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Year : 2019  |  Volume : 2  |  Issue : 2  |  Page : 23-35

Tackling immunotherapy resistance: Developing rational combinations of immunotherapy and targeted drugs

The Drug Development Unit, The Royal Marsden Hospital/Institute of Cancer Research, London, UK

Date of Web Publication22-Mar-2019

Correspondence Address:
Dr. Anna Minchom
Drug Development Unit, The Royal Marsden Hospital and The Institute of Cancer Research, London
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/JIPO.JIPO_24_18

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Mechanisms of resistance to immunotherapies are multiple and complex with components intrinsic to the tumor cell and within the immune microenvironment. We review evidence of the interaction of tumor cell signaling pathways with immune pathways and the role this plays in de novo and acquired resistance. The mitogen-activated protein kinase (MAPK) pathway activation and effects on T-cell function are discussed. Phosphoinositide 3-kinase (PI3K) pathway activation (including PTEN loss of function) correlates with T-cell inhibition and immunotherapy resistance. Wnt signaling has been implicated in T-cell function suppression. Key evidence from preclinical models exists for the role of these signaling pathways and is described. Clinical evidence is less advanced though correlation of mutations in key nodes with immune resistance provides a limited clinical correlation. Serial biomarker analysis in patients receiving targeted drugs has been attempted with notable examples including BRAF inhibition in melanoma patients resulting in dynamic changes in programmed death-ligand 1 (PD-L1) expression and tumor-infiltrating lymphocytes. Drug combinations aim to overcome mechanisms of resistance, and recent years have seen numerous combinations of targeted therapies and immune checkpoint inhibitors proposed. However, clear biological rationale and thoughtful trial designs with a translational focus are required to allow such combinations to achieve their full potential.

Keywords: Cellular pathways, combination, early-phase trials, immunotherapy, resistance

How to cite this article:
Cojocaru E, Scaranti M, Minchom A. Tackling immunotherapy resistance: Developing rational combinations of immunotherapy and targeted drugs. J Immunother Precis Oncol 2019;2:23-35

How to cite this URL:
Cojocaru E, Scaranti M, Minchom A. Tackling immunotherapy resistance: Developing rational combinations of immunotherapy and targeted drugs. J Immunother Precis Oncol [serial online] 2019 [cited 2020 Mar 29];2:23-35. Available from: http://www.jipoonline.org/text.asp?2019/2/2/23/254761

  Introduction Top

Cancer and the immune system

The complex interactions between cancer and the immune system have gained significant interest in cancer therapeutics in the last decade. The immune system consists of the innate and adaptive immune response in a highly orchestrated response to pathogens.[1] The innate immune response is a nonspecific immune reaction against pathogens, containing cellular components that recognize nonspecific chemical properties of foreign antigens. In the adaptive (acquired) immune response, pathogen-specific receptors are acquired during the lifetime of an organism on exposure to antigens.[2] The major histocompatibility complex (MHC) consists of cell surface proteins that bind antigens and display for recognition by T-cells. Class I MHC is present on all nucleated cells. MHC Class II is present on antigen-presenting cells (APCs) such as macrophages, B-cells, and dendritic cells (DCs). The T-cell receptor recognizes self or foreign antigens presented by the MHC molecules and activates T-lymphocytes.[3] Activated CD8+ T-lymphocytes mount a cytotoxic response; activated CD4+ T cells mediate the immune response through the secretion of specific cytokines. T-cell responses persist long-term, building immunological memory. To avoid constant activation of the immune system while antigens are displayed, multiple immune checkpoints are activated, maintaining immune homeostasis. Co-inhibitory receptors include TIM-3, LAG-3, programmed-death 1 (PD-1) or CTLA-4.[4] Inflammatory chemokines are responsible for the recruitment of the immune cells to peripheral tissues. FoxP3+ T-reg cells are regulatory lymphocytes that downregulate induction and proliferation of effector T-cells, therefore maintaining tolerance to self-antigens, and prevent autoimmunity.[5]

It is increasingly recognized that immune evasion is a “hallmark of cancer.”[6] Innate and adaptive immunity contributes to immune surveillance and tumor cell eradication.[7],[8] Permanent immune vigilance destroys cancer cells by identifying and eliminating cancerous or precancerous cells based on the expression of tumor-specific antigens on their surface.[9] At the time of diagnosis, cancers have suppressed, evaded, and developed resistance against immune control by multiple mechanisms.[8],[10]

Immunotherapy resistance

The development of CTLA-4 inhibitors (ipilimumab and tremelimumab) was a pivotal moment in cancer immunotherapy and led to significant antitumor responses, particularly in melanoma.[11],[12] PD-1 inhibitors such as nivolumab and pembrolizumab and the ligand 1 (PD-L1) inhibitors such as avelumab, durvalumab, and atezolizumab demonstrated antitumor activity and have received approval in 11 cancer indications including non-small cell lung, melanoma, renal, bladder, or microsatellite instability (MSI)-high tumors.[13]

Despite the impressive responses that led to their approval, immunotherapy resistance is common, and there is a need to temper the great enthusiasm for the promise immunotherapy shows with realism about, and dedicated research into, mechanisms of immunotherapy resistance.[14] Resistance can be classified as primary/ de novo resistance (where no drug response is achieved) or secondary/acquired resistance (when an initial response to a drug is followed by tumor growth). In terms of primary resistance, PD-1 inhibition leads to objective responses in 20%–30% of patients with solid tumors.[15] Combined with CTLA-4 inhibition, objective responses were seen in 42% renal tumors [16] and 57% of patients with metastatic melanoma.[17] Secondary resistance is inevitable. In renal cell carcinoma, the immunotherapy combination of nivolumab and ipilimumab showed a progression-free survival (PFS) of 11.6 months.[16] In other “immunogenic cancers,” similar benefits of PFS have been noted.[18],[19] Although exceptionally long PFSs of over 24 months have been published, these occur in a small subpopulation of patients.[20],[21]

The mechanisms behind resistance have been explored. In primary resistance, T-cells may fail to recognize the tumoral antigens. This occurs in the case of an absence of tumor antigens or inability of presenting these antigens on the tumoral cell surface via MHC.[22] Potential mechanisms of acquired resistance include loss of T-cell function, new escape mutation variants in cancer, and absence of T-cell recognition by downregulation in tumor antigen presentation.[2],[22],[23]

Resistance mechanisms can be intrinsic or extrinsic to the cancer cell. Intrinsic mechanisms include the absence of antigenic proteins (e.g. in tumors with low mutational burden) and the absence of antigen presentation in the case of deregulations of the transporter associated with antigen processing of the β2-microglobulin or human leukocyte antigen (HLA).[2],[24] Other intrinsic mechanisms of resistance described are genetic T-cell exclusion, as seen in β-catenin inhibition, STAT3 activation, p53, nuclear factor-kappa β (NF-kβ), and phosphatase and tensin homolog (PTEN) upregulations or mutations in the interferon-γ pathway signaling, which result in insensibility to T-cells.[23] Extrinsic mechanisms are complex and include external metabolic and endocrine environmental factors, immunoregulation within tumor microenvironment via checkpoint molecules, or presence of FoxP3+ T-reg cells.[25],[26],[27]

Key to understanding immunotherapy resistance is the concept of “immune exhaustion.” When an antigen persists, CD8+ T-cells develop a progressive loss of function. Mechanisms of immune exhaustion include upregulation of T-cell immune checkpoints (including via PD-1, CTLA-4, TIM-3, LAG-3, and TIGIT), enhanced FoxP3+ T-regs expression, or an increase in tumor-associated macrophages.[28] Epigenetic factors play a role in T-cell exhaustion, through demethylation of PD-1 and transcription factors such as Blimp-1, T-bet, Eomes, NFATC1, BATF, or Maf, described in preclinical models.[2],[29] Epigenetic mechanisms may influence immune infiltration, as in the case of CXCL9 and CXCL10 silencing in ovarian cancer cells, which was associated with reduced recruitment of T-cells.[30]

Understanding mechanisms of resistance to immunotherapy will pave the way to therapeutic approaches to overcome resistance. Causes of resistance are diverse and include cellular signaling pathway misregulation. These can potentially be drugged by existing and developing targeted agents. This review will further expand on the mechanisms of resistance associated with the cell signaling pathways and the potential therapeutic applications this brings.

  Methods Top

In this review, online search tools (including PubMed and clinicaltrials.gov) were employed to obtain published papers. The search was limited to English language articles. Recent reviews and research articles were searched for on the web, using the keywords: immunotherapy, resistance, early-phase trials, combination, and cellular pathways. Articles were retrieved and screened for relevance to the research questions mentioned above. Preclinical research on resistance mechanism to immune checkpoints and interaction of the immune system with different cellular pathways, translational research, clinical trial data of combination immunotherapy and targeted therapies (in abstract and published form), and reviews were obtained and methodology assessed. If felt to be of sufficient robustness, data were extracted and presented in the complete or partial form. Reference lists from included papers were also reviewed and data were extracted.

  Signaling Pathways and Immunotherapy Resistance Top

The MAPK pathway

The mitogen-activated protein kinase (MAPK) signaling pathway regulates cell proliferation and is deregulated in almost one-third of human cancers.[31] The MAPK cascade [Figure 1] is activated when extracellular ligands are bound to protein kinase receptors, including EGFR or PDGFR. Through adaptor proteins, RAF is recruited. RAF activation activates a cascade downstream including MEK1 and MAPK (ERK) molecules, responsible for regulation of the proliferation, apoptosis, and metabolism.[32],[33] BRAF inhibitors have an overall survival benefit in melanoma, and addition of MEK inhibitors has enhanced the BRAF inhibition results.[34],[35],[36],[37] Reactivation of signaling downstream to RAF is a major mechanism of resistance to BRAF inhibition.[38]
Figure 1: Main cellular pathways and implication of their inhibition on immune function. Grb2: Growth factor receptor-bound protein 2, SOS: Son of Sevenless, GTP: Guanosine triphosphate, MAPK: Mitogen-activated protein kinase, PD-L1: Programmed death-ligand 1, PI3K: Phosphoinositide 3-kinase, PIP2: Phosphatidylinositol 4,5-bisphosphate, PIP3: Phosphatidylinositol 3,4,5-trisphosphate, PTEN: Phosphatase and tensin homolog, TSC: tuberous sclerosis complex, AKT: Protein kinase B (PKB), mTOR: Mammalian target of rapamycin, PKC: Protein kinase C, NF-kb: Nuclear Factor kappa-light-chain-enhancer of activated B-cells, APC: Adenomatous polyposis coli.

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PD-L1 in context of BRAF/KRAS mutations

Khalili et al. demonstrated that the BRAF (V600E) mutation in melanoma drives production of interleukin-1α (IL-1α) and IL-1b, with the BRAF inhibitor vemurafenib reducing IL-1 expression in cell lines.[39] The upregulation was via upregulation of cyclooxygenase-2 and PD-L1/2 in tumor-associated fibroblasts.[39] Jiang et al. demonstrated that in this context, increased PD-L1 expression likely occurs via ERK 1/2 and JNK.[40]

Some clinical correlations have been found in retrospective clinical sample studies. In D'Incecco et al.'s study of postsurgical non-small cell lung cancer specimens, a correlation was shown between PD-1 and KRAS status (P = 0.005).[41] A study of >400 patient samples found PD-L1 expression more common in KRAS-mutated samples (P = 0.002).[42] A meta-analysis from Li et al. found a correlation with KRAS status and PD-L1 positivity, with those who were KRAS-mutant more likely to be PD-L1 positive (51% versus 36%, OR: 1.69, 95% CI: 1.01–2.84; P = 0.045).[43] In BRAF mutant melanoma, retrospective studies of PD-L1 expression differ in their conclusions, as to the potential impact PD-L1 has a predictive biomarker of response to BRAF inhibitors.[44],[45]

T-cell effects of BRAF and MEK inhibition

BRAF inhibitors cause hyperactivation of the MAPK pathway in BRAF wild-type cells including T-cells.[46] This could lead to upregulation in T-cell function. MEK inhibitions, however, have been postulated as being detrimental to T-cell function.[47],[48],[49],[50] Shindo et al. demonstrated that, in the context of postallograft transplant, MEK inhibitors inhibit cytokine production and CD-4+ and CD-8+ T-cell function though sparing more differentiated T-cell function.[47] Boni et al. further demonstrated MEK inhibition resulted in impaired T-cell function, which is contrast to BRAF inhibition sparing T-cell function.[48] Vella et al. further corroborate this with suppressed T-lymphocyte proliferation, cytokine production, and antigen-specific expansion with MEK inhibition but not BRAF inhibition, though no decrease in T-cell viability was seen.[49]

The differential effects of BRAF and MEK inhibition on T-cell function are less clear in clinical data. Frederick et al. collected biopsies from 16 patients with melanoma receiving a BRAF inhibitor (vemurafenib) or a BRAF inhibitor in combination with a MEK inhibitor (dabrafenib and trametinib) in BRAF mutant cancer.[51] They demonstrated that both BRAF inhibition alone and in combination with MEK inhibition led to increased melanoma antigen expression and CD8+ T-cell infiltrates, and decreased in immunosuppressive cytokines (IL6 and IL8). T-cell infiltrates and antigen expression decreased at progression on BRAF inhibitor and increased when combination of BRAF and MEK inhibitors was commenced. Dynamic changes in PD-L1, PD-1, and TIM-3 were seen.[51] Other groups report an increase in T-cell infiltrate with both BRAF inhibitors and combined BRAF and MEK inhibitors, though no difference in the degree of infiltration between single agent versus combination.

Immunotherapy and MEK/BRAF inhibitor combinations – preclinical data

Hu-Lieskovan conducted an experiment to evaluate if the addition of MEK inhibitor trametinib would improve the activity of BRAF inhibitor dabrafenib in combination with immunotherapy in a syngeneic BRAF-mutant melanoma mouse model (SM1).[53] Combination of dabrafenib and trametinib with an adoptive cell transfer (pmel-1 ACT) model showed complete tumor regression. Given the potential for upregulation of PD-L1, the team also tested dabrafenib and trametinib with anti-PD-1 therapy – with a corresponding increase in antitumor activity demonstrated.[53] Cooper et al. demonstrated that PD-1 blockade in combination with BRAF inhibition significantly delayed tumor growth and prolonged survival in mouse models treated with this combination compared to immunotherapy alone.[54]

Immunotherapy and MEK/BRAF inhibition – clinical trials

A Phase 1 of vemurafenib in combination with ipilimumab was closed prematurely due to liver toxicity.[55] PD-1 inhibition (pembrolizumab) combined with dabrafenib and trametinib resulted in Grade 3–4 toxicities in 73% of patients enrolled in the KEYNOTE-022 study of patients with BRAF-mutant-advanced melanoma, although antitumor activity (overall response rate) was seen in 67% of patients.[56],[57] Phase 2 of this trial is ongoing.[56]

In another Phase 1 trial, patients with metastatic colorectal cancer were treated with escalating doses of MEK inhibitor cobimetinib and a PD-L1 inhibitor atezolizumab.[58] All patients had MSI-stable disease; 4 out of 23 had a partial response to this treatment, while the other 5 patients had stable disease. In MSI-high colorectal cancer, the number of genetic mutations (tumor mutational burden), thus potential response to PD1/PD-L1 inhibition is considered to be high. Pembrolizumab is now FDA-approved in MSI-high colorectal cancer.[59],[60] The responses may, therefore, demonstrate that MEK inhibition might sensitize an otherwise immune-resistant cancer.[58]

A recently published Phase 2 trial evaluated the combined therapy with vemurafenib and IL-2 (aldesleukin) in patients with metastatic melanoma. The combination was associated with an impressive overall response rate of 83.3% and manageable toxicities.[61] However, the IL-2 induced an increase in regulatory T-cells that may override potential synergy. Other clinical studies are ongoing in order to help us to clarify the toxicity profile, maximum tolerated doses (MTDs), and the efficacy of these combinations [Table 1].[62],[63],[64],[65]
Table 1: Immunotherapy and targeted drug combinations in early.phase trials

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The PI3K/AKT pathway

The PI3K/AKT pathway plays an important role in cell growth, proliferation, survival, and migration [Figure 1]. When activated, PI3K phosphorylates phosphoinositides (PIP3). PIP3 recruits to a range of signaling proteins including AKT; this activates a cascade involved in cell growth and survival. PI3K cascade activation is inhibited by the action of the PTEN, of which loss of function is commonly observed in cancers.[78],[79] Tuberous sclerosis complex (TSC) negatively regulates the mammalian target of rapamycin (mTOR).[80],[81]

PD-L1 upregulation with mTOR, PI3K, and AKT

PIK3CA mutations or PTEN loss in breast cancer, prostate cancers, and gliomas are associated with hyperactivity of the PI3K/AKT pathway and demonstrate an increased PD-L1 expression in cell line models.[82],[83],[84] In a cell line study, immunoblotting of EGFR and KRAS mutant lung cancer cell lines showed increased activation of AKT/mTOR signaling and expression of PD-L1 compared to the wild-type models. Furthermore, inhibition of PI3K, AKT, or mTOR decreased PD-L1 expressionin vitroandin vivoin mice with lung adenocarcinoma, when using the mTOR inhibitor rapamycin or AICAR, an activator of AMPK, which inhibits mTOR independently of PI3K or AKT.[85] Chen et al. have shown upregulation of PD-L1 in KRAS mutant cell lines via ERK rather than AKT.[86] Others have demonstrated an increase in PD-L1 in the context of KRAS mutant cancer on prolonged exposure to MEK and AKT although the PD-L1 changes were inconsistent.[87]

PI3K and immune-cell function

PI3K function is felt to be important in lymphocyte function where different catalytic isoforms of PI3K such as p110α and p110 δ exert different influences over the lymphocytes.In vitro inhibition of p110α has a minimal impact on lymphocyte proliferation and survival.[88] In mice models, p110α did not interfere with T-cell-dependent antibody secretion. However, the pan-class PI3K inhibitor and the selective p110 δ inhibitor strongly impaired B-cell division and survivalin vitroand germinal center responses in mice.[88]

The PI3K signaling pathway is also involved in macrophage functioning. Macrophage PI3K signaling inhibits NF-kβ activation and promotes suppression of the immune system and tumor cell proliferation in mice.[89] Conversely, blocking the subunit PI3Kγ activates NF-kβ and reestablishes CD8+ T-cell cytotoxicity.[89],[90],[91],[92]

The mTOR inhibitor rapamycin is associated with inflammatory adverse events such as pneumonitis and glomerulonephritis.[93],[94],[95],[96] Weichhart et al. have shown that inhibition of mTOR was associated with pro-inflammatory cytokines secretion via the transcription factor NF-kβ and impairment of the release of the anti-inflammatory cytokine IL-10. Deletion of TSC2, which negatively regulates mTOR, reversed pro-inflammatory cytokine release.[97]

Using the BRAF-mutant melanoma cell line A375, Peng et al. inhibited PTEN expression and evaluated antitumor response to immunotherapy.[98] Inhibition of PTEN resulted in decreased infiltration of CD8+ T-cells in melanoma cells regardless of their BRAF status; moreover, when melanoma cells were cultured with the tumor-reactive PMEL-1 T-cells in vitro, tumor cell lysis was reduced. In an ACT mouse model, PTEN loss was correlated with reduced antitumor responses, suggesting that PTEN loss can cause resistance to T-cell-mediated immune response. In the same experiment, using a selective PI3Kb inhibitor, the tumoral growth of PTEN-loss melanoma cells was significantly reduced; therefore, inhibition of PI3Kb could sensitize PTEN-loss melanoma cells to immunotherapy.[98]

HER-2, a receptor tyrosine kinase involved in cell proliferation and survival via MAPK and PI3K/AKT pathways, is expressed in 20% to 30% of breast cancers. Trastuzumab, a humanized monoclonal antibody against the HER-2 extracellular domain, is widely used in breast cancer treatment.[99],[100],[101] In two different PTEN-loss-mediated trastuzumab-resistant mammary tumor mouse models, the combination of HER-2/Neu antibody with triciribine, an AKT inhibitor, impaired tumor growth and increased T-cell infiltration in the tumor microenvironment, by inducing Th1 polarization and Neu-specific CD8+ T-cell response.[102]

The WNT/β-catenin pathway

The canonical WNT pathway mediates cell proliferation and growth through β-catenin. β-catenin activates cyclin D1 and MYC through transcriptional mechanisms, which controls the transition of G1 to S phase in the cell cycle. Misregulation of these processes, in the case of Wnt hyperactivation, leads to carcinogenesis.[103] Increased levels of β-catenin have been correlated with poor prognosis in breast cancer and many other tumors present high levels of Wnt proteins.[104],[105],[106] β-catenin and APC gene are involved in the development of colorectal cancer, and APC involvement in familial adenomatous polyposis is well known.[107] Wnt inhibitors are in early drug development (ClinicalTrials.gov identifier NCT03264664).[108]

Wnt/β-catenin pathway and immune activation – clinical correlation

In tumors with known Wnt pathway activation, such as colorectal and ovarian cancer, analysis of tumor microenvironment revealed a lack of T-cell infiltration and poor response to immune checkpoint blockade.[109],[110] In a transcriptome analysis of 703 primary cutaneous melanoma samples, β-catenin immune evasion was identified in 42% of samples.[111] In an analysis of baseline melanoma samples, tumoral β-catenin expression was inversely correlated with FoxP3 and CD8 positivity).[112]

Wnt/β-catenin pathway in dendritic cells

Malignant tumors activate the Wnt/β-catenin pathway in DCs to induce immune tolerance through suppression of T-cell effector response and promotion of T-regs.[113] Decreased expression of β-catenin in DCs upregulates DCs in the tumoral microenvironment, leading to an enhanced immune response in mouse models.[114],[115] Retinoid acid (RA) plays a key role in DC regulation and immune tolerance.[116] Studies show that RA induces FoxP3 in CD4+ T-cells in vitro, in the presence of TGF-β.[117] Moreover, RA can suppress Th-1 and Th-17 by suppressing interferon-gamma and IL-17. β-catenin overexpression induces RA and IL-10 production via DCs, thus promoting immune suppression.[118] One of the possible mechanisms of the T-regs responses through β-catenin pathways is the binding of the β-catenin to the promoter of Aldh1 (aldehyde dehydrogenase) to drive RA synthesis. When expressed in intestinal DCs, β-catenin was correlated with IL-10 secretion and TGF-β and T-reg cell stimulation.[116]

In a genetically engineered mouse model of β-catenin-positive melanoma, T-cell infiltration was minimal and resistance to immune checkpoint blockade was noted. This was in part due to reduced CCL4, a chemokine responsible for BAT3 DC recruitment into the tumor microenvironment.[119],[120],[121] The absence of BAT3 DCs in the case of β-catenin-positive tumors can cause T-cell dysfunction by poor homing and trafficking to T-cell into the tumor microenvironment. Spranger et al. showed that intratumoral upregulated Wnt/β-catenin signaling is associated with poor immune infiltration and ineffective cytotoxic T-cells, due to a decrease in the recruitment and frequency of CD103+DCs.[122] Wnt/β-catenin signaling was correlated with increased ATF3 levels and decreased production of CCL4.[123]


MYC activation through MYC amplification is one of the most highly amplified oncogenes and is regularly present in aggressive cancers, such as breast, lymphoma, or ovarian cancer.[124] MYC inactivation in mice models resulted in decreased mRNA and protein expression of PD-L1 and CD47 and is associated with tumour shrinkage in these models.[125],[126]

The JAK-STAT pathway

The Janus family of cytoplasmic tyrosine kinase (JAK) and its associated transcription factors STAT3 and STAT5 regulate T-regs through enhancement of FoxP3 expression. In T-reg cells, activated STAT3 and FoxP3 regulate IL5 and TGFB1 genes and result in suppression of Th17 cell-mediated inflammation.[127] In a prostate cancer mice model, depletion of STAT3 signaling resulted in the elimination of macrophage accumulation and restoration of T-cells within the tumor microenvironment.[128] In gliomas, reduction of STAT1 was associated with reduced CXCL10 expression, an important cytokine for T-cell recruitment into the TME.[129] Inhibition of cytokine-dependent JAK-STAT3 pathway activation may potentially overcome the suppression of antitumor immunity.[130]

  Drug Development of Targeted Drugs and Immunotherapy Combinations Top

There are currently many ongoing Phase 1 or 2 trials of combination immunotherapy with targeted agents [Table 1]. The challenges of early-phase clinical trial design with immune checkpoint blockade have been previously described, emphasizing the focus on safety, selection of the trial population, and MTD definition. Parallel expansion cohorts have been established as part of Phase 1/2 design and have led to successful early licensing and drug approval.[131]

The traditional paradigm of escalating dose to MTD, based on the assumption that greater efficacy is linked to a higher dose, should be reviewed in the context of immunotherapy trials, given that most immunotherapy trials failed to identify an MTD. Phase 2 dose decision should be taken based on pharmacokinetic and pharmacodynamic data. This challenge is exemplified by the number of dosing and scheduling investigated for immune checkpoint-targeted antibodies.[132],[133],[134] In designing a trial combining immunotherapy with a targeted drug, it is often felt logical to use the licensed or recommended Phase 2 dose of the immunotherapy and to dose escalate the targeted drug from a dose level below that of the recommended Phase 2 dose. This is supported by an analysis of 22 combination trials indicating that both targeted drug and immunotherapy drug can be given at full dose in 59% of studies with a dose reduction generally of the targeted drug.[135] The standard 3 + 3 dose escalation design is most frequently used. An alternative could be a model-based design, aiming to reduce the number of patients exposed to subtherapeutic doses and speed up dose escalation. This is of particular relevance in immunotherapy and targeted agent combinations where this model-design approach might treat a higher number of patients at a near-optimal dose and provides a broader characterization of the dose-toxicity profile.[136]

Many early trials of immunotherapy combined with targeted agents showed an unacceptable toxicity profile.[57],[137],[138] Such toxicity can be difficult to predict given the mechanistic differences in the pharmacodynamic effects of immunotherapy and targeted drugs. Pharmacokinetic measures and drug interaction play a key role in establishing dose and schedule. Drug interactions are seldom studied formally in immunotherapy and targeted agent combination trials, and unfortunately, current models are poor predictors. It is known that immunotherapy influences cytokine levels, which affect selected cytochrome P450 enzymes.[139]

Pharmacodynamic assessment is of particular importance in the context of immunotherapy and targeted drug combination trials given the complexity of effects from a targeted drug. Pharmacodynamic measurement of targeted agents is relatively established, with key downstream targets measured in tumor or circulating cells. Circulating biomarkers have an advantage in terms of ease of sample collection and include circulating tumor cells (CTCs) or peripheral blood mononuclear cells for target modulation. Circulating markers for immune activation include RNA profiling for cytokine profile, flow cytometry for T-cell subsets or CTCs for PD-L1 expression. Direct visualization of tumor inflammatory infiltrates is preferable given that the peripheral immune cell activation may not fully correlate with intratumoral immune effect, though serial invasive biopsies are challenging and not always feasible.[140],[141]

Regardless of the trial design and population selected, a clear biological rationale is needed, based on preclinical and pharmacodynamic data. If an effective immunotherapy and targeted drug combination are established in the context where both the first and the second are licensed individually, the timing and sequencing of the combination will become important. An important question that arises is the sequence of treatment when using two classes of drugs. Is it preferable to treat with immunotherapy and targeted drug upfront or blocking the immune checkpoint first then followed by targeted drug or vice versa? This relies on the answer to fundamental questions of the nature of immune exhaustion and the ability to overcome it. Though this scenario is not yet with us, a forward-thinking approach to consider future treatment paradigms is beneficial to successful drug development.

There is a critical need for validated biomarkers that predict response (and resistance) to immunotherapy in the cancer population.[142],[143] Much effort has been put to integrate the immunohistochemistry expression of PD-L1 with the response to immune checkpoint inhibitors. Although some studies reported positive correlations between the PD-L1 expression with the anticancer response, PD-L1 remains an imperfect biomarker.[144] Tumoral mutational burden (TMB) is a quantified measure of the number of acquired mutations carried by tumor cells derived from genomic sequencing data.[145],[146] Tumor cells with high TMB present a larger range of neoantigens that are recognized by T-cells, encouraging a stronger antitumoral reaction. TMB seems to correlate with response to immunotherapy in some settings.[147],[148] Scoring systems to quantify the number of tumor-infiltrating lymphocyte (TILS) within a tumoral microenvironment have been developed. The “immunoscore” quantifies CD3+/CD8+, CD3+/CD45RO+, or CD8+/CD45RO+ T-cells and can predict outcomes in patients with early-stage colorectal cancer.[149] The presence of TILS in the tumoral microenvironment has been correlated with improved survival in melanoma or ovarian cancer.[150],[151] The presence of cytotoxic CD8 T-cells correlated with response to pembrolizumab in melanoma patients.[152] Recent research focuses on the extent of the T-regs in the TME and its immune suppressive role, as is well known that T-reg expansion supports tumoral immune escape.[153] What is clear is that though these proposed biomarkers are of use in some clinical settings they are not absolute indicators of immunotherapy response. They are also limited by requiring invasive biopsies, and the role of circulating biomarkers is an important area of future research.[154]

  Conclusion and Future Directions Top

The development of cancer immunotherapy has been the largest breakthrough in cancer treatment of the last decade. Understanding the resistance mechanisms to immunotherapy should be a priority.

It has been appreciated for some time that immune markers (classically PD-L1) demonstrate a correlation with mutations in key node in signaling pathways. Detailed preclinical and translation work has unearthed the vast complexity of the interaction of signaling pathways with the immune system. Building on this, combination therapies of targeted agents and checkpoint inhibitors showed promising results. Of particular note is the role of BRAF and MEK inhibitors. Further emerging data should help to clarify, in particular, the nuances of MAPK pathway modulation in immune cells versus cancer cells and the overall impact on tumor and immune response. Given the PI3K pathway plays a pivotal role in both cancer and myeloid and lymphoid subsets, it can be expected that further drug development in this area will yield therapeutic effects; emerging data are eagerly anticipated. The Wnt-β-catenin pathway has a key role in DC function. Pharmacological modulators of this pathway are less developed and once successful pathway modulation is shown combination trials with immunotherapy will need to be developed.

In clinical practice, targeted agent and immunotherapy drug combinations may have high toxicity profiles, and a clear early drug development plan specific for these combinations is required.

Other immunological agents have shown activity against malignant tumors. Antigen-specific vaccines or DC vaccines are developed individually ex vivo and aim to elicit cellular immunity when re-introduced into the patient's blood stream.[155],[156],[157] This approach is currently under development and alongside ACT therapy could illustrate the future of cancer treatment. We have yet to see how these combine with targeted agents.

We are still in the very early stages of understanding the ideal combination of treatment for each patient and the most effective sequence of therapies [Figure 2]. The benefits of immunotherapy and targeted drug combinations have yet to realize their full potential.
Figure 2: Drug development of combination trials of immunotherapy agents and targeted agents. PK: Pharmacokinetics, RP2D: Recommended Phase 2 trial, DLT: Dose-limiting toxicities.

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Financial support and sponsorship

The authors disclosed no funding related to this article.

Conflicts of interest

The authors disclosed no conflicts of interest related to this article.

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  [Table 1]


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