Keywords

1 Introduction

Aberrant PI3K signalling is known to drive cancer progression and common cancers often comprise mutations in PIK3CA and loss of PTEN function (reviewed in Thorpe et al. (2015)). A huge effort has been made to develop inhibitors of three broad types; dual PI3K-mTOR inhibitors, pan-class I inhibitors and isoform-specific inhibitors, developed to directly inhibit cancer cell proliferation and survival (reviewed in Janku et al. (2018)), with several currently undergoing testing in clinical studies either alone or in combination with other therapies (Fig. 1). Whilst there is extensive data indicating cancer cell-intrinsic effects of inhibiting PI3K signalling, it is becoming increasingly clear that indirect effects of inhibiting the pathway also contribute to control of tumour growth.

Fig. 1
It depicts a schematic diagram of PI3K isoform-specific inhibitors. It also depicts Gain of function mutation, P13Ka Alpelisib Serabelisib Inavolisib.

PI3K isoform-specific inhibitors. Schematic of the PI3K isoform-specific inhibitors currently in various stages of development and their tumour-specific targets

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Immunotherapy as a cancer-targeting approach dates back to 1891 when William B. Coley first sought to harness the power of the immune system to control solid tumours (Kienle 2012). The administration of Coley’s toxins, a bacterial vaccine, designed to induce a large infection at the tumour site, resulted in clearance of both the infection and regression of the tumour in a proportion of patients. Despite these striking results, the lack of understanding at the time of how the immune system may control cancer, led to these first immunotherapy approaches falling from favour, as surgical, chemotherapy and radiotherapy treatments began to significantly improve cancer outcomes.

The field of immunotherapy has been revived in recent years, with the advent of new treatment strategies such as immune checkpoint blockade (ICB), adoptive transfer T cell therapy (ACT) and tumour vaccine therapy (Reviewed in Waldman et al. 2020). These therapeutic modalities are designed to reinvigorate the anti-tumour immune response, by either directly targeting the T cells themselves, to improve their anti-tumour response, or indirectly, by targeting immuno-suppressive populations, such as regulatory T cells (Tregs), tumour-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs), within the tumour microenvironment that typically subdues the T cell response to cancer. Whilst immunotherapy can result in striking control of tumour burden, the number of patients who successfully respond to these therapies remains small. Typically, patients fail to mount a sufficient response to therapy as a consequence of T cells becoming exhausted and dysfunctional prior to or during treatment. As the scientific field continues to elucidate the impediments to T cell anti-tumour responses it is becoming clear that different approaches are still needed, and that a combination of therapies may offer the greatest potential for tumour control.

As discussed in previous chapters, the different PI3K isoforms have critical roles in the signalling of T cells, B cells and innate cells of myeloid origin, such as macrophages. Given the success and tolerability of isoform-specific inhibitors such as the PI3Kδ inhibitor, Idelalisib, for haematological malignancies (Yang et al. 2015), the focus of much research and drug development programmes in recent years has been to understand how other immune cell populations can be targeted via isoform-specific PI3K inhibitors.

Herein, we firstly discuss the data surrounding PI3K isoform-specific inhibition, through genetic inactivation and pharmacological blockade, to target immune cell populations within the tumour microenvironment in preclinical models (Fig. 1). Finally, we discuss the data reported from clinical trials to date, using different inhibitors in solid malignancies.

2 Immune Mechanisms of Action in Preclinical Studies

2.1 PI3Kδ Inhibitors as a Treg Targeted Therapy

FoxP3+ Tregs suppress CD8+ and conventional CD4+ T cells thereby helping to maintain tolerance and prevent autoimmunity. However, these mechanisms in the context of cancer can prevent the host from generating a successful anti-tumour T cell response. Shimizu et al. were the first to demonstrate that removal of regulatory T cells led to the development of potent anti-tumoural CD8+ T cell responses and control of tumour burden (Shimizu et al. 1999). However, complete depletion of regulatory T cells is not considered feasible as a clinical therapy due to the significant presentation of autoimmune side effects and so approaches to selectively inhibit Treg function have been sought. Due to the contrast in reliance on the PI3Kδ isoform for effector T cell and Treg signalling (discussed in Chap. 8 and by Ahmad et al. 2017), PI3Kδ inhibition has been suggested as an attractive approach for Treg-specific cancer immunotherapy.

Initial preclinical studies utilised a strain of mice with either a global or a Treg-specific inactivation of the PI3Kδ isoform (δD910A) to examine the effect that impaired T cell signalling had upon tumour growth. A seminal study by Ali and colleagues demonstrated that δD910A mice were better able to control tumour growth in a number of mouse tumour models (Ali et al. 2014). Adoptive transfer experiments of PI3Kδ-inactive Tregs into wild type tumour-bearing hosts established that the reduced immunosuppression mediated by δD910A Tregs was responsible for reduced tumour growth. The deletion of the CD8+ effector T cell population abrogated tumour control in δD910A mice, demonstrating that tumour control is dependent on both the loss of Treg-mediated immunosuppression and the generation of a robust anti-tumour CD8+ T cell response as a consequence (Ali et al. 2014; Lim et al. 2018). The authors confirmed the therapeutic potential of PI3Kδ, by treating mice with a small molecule inhibitor of PI3Kδ, PI-3065 and demonstrated partial tumour control in both breast and pancreatic cancers in vivo. This proof-of-concept study in solid tumours has been strengthened by a number of other groups (Carnevalli et al. 2018; Lauder et al. 2020) who have sought to delineate the mechanism by which PI3Kδ inhibition confers tumour control either alone or in combination with other therapeutic modalities.

Idelalisib, also known as CAL-101 or GS-1101, an approved PI3Kδ inhibitor for haematological cancers (discussed in Chap. 23) and similar PI3Kδ inhibitors are being redeployed by several groups as a therapy designed to specifically target Tregs in solid malignancies. Ahmad and colleagues reported that CAL-101 blockade of PI3Kδ signalling in vitro was critical for Treg suppression and survival, whereas effector CD4+ T cells could utilise PI3Kα and PI3Kβ to maintain their function (Ahmad et al. 2017). Using the Treg-dependent tumour model, TC-1 (lung carcinoma), the authors demonstrated similar findings with modest control of tumour burden and partial improvement in survival time observed following CAL-101 treatment. However, when they boosted the antigen-specific response using an E7 tumour-specific vaccine in combination with CAL-101, they observed a significant reduction in tumour burden and improved long-term survival compared to either treatment strategy alone. Phenotypic analysis of the anti-tumour immune response in combination-treated animals demonstrated similar findings to the original study by Ali and colleagues (Ali et al. 2014), collectively pointing to a reduction in Treg response and the generation of a robust CD8+ T cell response is necessary for potent tumour control.

Lauder and colleagues expanded on previous studies and demonstrated that whilst all treated mice exhibited a level of tumour control following PI-3065 treatment, there was a dichotomy in the response to therapy, with complete tumour regression occurring in a small proportion of treated animals (Lauder et al. 2020, 2021). Detailed analysis of the anti-tumour immune response generated following PI-3065 treatment supported the previous studies that eradication of tumour burden was reliant on the dampening of the Treg response and the generation of a robust antigen-specific CD8+ T cell response. Whilst all treated mice had a reduced number of tumoural Tregs, those that exhibited only partial control developed a pool of dysfunctional Tregs characterised by increased Ki67, CD69 and LAG3 expression and a reduced number of tumour antigen-specific CD8+ T cells (Fig. 2). Combination treatment with PI-3065 and subsequent anti-LAG3 antibody therapy resulted in significant tumour control in all treated mice (Lauder et al. 2020). However, the greatest significance of the aforementioned study is that it highlighted the tumour-specific impediments to PI3Kδ inhibitors as a therapy. An essential requisite for tumour control was the development of an increased CD8+ T cell: Treg ratio in PI3Kδ responsive tumours. In the absence of an increased ratio, PI3Kδ unresponsive tumours, such as the MC38 colon cancer model, remained unresponsive despite treatment with a secondary immune checkpoint therapy (Lauder et al. 2020, 2021).

Fig. 2
It depicts three structures of Immune mechanisms of PI3K specific isoform action.

Immune mechanisms of PI3K specific isoform action. a Pharmacological blockade of PI3Kδ in vitro leads to the development of CD8+ T cells with superior anti-tumour activity when adoptively transferred into tumour-bearing hosts. b In vivo therapeutic targeting of PI3Kδ results in a reduced Treg: CD8+ T cell ratio and an enrichment in antigen-specific CD8+ T cells with improved anti-tumour function. c Specific targeting of PI3Kγ controls tumour growth by inducing immune gene signature switching of macrophages in vivo, from pro-tumoural to anti-tumoural

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In preclinical models that are resistant to Treg-specific inhibition such as MC38, genetic inactivation of PI3Kδ signalling has been reported to enhance tumour growth as a consequence of reduced CD8+ T cell function in vivo (Putz et al. 2012). These conflicting data indicate that simply targeting Tregs may not be sufficient in every tumour type. Indeed, combination immunotherapy approaches designed to target multiple tumour resident populations namely Tregs and TAMs were able to control tumour growth in the previously unresponsive MC38 model (Gyori et al. 2018). Dual PI3K isoform inhibitors offer the potential to target multiple cell types within the tumour microenvironment with a single treatment. Carnevalli et al. compared tumour control in PI3Kδ responsive (CT26 and 4T1) and unresponsive (MC38) tumours to either a PI3Kδ single inhibitor (PI-3065) or a dual PI3Kα/δ inhibitor (AZD8835) (Carnevalli et al. 2018). In all models, AZD8835 offered superior control of tumour growth and prolonged survival. Unlike the previously discussed studies where PI3Kδ isoform inhibitors were routinely administered daily for the duration of the study, the robust anti-tumour response reported with AZD8835 was a consequence of an intermittent dosing regimen, with 2 days on treatment/5 days off treatment. Although not as pronounced as the changes with continual PI-3065 treatment, this intermittent therapeutic approach resulted in a reduction in Tregs and increased CD8+ T cell: Treg ratio within the tumour. However, AZD8835 conferred enhanced transcriptional and phenotypic changes resulting in CD8+ T cells with increased expression of Ki67, CD25, Granzyme B, IFNγ and a reduced susceptibility to exhaustion as determined by reduced PD1 expression. Furthermore, the intermittent interruption of PI3K signalling in CD8+ T cells appears to promote an IL-2 autocrine signalling loop within the tumour that drives T cell effector function and survival, which ultimately supports tumour control (Carnevalli et al. 2018).

2.2 PI3Kδ Inhibitors as an Adjuvant to Improve T Cell Therapy in Cancer

The role of the PI3Kδ isoform in CD8+ T cell signalling during proliferation and effector functions are described in detail in Chap. 12. It is known that pharmacological inhibition of PI3Kδ skews activated CD8+ T cells to develop progeny with a self-renewing phenotype characterised by the expression of the transcription factor TCF1 (Lin et al. 2015; Nish et al. 2017). Several recent studies have demonstrated that the presence of CD8+TCF1+ stem-like T cells within the tumour microenvironment is associated with tumour control and successful responses to checkpoint therapy (Siddiqui et al. 2019; Kurtulus et al. 2019; Sade-Feldman et al. 2018; Baharom et al. 2021).

Using a preclinical model of melanoma, Bowers and colleagues demonstrated that expansion of CD8+ T cells in the presence of CAL-101 prior to adoptive cell transfer (ACT) into B16F10 tumour-bearing hosts, resulted in significantly reduced tumour burden and prolonged survival (Bowers et al. 2017). RNA-sequencing revealed that CAL-101-treated T cells had improved anti-tumour capacity driven by enhanced expression of TCF1 and a stem-like memory phenotype characterised by increased expression of CD62L, CD127 and CCR7 (Fig. 2). These findings have the capacity to significantly improve cellular immunotherapy approaches such as ACT or chimeric antigen receptor (CAR) T cell therapy, which despite their potential to reinvigorate the anti-tumour immune response, typically fail as a consequence of the transferred cells becoming exhausted and dysfunctional. The generation of human CAR-T cells in the presence of the PI3Kδ inhibitors TGR-1202 or CAL-101 resulted in cells with a less differentiated phenotype compared to untreated cells but with increased cytotoxic capacity in vitro (Dwyer et al. 2020). Such encouraging findings in the preclinical setting warrant the development of clinical trials in patients to determine if PI3Kδ blockade during the manufacture of CAR-T cells can result in a superior tumour control following transfer into patients.

PI3Kδ inhibitors have been also shown to reinvigorate the existing T cell population independently of ACT. Therapeutic administration of the PI3Kδ inhibitor, PI-3065, to mice bearing 4T1 breast tumours promoted the development of a population of stem-like memory T cells, identified by their expression of TCF1, that had superior anti-tumour capacity (Lauder et al. 2020).

2.3 Inhibition of Myeloid Populations Within the Tumour Microenvironment via Specific PI3K Isoform Inhibitors

As discussed in Chap. 6, cells of myeloid origins, such as macrophages, monocytes and neutrophils are reliant on the PI3Kγ isoform for signalling and downstream functions. Within solid tumours, TAMs and MDSCs are typically considered to elicit a pro-tumoural role as they mediate immunosuppression, promote angiogenesis and aid tumour invasion and metastasis (reviewed in Cassetta and Pollard 2018; Groth et al. 2019). High frequencies of TAMs and/or MDSCs within the tumour microenvironment are routinely associated with poor clinical prognosis in a number of cancers (Zhang et al. 2012). Using PI3Kγ specific blockade to target these populations and improve tumour outcomes has been widely studied in recent years with favourable outcomes at the preclinical stage.

Schmid and colleagues were the first to demonstrate that PI3Kγ promotes myeloid cell recruitment to a range of murine tumours driven by the expression of the integrin α4β1 (Schmid et al. 2011). This macrophage recruitment to tumours could be blocked through genetic inactivation (PI3Kγ−/−) or pharmacological inactivation with the PI3Kγ specific inhibitor, TG100-115. Consequently, reducing the infiltration of macrophages into the tumour significantly reduced tumour burden. Subsequent studies have sought to delineate further how PI3Kγ inhibition of TAMs contributes to tumour control. Using a mouse model of pancreatic ductal adenocarcinoma (PDAC), TG100-115 was employed to successfully control both primary tumour burden and metastasis (Kaneda et al. 2016a). Kaneda and colleagues demonstrated that inhibition of PI3Kγ altered the immune signature of TAMs, shifting them from an immune-suppressive phenotype by reducing arginase-1, TGFβ, IL-10 and PDGF-BB expression, to a pro-inflammatory phenotype by increasing IFNγ and IL-12. These phenotypic changes to the TAMs resulted in an elevated CD8+ T cell infiltrate into PDAC tumours that promoted tumour control. A parallel study by the same group demonstrated that inhibition of macrophage PI3Kγ promoted NFκB activation whilst impeding C/EBPβ activation. This alternation in transcriptional programme resulted in a switch away from the normal immune suppression driven by TAMS to an anti-tumour immune-stimulatory phenotype (Kaneda et al. 2016b). Whilst PI3Kγ inhibition does not directly target T cells, the switch to a pro-inflammatory phenotype in PI3Kγ-inhibited macrophages indirectly augments the development of a robust anti-tumour T cell response, characterised by increased CD8+ T cell cytotoxicity and Th1 responses driven by increased granzyme B and IFNγ expression and reduced IL-10 (Fig. 2). This enhanced anti-tumour T cell response increased sensitivity to immune checkpoint blockade, with combination therapy (TG100-115 and anti-PD1 antibodies) inducing significantly greater tumour regression (≥80% of treated mice) and long-term survival in comparison to either therapy alone. This sensitivity to immune checkpoint blockade was recapitulated in a study by De Henau and colleagues who demonstrated that high tumoural infiltration of MDSCs was associated with resistance to immunotherapies such as anti-PD1 and anti-CTLA4 antibodies in a number of preclinical models (Henau et al. 2016). Targeting the MDSCs using the specific PI3Kγ inhibitor, IPI-549, restored sensitivity to checkpoint immunotherapy and improved tumour control. Gene expression analysis revealed that IPI-549 treatment resulted in MDSCs with a reduced immune-suppressive phenotype that promoted CD8+ T cell infiltration and an increased CD8+ T cell: Treg ratio within the tumour.

As the studies by Kaneda and colleagues have indicated, not all macrophages within the TME are pro-tumoural. Macrophages with a pro-inflammatory phenotype are considered to be anti-tumoural and contentiously referred to as M1 macrophages whilst M2 macrophages elicit anti-inflammatory effects that promote tumour growth (reviewed in Mantovani et al. 2021). With the previously discussed studies demonstrating that PI3Kγ blockade could switch the transcriptional profile of TAMs from pro-tumoural to anti-tumoural, Lee and colleagues expanded these findings in colon cancer whereby TG100-115 treatment of mice bearing CT26 tumours resulted in a significant reduction in tumour growth (Lee et al. 2020). Analysis of the tumour microenvironment demonstrated an increased infiltration of M1 macrophages and a reduction in M2 macrophages. Retrospective analysis of a cohort of colorectal cancer patients found that patients with an increased ratio of M1 to M2 macrophages had significantly improved progression-free and overall survival (Lee et al. 2020).

Taken together these studies support the use of PI3Kγ-specific inhibitors as an immunotherapy to reduce both the total number of tumoural TAMS, but also to skew the resident TAM population in favour of M1 macrophages. As PI3Kγ inhibitors reach clinical trials, elucidating the immune contexture of different tumours will highlight the potential for combination therapy with other immunotherapies such as checkpoint inhibitors that could significantly potentiate the effect of either treatment alone.

Despite their preference for PI3Kγ signalling, TAMs and MDSCs can also be therapeutically targeted by blocking the PI3Kδ isoform. In vitro studies demonstrated that CSF-1-induced migration and degradation of the extracellular matrix by TAMs was reduced in the presence of the PI3Kδ inhibitor GS-1101 (Mouchemore et al. 2013). Furthermore, Ali et al. demonstrated that the MDSC expansion driven by the breast tumour cell line, 4T1, is significantly abrogated in D910A mice and the ex vivo capacity of D910A MDSCs to suppress CD8+ T cell proliferation is also potently reduced (Ali et al. 2014). We have found that the therapeutic administration of PI-3065 to 4T1 tumour mice, results in a significant reduction in peripheral expansion of MDSCs (unpublished findings, manuscript in preparation). Furthermore, genetic inactivation of PI3Kδ or oral administration of the PI3Kδ inhibitor IC87114 resulted in reduced recruitment of TAMs to the breast tumour microenvironment, conferring partial tumour control (Goulielmaki et al. 2018). A note of caution when considering these findings is that tumours can drive expansion of MDSCs, therefore, reduced MDSCs in treated animals may be an indirect effect of PI3Kδ inhibition resulting in better control of tumour growth rather than inhibition of PI3Kδ in MDSCs. In addition, Tregs can also drive MDSC expansion hence a reduction in MDSCs may reflect inhibition of Tregs rather than direct effects of the PI3Kδ inhibitor on MDSCs.

Oncolytic viral therapy is a novel cancer immunotherapy approach, whereby viruses are genetically manipulated to specifically target and kill the tumour (reviewed in Harrington et al. 2019). However, despite their high specificity when administered intratumorally, their clinical promise falls short due to low levels of virus reaching the tumour when delivered intravenously (reviewed in Cook and Chauhan 2020). A ground-breaking study by Ferguson and colleagues demonstrated that in vitro treatment of macrophages with the PI3Kδ inhibitor, IC87114 prevented oncolytic viral attachment to the macrophages (Ferguson et al. 2020). Pre-treatment of tumour-bearing mice with IC87114 3 hours prior to administration of the tumour-specific oncolytic virus, resulted in significantly reduced tumour burden, prolonged survival and the development of enhanced anti-tumour immunity as characterised by increased CD4+ and CD8+ T cell tumoural infiltration and elevated numbers of IFNγ+ CD8+ T cells.

3 Clinical Trials and Human Studies

Following the success of the first-in-class PI3Kδ isoform-specific inhibitor Idelalisib as a therapy for B cell-derived haematological malignancies, and the preclinical data showing efficacy in solid tumours, multiple clinical trials are now underway with a range of PI3K specific inhibitors in both haematological and solid cancers. Many of the trials for solid tumours use pan-PI3K or isoform-specific inhibitors to treat cancers with activating mutations in the PI3K pathway or loss of PTEN. Details of all trials can be found using the clinical databases: clinicaltrials.gov or eudract.ema.europa.eu; the key trials for isoform-specific inhibitors are also listed in Table 1. However, several trials are now utilising either PI3K inhibitors as an immunotherapy, where the target is the immune cells within the tumour rather than the cancer itself, or as an adjuvant to improve existing immunotherapy approaches, and these trials are discussed in detail below.

Table 1 PI3K isoform-specific inhibitor clinical trials in solid tumours
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3.1 PI3Kδ Inhibitor: Idelalisib

Given the clear role of PI3Kδ in Treg-mediated suppression within the tumour, the potential for repurposing Idelalisib as a therapy for solid tumours has been an attractive prospect. A study sponsored by Gilead (NCT02468557) recently reported its findings from a phase 1 study in pancreatic ductal adenocarcinoma (PDAC). The study intended to primarily assess the safety and adverse event incidence of Idelalisib alone and in combination with other chemotherapy drugs. The study also sought to determine the efficacy of treatment as determined by overall response rate, progression-free and overall survival and immune phenotyping of the tumour environment, specifically the effect Idelalisib had on CD8+ T cells and FoxP3+ Tregs within the tumour. However, the study was terminated early due to two progression-associated deaths and three serious adverse events in the 12 participants enrolled and treated in the Idelalisib only arm (Borazanci et al. 2020). All 12 participants reported adverse events. The toxicity reported is unlikely to be specific to PDAC patients, as serious off-target effects have been widely reported in patients treated with Idelalisib in haematological cancers (reviewed in Cuneo et al. 2019; Hanlon 2020).

A second trial aims to reinvigorate the T cell response in patients who have failed on immunotherapy by using Idelalisib to target immunosuppression within the tumour. Patients with non-small cell lung cancer who have become refractory to anti-PD1 immunotherapy will be treated with a combination of Idelalisib and the anti-PD1 monoclonal antibody, pembrolizumab, to determine if response rates can be improved by dual therapy. As a phase 2 study (NCT03257722), the safe and tolerable dose of Idelalisib in combination with a standard dose of pembrolizumab that results in optimal Treg suppression will be established and the efficacy of dual therapy will be measured by the overall response rate to treatment. This study is still in the early stages of recruiting patients and is yet to report any data or safety concerns.

Given its unquestionable clinical success for CLL and NHL, many drug companies have invested heavily in developing next-generation PI3Kδ inhibitors that replicate Idelalisib’s efficacy but with reduced toxicity. Many of these inhibitors are now being trialled with success for haematological cancers; those that are now being tested in solid tumours are discussed in detail below.

3.2 PI3Kδ Inhibitor: Parsaclisib

Parsaclisib (INCB050465) is an Incyte-developed structurally unique, next-generation PI3Kδ inhibitor, that offers significantly less side effects than first-generation PI3Kδ inhibitors such as Idelalisib (Yue et al. 2019). As a therapy, parsaclisib has shown efficacy in the preclinical Pfeiffer DLBCL model of B cell lymphoma (Shin et al. 2015). A number of clinical trials have reported both efficacy and improved tolerability in haematological cancers (Forero-Torres et al. 2019; Coleman et al. 2021) so its therapeutic potential in solid cancers is now being trialled in patients.

The Incyte sponsored phase 1 trial (NCT02646748), is a two-stage combination therapy trial. In the first stage, escalating doses of parsaclisib were given alongside pembrolizumab to evaluate the safety and tolerability in patients with a range of solid cancers (colorectal, endometrial, breast, pancreatic, lung, head and neck cancer, melanoma). In stage two, efficacy will be assessed in patients with either small cell or non-small cell lung cancer and urothelial cancer. Secondary outcomes will examine how treatment alters the immune contexture of the tumour, specifically examining how the intra-tumoural CD8+ T cell: Treg ratio is altered. The study is due for completion in December 2021, however, preliminary findings indicate that combined therapy significantly reduces the number of intra-tumoural Tregs and increases the CD8+ T cell: Treg ratio ( Kirkwood et al. 2018). Furthermore, analysis of both tumour and PBMCs showed increased T cell activation in patients administered combination therapy.

A parallel trial (NCT03589651), also seeks to determine the efficacy of parsaclisib as combination therapy, with the Incyte-developed anti-PD1 monoclonal antibody therapy, retifanlimab, which has shown promise in other clinical trials for solid tumours (Berton-Rigaud et al. 2020). A third trial by the same sponsors (NCT02559492), was designed to determine both the tolerability and efficacy as measured by tumour response rate, progression-free survival and duration of response, to a combination therapy of parsaclisib and the JAK1 inhibitor, itacitinib, in patients with metastatic cancer. After the primary outcome of measuring safety and tolerability of combined treatment groups was completed the study was terminated early, although no data has been published to date.

3.3 Novel PI3Kδ Inhibitors

A Cancer Research UK sponsored phase 2 trial (NCT02540928), sought to examine changes to the CD8+ T cell infiltrate of head and neck squamous cell carcinoma before and after treatment with the PI3Kδ inhibitor, AMG319. An initial report published in 2018, demonstrated between 50 and 88% inhibition of pAKT following treatment with AMG319 (Ottensmeier et al. 2018). However, of the 22 participants recruited, 10 patients reported skin and gut-associated adverse events, with nine participants terminating treatment early. These adverse events are similar to those seen in an earlier clinical trial in CLL and NHL patients treated with AMG319 (Lanasa et al. 2013). As a consequence of the adverse events reported, this study has subsequently been terminated early due to safety concerns, with full results yet to be reported.

The Shanghai Yingli Pharmaceutical developed inhibitor, YY-20394 or Linperlisib, is currently being studied in a number of clinical trials for lymphoma and leukaemia (NCT04108325, NCT04379167, NCT04370405, NCT04279405, NCT04705090, NCT04500561). Interim results from these studies suggest YY-20394 may offer an improved safety profile with less adverse events reported to date (Qiu et al. 2019). YY-20394 is reported to be structurally unique to other PI3Kδ inhibitors, such as Idelalisib. Patients with advanced cancers are currently being recruited to a phase 1 trial (NCT04049929), designed to assess primarily the safety profile of YY-20394 and secondly the efficacy as determined by tumour progression rate.

Finally, the iOnctura developed inhibitor, IOA-244, has reportedly shown great therapeutic potential as a Treg and MDSC targeting therapy in a preclinical model of colon cancer with high Treg: CD8+ T cell ratio (Johnson et al. 2019). IOA-244 combination therapy with either anti-PD1 or anti-PD-L1 significantly inhibited tumour growth. IOA-244 is now being studied in a phase 1 trial (NCT04328844) as a monotherapy and in combination with the chemotherapeutics pemetrexed/cisplatin in a range of advanced solid tumour indications. This first-in-human study will involve a dose escalation to determine the safety profile of IOA-244. The second stage of the study will determine both tolerability and efficacy of IOA-244 as either a mono or combined therapy and will examine changes to the immune phenotype of lymphocytes in peripheral blood. Results from this study are not expected until the middle of 2023 at the earliest.

3.4 PI3Kγ Inhibitor: IPI-549

Given the success of targeting macrophages via PI3Kγ and improving tumour control in the preclinical models discussed earlier, a number of drug companies have advanced PI3Kγ inhibitors into human trials. Infinity pharmaceuticals were the first to test IPI-549, eganelisib, in a cohort of over 200 patients with a range of solid tumours. The first part of the MARIO-1 (Macrophage Reprogramming in Immuno-Oncology) phase 1 trial (NCT02637531) sought to test the safety and tolerability of eganelisib, in a dose-escalation study, with the efficacy of eganelisib as either a monotherapy or in combination with nivolumab measured in stage two. Full data sets from the study which was due to finish mid-2021 are yet to be released, however, preliminary findings were reported in 2017 and 2018. These preliminary data suggest that eganelisib and nivolumab combination therapy was generally well tolerated with patients typically only experiencing grade 1–2 adverse events (Sullivan et al. 2018; Tolcher et al. 2017). Blood samples taken during the treatment phase indicated T cell activation and reduced immune suppression in peripheral blood. The study sponsors have subsequently commenced two additional trials with eganelisib in 2019. The MARIO-3 phase 2 trial (NCT03961698) is a multi-arm trial in patients with triple-negative breast cancer (TNBC) or renal cell cancer, designed to test the efficacy of targeting macrophages with eganelisib. Patients will receive eganelisib, in combination with either an anti-PD1 (Atezolizumab) or anti-VEGF therapy (Bevacizumab) and the primary outcome of complete response to therapy will be measured over an 18-month period. The secondary outcomes will determine the safety profile, progression-free survival and duration of response. The third Infinity pharmaceuticals sponsored trial is the MARIO-275, phase 2 trial (NCT03980041). Similarly to the MARIO-1 and -3 studies, the efficacy of eganelisib as a monotherapy or in combination with nivolumab will be tested in immunotherapy-naïve advanced urothelial cancer patients.

The efficacy of IPI-549 in a checkpoint inhibitor-independent setting will be tested in patients with TNBC or ovarian cancer (NCT03719326). This two-part dose escalation or expansion study will measure the safety and tolerability of IPI-549, in combination with the dual adenosine receptor antagonist, etrumadenant and the chemotherapy, doxorubicin. Secondary outcomes intend to determine the efficacy of therapy with respect to progression-free and overall survival, duration of response and immune phenotyping of peripheral blood during the study to determine the effect of treatment on the immune response.

A further study, independent of Infinity pharmaceuticals, is designed as a ‘poof-of-concept’ study to test the hypothesis that macrophage phenotype switching occurs in humans in response to the PI3Kγ inhibitor, IPI-549, as previously reported in preclinical models (Kaneda et al. 2016a, 2016b; Henau et al. 2016). This phase 2 window trial (NCT03795610) in a small cohort of patients with head and neck cancer will take tumour biopsies before and after IPI-549 treatment to allow comparison of the immune signature of TAMs. The secondary objectives aim to determine the safety and tolerability of IPI-549 and to examine changes to the myeloid and T cell tumoural infiltrate following treatment.

3.5 Dual PI3Kδ/γ Inhibitor: Duvelisib

IPI-145, Duvelisib, is one of the next-generation PI3K isoform inhibitors, designed to target both the immune cell dominant PI3Kδ and γ isoforms. A number of clinical trials have reported encouraging results for haematological cancers (O'Brien et al. 2018; Flinn et al. 2019, 2018). Given the success shown with individual δ and γ isoform inhibitors in preclinical models described earlier, the ability to target immunosuppressive Tregs, MDSCs and TAMs with a single agent may offer advanced efficacy but without the toxicity of pan-PI3K inhibitors.

Two parallel studies are currently underway to determine the incidence of adverse events and overall efficacy of Duvelisib and anti-PD1 treatment in patients with head and neck cancer (NCT04193293) or unresectable melanoma (NCT04688658). The effect of Duvelisib and Nivolumab treatment on immune cell function and phenotype in both the tumour and the periphery will be established before, during and after treatment in melanoma patients. Furthermore, the development of PD1 resistance mechanisms will be established by examining the gene signature of tumour-infiltrating immune cells. To date neither study has reported any findings, however, a recent study demonstrated that treatment with IPI-145 provided no control of tumour growth in a mouse model of melanoma (Dwyer et al. 2020). In comparison to treatment with the single δ inhibitors CAL-101 or TGR-1202 or the γ inhibitor IPI-549, T cells treated with IPI-145 were functionally impaired, with less cytokine production and reduced persistence in vivo, suggesting that IPI-145 treatment prevented the generation of a sufficient anti-tumour T cell response capable of controlling tumour burden.

4 Future Perspectives

Targeting the PI3K pathway using isoform-specific inhibitors has demonstrated promise at the preclinical stage, either as a novel monotherapy or in combination with other treatments. Similar to the first-generation immune checkpoint inhibitors that target PD1 and CTLA4 (Nivolumab, Pembrolizumab and Ipilimumab), only tumours that have high numbers of immunosuppressive cells can be successfully treated with PI3K isoform inhibitors. As PI3K isoform-specific inhibitors move into the clinical phase of testing, elucidating the effect of PI3Kδ or PI3Kγ inhibition on the immune response within each tumour type will be critical in determining which patient cohorts are likely to be responsive to PI3K specific therapy. A detailed phenotypic analysis of the tumour-infiltrating lymphocytes has the potential to identify which co-inhibitory receptors could be targeted with additional immunotherapies that could potentiate the clinical response to PI3K inhibitors. However, given the immune-related toxicity reported with both checkpoint inhibitors and first-generation PI3K inhibitors such as Idelalisib, it remains to be seen if PI3Kδ- and PI3Kγ-specific inhibitors will be tolerated sufficiently to enable their future use as a first-line treatment regimen.