1 expression was rarely detected in germinal center B cell-like (GCB) subtype, while 57% of activated B cell-like DLBCL samples were PD-L1 positive [60]. Other studies showed similar low expression of surface PD-L1 and soluble PD-L1, and the surface PD-L1 expression was positively associated with the number of PD-1⁺ TILs and inversely correlated with the number of Tregs in GCB-DLBCL [61, 62]. In a small number of follicular lymphoma (FL) patients, PD-L1 expression was high [63]. PD-1 expression on TILs of FL was abundant but with complicated expression patterns: many cell types, including CD4⁺ Th1 cells, CD8⁺ cytotoxic T cells, and Tregs, expressed PD-1 [64]. In CLL, histiocytes, not tumor cells, were the main source of PD-L1 expression within the TME [65]. Low numbers of PD-1⁺ TILs were observed, which had controversial association results among different contexts [56]. One study has shown that PD-1 expression was increased on CD4⁺ and CD8⁺ T cells, and the crosstalk between PD-L1 on CLL and PD-1 on CD8⁺ T cells resulted in decreased IFN-γ production [66].

Although PD-L1 expression is heterogeneous among MM patients, many studies have shown that PD-L1 expression is limited to malignant plasma cells (PCs), and PD-L1 overexpression is associated with increased risk of progression from smoldering multiple myeloma to MM [62, 67]. However, other groups detected very low PD-L1 expression on normal PCs and did not observe significant upregulation of PD-L1 on malignant PCs from MM patients, which could explain why nivolumab monotherapy and in combination with ipilimumab had no significant therapeutic activity in a phase I CheckMate-039 study treating RR MM patients [68]. PD-1 was upregulated on CD4⁺ T cells, CD8⁺ T cells, and NK cells within the BM of MM [68‐70]. PD-1⁺ NK and T cells were less proliferative and cytotoxic, which could be reversed by anti-PD-L1/PD-1 blocking in vitro and in vivo [68, 70]. Furthermore, lenalidomide, an immunomodulatory drug (IMiD), reduced expression of PD-1 on T and NK cells and downregulated PD-L1 expression on PCs and myeloid-derived suppressive cells (MDSCs) [67, 69, 71]. As a result, combined blockade of PD-L1/PD-1 and lenalidomide enhanced granzyme B and IFN-γ production by T and NK cells and inhibited MDSC-mediated MM progression [67, 71].

Pembrolizumab is approved for RR primary mediastinal B cell lymphoma (PMBCL) based on ORRs of 48% (CR 31%) in KEYNOTE-13 and ORRs of 45% in phase II, KEYNOTE-170 studies (Table 1) [44]. CheckMate-039 also evaluated the efficacy of single agent nivolumab in NHL and demonstrated ORRs of 40% in FL, 36% in DLBCL, 15% in mycosis fungoides, and 40% in peripheral T cell lymphoma (PTCL) (Table 1) [39]. Furthermore, the nivolumab plus ipilimumab cohort of CheckMate-039 reported ORRs of 20% in FL/DLBCL and 9% in transplant-naïve T-NHL patients in 65 patients who had a median number of 4 prior therapies [40]. Nivolumab monotherapy in patients with RR DLBCL who were ineligible for auto-SCT and those with post auto-SCT relapse produced ORRs of 3% and 10%, respectively. Furthermore, median progression-free survival (PFS) and OS were 1.9 and 12.2 months in the post auto-SCT relapse cohort and 1.4 months and 5.8 months in the auto-SCT ineligible group, respectively [72]. Similarly, pembrolizumab maintenance in post auto-SCT chemosensitive patients failed to meet its primary end point as well [73]. In RR FL, pembrolizumab plus rituximab (chimeric anti-CD20 IgG1 mAb) showed ORR of 67% (CR 50%, PR 17%) in patients and a median PFS of 11.4 months. Interestingly, PDL-1 expression was not associated with response [74]. Nivolumab plus ibrutinib demonstrated responses 61% in patients with high-risk CLL/small lymphocytic leukemia (SLL), 33% with FL, 36% with DLBCL, and 65% of patients with Richter’s transformation [75]

Unlike single agent PD-1 blockade that produced minimal responses in RR MM, the combination of PD-1 inhibition with IMiDs was efficacious and produced ORRs of 50–60% [76]. Pembrolizumab monotherapy in patients who did not achieve CR prior to transplant produced a CR rate of 31% and MRD-negative rate of 41% [77]. In addition, pembrolizumab plus lenalidomide and dexamethasone in high-risk patients within 3–6 months of auto-SCT led to stringent CR in 33% patients and 4 patients achieving MRD-negative remission [78]. Despite the obvious preclinical anti-tumor effects of PD-1 blockade and positive results from earlier trials in MM, phase III clinical trials involving combination therapy of PD-1 blockade with IMiDs were placed on hold by the FDA in 2017 due to increased rate of adverse effects observed in KEYNOTE-183 (NCT02576977) and KEYNOTE-185 (NCT02579863) [79]. The pembrolizumab arm in KEYNOTE-183 (pembrolizumab plus pomalidomide and low-dose dexamethasone) experienced myocarditis, hepatitis, Steven Johnson syndrome, hyperthyroidism, pneumonitis, and 2 treatment-related deaths [79]. KEYNOTE-185 (lenalidomide plus pembrolizumab plus low-dose dexamethasone) reported 6 treatment-related deaths, with 4 being due to either cardiac arrest (1), pneumonia (1), myocarditis (1), and cardiac failure [79].

Several ongoing trials are assessing the combination of PD-1 or PD-L1 inhibition with conventional chemotherapy in untreated DLBCL (NCT 03003520) or as consolidation therapy in NHL (NCT03620578) (Table 2). The JAVELIN study (NCT 02951156) is a phase Ib trial assessing efficacy and safety of immunotherapy-based regimens containing avelumab (human anti-PD-L1 IgG1 antibody) in combination with utomilumab (4-1BB agonist), azacytidine (AZA), rituximab, and/or conventional chemotherapy in patients with RR DLBCL (Table 2) [80].

Currently, bispecific antibodies, which recruit patient’s T cells or NK cells against cancer cells expressing tumor-associated antigens, have been attracting attention for treating hematologic malignancies. A typical example is CD33/CD3 bispecific T cell engager (BiTE). Given that CD33 is overexpressed in AML blasts, a BiTE antibody against both CD3 and CD33 has been developed to recruit T cells to kill CD33⁺ AML cells [115]. Similarly, bispecific antibody targeting both CD3 and CD123 has been designed as CD123 is overexpressed in a wide range of hematologic malignancies, particularly on LSCs [116, 117]. However, ongoing clinical trials have showed that only a small fraction of patients could benefit from bispecific antibody treatment. A major mechanism limiting the therapeutic efficacy is due to T cell anergy and exhaustion driven by inhibitory immune checkpoint pathways, such as PD-L1/PD-1 axis [118]. For example, T cells recruited to CD33-positive cells showed impaired cytotoxicity due to high expression of PD-L1 on AML cells, which was induced by CD33/CD3 BiTE antibody treatment. Inspired by the inhibitory role of PD-L1/PD-1 pathway in AML, combining PD-L1/PD-1 blockade with CD33/CD3 BiTE antibody showed enhanced T cell proliferation and IFN-γ production [119].

The expression of PD-L1, PD-L2, PD-1, and CTLA-4 was upregulated in a cohort of MDS, CMML, and AML patients treated with epigenetic therapy, suggesting inhibitory immune checkpoint signaling pathways might be involved in hypomethylating agent (HMA) resistance [13]. HMAs triggered demethylation of the PD-1 promoter leading to increased expression of PD-1 on T cells, which promoted exhaustion of tumor-specific T cells and therefore resulting in immune escape [32]. Therapeutically, number of ongoing clinical trials have been designed to combine HMAs with ICB (Tables 1 and 2). Notably, AZA plus nivolumab showed better OS (16.1 months vs 4.1 months) and better ORR (33% vs 20%) in heavily treated RR AML patients compared to a historical cohort with AZA-based salvage therapy. A second cohort in this trial treated with nivolumab and ipilimumab plus AZA led to 6 of 14 patients achieving CR/CRi [32]. Responders had a progressive increase of CD4⁺ and CD8⁺ TILs in the BM, demonstrating that AML patients could benefit from PD-1 blocking therapy. Furthermore, CTLA-4⁺CD8⁺ cell numbers were increased in both responders and non-responders, indicating a dual combination of PD-1 blockade and CTLA-4 blockade with AZA might be able to further improve response rates [32].

Cytokines like IFN-α was approved for the treatment of hairy cell leukemia in 1986 and IL-2 for the treatment of metastatic renal cell carcinoma (1992) and advanced melanoma (1998) [120]. Although being one of the first forays in immunotherapy, nowadays, cytokine therapy is mainly used in combination with other anti-tumor treatments. For example, recently bempegaldesleukin (NKTR-214), an IL2Rβ (CD122)-biased agonist, has shown capabilities of enhancing the proliferation and activation of CD8⁺ T cells and NK cells without increasing the number of Tregs [121]. Results of PIVOT-02 trial, combination of NKTR-214 and nivolumab, has shown that this combination is safe and efficacious (ORR 48% in 23 patients) in metastatic urothelial carcinoma [122]. Aside from IL-2, IL-15 has also been evaluated in stimulating NK cells and T cells. Combination therapy with IL-15 and blocking antibodies against PD-1 and CTLA-4 has been shown to synergistically activate T cells and prolong the survival of tumor-bearing mice [123]. In addition, a recent study has demonstrated that DC-derived IL-12 is necessary for successful anti-PD-1 cancer therapy, suggesting that IL-12 and PD-1 blockade could be rationally combined [124]. In an earlier study, synergistic effects were observed when tumor-bearing mice were treated with Semliki Forest virus-based vector encoding IL-12 and anti-PD-L1 mAb [125]. Currently, there are limited pre-clinical and clinical trials based on the combination of ICB and cytokine therapy in hematologic malignancies although much more trails are ongoing in solid tumors.