Beyond the Barrier: Unraveling the Mechanisms of Immunotherapy Resistance

Abstract

Immune checkpoint blockade (ICB) induces a remarkable and durable response in a subset of cancer patients. However, most patients exhibit either primary or acquired resistance to ICB. This resistance arises from a complex interplay of diverse dynamic mechanisms within the tumor microenvironment (TME). These mechanisms include genetic, epigenetic, and metabolic alterations that prevent T cell trafficking to the tumor site, induce immune cell dysfunction, interfere with antigen presentation, drive heightened expression of coinhibitory molecules, and promote tumor survival after immune attack. The TME worsens ICB resistance through the formation of immunosuppressive networks via immune inhibition, regulatory metabolites, and abnormal resource consumption. Finally, patient lifestyle factors, including obesity and microbiome composition, influence ICB resistance. Understanding the heterogeneity of cellular, molecular, and environmental factors contributing to ICB resistance is crucial to develop targeted therapeutic interventions that enhance the clinical response. This comprehensive overview highlights key mechanisms of ICB resistance that may be clinically translatable.

1. INTRODUCTION

Immune checkpoint blockade (ICB) has revolutionized the medical treatment of cancer patients by providing durable responses in a subset of tumor types. However, most cancer patients are not eligible for ICB approaches, and even in those who are eligible, response rates remain low due to primary and acquired resistance (1–3). Most approved ICB drugs target cytotoxic T lymphocyte associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1)/PD-ligand 1 (PD-L1) (B7-H1)to disruptinhibitory signalingto Tcells andreactivate animmune responseto tumors(4). CTLA-4 is a competitive antagonist of the cluster of differentiation 28 (CD28)–B7 interaction that blocks costimulation of T cells by antigen-presenting cells (APCs) (5). CTLA-4 inhibition may also block CTLA-4-mediated inhibitory signaling to effector T cells and deplete regulatory T cells. This permits increased antitumor T cell activity in the tumor microenvironment (TME) (6). PD-1 is an inhibitory receptor expressed on T cells that limits pathological immunity (7). In contrast, PD-L1 can be expressed on APCs, including dendritic cells (DCs) and macrophages, tumor cells, fibroblasts, and epithelial cells. PD-L1 is induced by IFN-γ (7). A multitude of small molecules and antibodies targeting other inhibitory immune checkpoints, such as lymphocyte activation gene 3 (LAG-3), T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT), T cell immunoglobulin mucin 3 (TIM-3), adenosine A2A receptor, B7-H3, CD39, and CD73, are in preclinical or clinical development. Developing these alternative immune checkpoint approaches will hopefully synergize with or overcome resistance to the current ICB options (8). Many patients are innately resistant to ICB, while some responders acquire secondary resistance after an initial response, often due to either evolution of the cancer cells or alterations in the TME. Late acquired resistance and relapse are also common.

Primary and secondary resistance to ICB are the major obstacles to large-scale deployment of immunotherapy. These resistances are driven by both tumor-intrinsic and tumor-extrinsic mechanisms, including patient demographic factors, microenvironmental interactions, and genomic alterations. Certain histological and genetic features can predict immunotherapy response, and microsatellite-instable tumors have good ICB response rates (9). However, large-scale biomarkers remain elusive and predicting patient response for most tumor types remains more of an art than a science. T cell tumor infiltration largely correlates with favorable patient outcomes. The levels of tumor-infiltrating lymphocytes can be used as a prognostic signature in some cancers, but this is not entirely generalizable (10–12).

The therapeutic response is extremely heterogeneous and likely requires personalized approaches to extend ICB response to resistant patients. Preclinical research on the modulation of the composition and metabolic environment of the TME provides tantalizing mechanistic clues to resistance, but translational approaches are still not well developed (13). How to counteract the combination of low nutrient and oxygen availability in the TME with the buildup of extracellular tumoral waste and infiltration of protumor immune cells contributes to a complicated and multifaceted picture difficult to distill into its principal components. Both basic science investigation and multifactorial clinical analysis are required to divine what predicts resistance and potential ways to counteract its development.

2. COMMON CLASSES OF IMMUNE CHECKPOINT BLOCKADE DRUGS

CTLA-4 was first described as a negative regulator of T cells in 1987, and PD-1 was identified in 1992 (14) (Table 1). Blocking either the CTLA-4 or the PD-1/PD-L1 pathway enhances T cell antitumor activity. In 2011, ipilimumab, which targets CTLA-4, became the first immune checkpoint inhibitor approved in the United States (15). It became rapidly apparent that ICB can induce durable regression in a subset of patients. In 2014, high-affinity monoclonal G4 antibody inhibitors of PD-1, nivolumab and pembrolizumab, were approved to treat melanoma (16).There are now five approved PD-1 or PD-L1 inhibitors, and the indications for these drugs are rapidly expanding. T cell receptor engagement can result in T cell activation and the production of IFN-γ, which stimulates PD-L1 expression, thereby potentially contributing to T cell dysfunction in the TME (17, 18). Nivolumab was later approved for all microsatellite-instable solid tumors (19). The KEYNOTE trial series revealed that pembrolizumab, a selective IgG4-kappa monoclonal antibody against PD-1, is superior to chemotherapy for certain cancers. Current disease indications of pembrolizumab include melanoma, non-small-cell lung cancer (NSCLC), Hodgkin lymphoma, urothelial cancer, gastric cancer, cervical cancer, hepatocellular carcinoma, and renal cell carcinoma (20, 21). Atezolizumab is a human IgG1 monoclonal anti-PD-L1 antibody. This drug is approved for NSCLC, small-cell lung cancer, and triple-negative breast cancer (22, 23). Avelumab is a human IgG1 recombinant PD-L1 antibody that is approved for metastatic Merkel cell carcinoma, renal cell carcinoma, and urothelial carcinoma (24). Durvalumab is a human IgG1 kappa monoclonal antibody of PD-L1 that improves survival in small-cell lung cancer with platinum combination therapy and extends survival in NSCLC after chemoradiotherapy (25, 26).There are over 15 ICBs in various stages of clinical trial approval targeting both the checkpoints described above and a variety of other inhibitory checkpoints. Overall, ICB approaches seem to function best in infection-associated tumors, tumors with high expression of PD-L1 in the TME, tumors with a high mutational burden, and tumors with an active T cell infiltrate (27). For example, the virally induced Merkel cell carcinoma and the ultraviolet-induced desmoplastic melanoma have immunotherapy response rates over 50% (28). Other cancers with relatively high genetic mutations, such as melanoma, lung cancer, esophageal cancer, bladder cancer, and urothelial cancers, are also responsive to ICB, with response rates around 25% (27). However, some tumor types, such as breast cancer, brain cancer, prostate cancer, and pancreatic cancer, have vanishingly small response rates.

Table 1.

Selected approved immune checkpoint blockade agents and combinations

Drug	Approval	Indication
CTLA-4 inhibitors
Ipilimumab	2011	Melanoma
PD-1 inhibitors
Nivolumab	2014	Classical Hodgkin lymphoma Esophageal squamous cell carcinoma Melanoma Renal cell carcinoma Head and neck squamous cell carcinoma Urothelial carcinoma
Pembrolizumab	2014	Bladder cancer Cervical cancer Classical Hodgkin lymphoma Cutaneous squamous cell carcinoma Esophageal or gastroesophageal junction adenocarcinoma Head and neck squamous cell carcinoma Merkel cell carcinoma MSI-high or mismatch repair-deficient cancers NSCLC Primary mediastinal large B cell lymphoma Renal cell carcinoma Triple-negative breast cancer Tumor mutational burden high tumors Urothelial carcinoma
Cemiplimab	2018	Basal cell carcinoma Cutaneous squamous cell carcinoma NSCLC
PD-L1 inhibitors
Atezolizumab	2016	NSCLC
Avelumab	2017	Merkel cell carcinoma Renal cell carcinoma Urothelial carcinoma
Durvalumab	2017	NSCLC
Combination therapies
Ipilimumab and nivolumab	2015	Esophageal cancer Hepatocellular carcinoma Melanoma MSI-high or mismatch repair-deficient metastatic colorectal cancer NSCLC Pleural mesothelioma Renal cell carcinoma
Pembrolizumab and pemetrexed (folate analog)	2017	NSCLC
Pembrolizumab and pemetrexed and carboplatin (platinum chemotherapy)	2018	NSCLC
Durvalumab and chemoradiation	2018	NSCLC
Pembrolizumab and chemoradiation	2018	NSCLC
Atezolizumab and bevacizumab (VEGF inhibitor) and paclitaxel (microtubule inhibitor) and carboplatin	2018	NSCLC
Pembrolizumab and axitinib (receptor tyrosine kinase inhibitor)	2019	Renal cell carcinoma
Pembrolizumab and lenvatinib (VEGF inhibitor)	2019	Endometrial carcinoma
Avelumab and axitinib	2019	Renal cell carcinoma
Atezolizumab and chemotherapy	2019	Small-cell lung cancer
Atezolizumab and paclitaxel	2019	Metastatic triple-negative breast cancer
Pembrolizumab and chemotherapy	2019	Head and neck squamous cell carcinoma
Atezolizumab and paclitaxel and carboplatin	2019	NSCLC
Durvalumab and chemoradiation	2020	NSCLC
Ipilimumab and nivolumab and platinum chemotherapy	2020	NSCLC
Pembrolizumab and chemotherapy	2020	Triple-negative breast cancer
Avelumab and chemotherapy	2020	Urothelial carcinoma
Atezolizumab and bevacizumab	2020	Hepatocellular carcinoma
Atezolizumab and cobimetinib (MAPKK inhibitor) and vemurafenib (BRAF V600E inhibitor)	2020	BRAF V600+ advanced melanoma
Nivolumab and cabozantinib (tyrosine kinase inhibitor)	2021	Renal cell carcinoma
Pembrolizumab and chemotherapy	2021	Gastroesophageal junction adenocarcinoma
Pembrolizumab and chemotherapy	2021	Cervical cancer
Nivolumab and relatlimab-rmbw (LAG-3 inhibitor)	2022	Melanoma
Durvalumab and tremelimumab (anti-CTLA-4)	2022	NSCLC

Abbreviations: BRAF, V-raf murine sarcoma viral oncogene homolog B1; CTLA-4, cytotoxic T lymphocyte associated protein 4; LAG-3, lymphocyte activation gene 3; MAPKK, mitogen-activated kinase kinase; MSI, microsatellite instability; NSCLC, non-small-cell lung cancer; VEGF, vascular endothelial growth factor.

Over 5,000 trials that include a PD-1/PD-L1 blockade agent are initiated each year. Over one-third of these are testing investigational agents that are not yet approved for any indication, including alternative immune inhibitory checkpoints (29). Combination therapy is becoming increasingly common, and many active trials combine immunotherapy, chemotherapy, targeted therapy, radiation, or dietary intervention (29) (Table 1).TIM-3, expressed on most immune cells, mediates CD8⁺ T cell exhaustion. Animal studies have shown that targeting PD-1 and TIM-3 together boosts antitumor immunity, and there are many current phase I and II trials exploring coblockade of TIM-3 and PD1 (30). LAG-3 is an inhibitory molecule expressed on activated T cells. LAG-3 binds stable peptide-MHC-II complexes and transduces an inhibitory signal (31). LAG-3 blockade is synergistic with PD-1 in many mouse models and has been used to treat cancer patients in combination with ICB with modest effects (31, 32) (Table 1). TIGIT is an inhibitory molecule on T cells that mediates T cell dysfunction. TIGIT is a ligand for CD155 on DCs and promotes secretion of anti-inflammatory IL-10 (33). TIGIT is upregulated on tumor-infiltrating cells, and TIGIT blockade alone or in combination with PD-1 blockade has shown efficacy in murine glioblastoma tumors (34). PD-1 and TIGIT regulate different costimulatory receptors, but PD-1 inhibits the phosphorylation of the TIGIT receptor CD226, leading to blockade of both TIGITandPD-1foroptimalantitumorTcellresponses(35).TheARC7trialiscurrentlyevaluatingTIGITblockadecombinedwithanti-PD-L1 therapyforNSCLC(36).Manyotheragentsthat block immune inhibitory molecules are under preclinical investigation.Significant work is needed to identify the optimal combination therapies with both currently clinically approved inhibitors and novel ICB targets.

3. RESPONSES TO IMMUNOTHERAPY

Evaluating the response to ICB requires different criteria than those used for chemotherapy and targeted molecular therapies. The criteria outlined in Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 are the most common criteria used to evaluate immunotherapy (Figure 1). RECIST 1.0 was defined in the 1990s and used the unidimensional measurement of the tumor’s maximum diameter as evaluated by computerized tomography imaging scan (37). RECIST 1.1 increases the number of target lesions to two lesions per organ, for five in total. It indicates that short-axis measurements of lymph nodes should be used for classification and that nonpathological lymph nodes are less than 10 mm. Disease progression is classified as an increase greater than 20% in the sum of target lesions, with an absolute increase of over 5 millimeters, and progression must be confirmed with repeat imaging at 8 weeks (37). Another criterion often used is the immune-related RECIST (iRECIST), which allows for new metastases and requires confirmation of progression to allow patients with atypical responses to continue treatment (38). This criterion defines complete response as the disappearance of all lesions, partial response as a decrease greater than 30–50% from baseline, and stable disease as where neither complete response nor partial response criteria are met (37). Progressive disease is defined as an increase greater than 25% in the sum of all lesions with secondary radiologic confirmation, as some patients undergopseudoprogression, wherepatients’diseaseinitiallyprogressesandthenrespondstotherapy. Identifying pseudoprogression is critical, as these patients could eventually respond durably to immunotherapy if treatment is continued. Pseudoprogression is attributed to the recruitment of activated T cells to the tumor, which leads to an inflammatory reaction at the tumor site that leads to increased tumor volume. However, most of this evidence stems from case reports of biopsies with isolated tumor cells surrounded by activated T cells and inflammation (39,40).Pseudoprogression has been observed in melanoma, NSCLC, Merkel cell carcinoma, mesothelioma, Hodgkin lymphoma, and head and neck squamous cell carcinoma (41, 42). However, patients with pseudoprogression have longer survival rates than do patients with true progressive disease, indicating that treatment has clinical utility in these populations (43). Another category of immunotherapy response is the dissociated response, where some lesions increase in size, or new lesions are imaged, while other lesions regress (44). This type of response may be due to tumor heterogeneity and genomic instability. Dissociated response ranges from 7.5% to 10% in metastatic tumors, and this type of response is associated with increased survival compared with patients with progressive disease (45, 46).

Figure 1.

Immunotherapy response. Consensus guideline iRECIST defines how to evaluate clinical response in cancer immunotherapy trials to maintain consistent trial design and validation. iRECIST requires the confirmation of progressive disease using imaging 4–8 weeks after initial assessment. New lesions result in confirmed progressive disease if there is an increase of size greater than 5 mm in any new lesions. Clinical status is considered when deciding whether to continue treatment in unconfirmed progressive disease. Abbreviations: CT, computed tomography; iRECIST, immune-related Response Evaluation Criteria in Solid Tumors.

Recent studies have also reported hyperprogression, or rapid progression, after immunotherapy in cancer patients (47,48).It remains a point of debate whether rapid progression is the natural outcome of tumor growth or whether immunotherapy directly triggers acceleration of growth rate. In both NSCLC and urothelial carcinoma, immunotherapy-treated patients show decreased survival compared with chemotherapy-treated patients in the initial phase of treatment, although immunotherapyismoreeffectiveinthelongterm(49,50).Thisfindingsuggeststhatimmunotherapy can initially lead to an increase in tumor size due to inflammatory infiltrate in a subset of patients. This was validated by a study that compared tumor growth rates before and during immunotherapy in head and neck squamous cell carcinoma. This study defined hyperprogression as a twofold increase in tumor growth rate after ICB treatment. Hyperprogression occurred after immunotherapy treatment and was associated with shorter progression-free survival (51).

Hyperprogression is associated with older age and murine double minute 2 (MDM2) and MDM4 amplification (52, 53). One potential method to discriminate between pseudoprogression and hyperprogression is the cell-free DNA chromosomal number instability score, which drops after successful treatment with ICB in responding and pseudoprogressing tumors but not inhyperprogressing tumors (54).Predictingand defining hyperprogression rapidly iscritical to reducing harm induced by immunotherapy agents.To this end, a full understanding of the molecular and cellular underpinnings of hyperprogression is essential and urgent. A recent study identified that patients with hyperprogressive disease and responding patients have comparable levels of infiltrating T cells and IFN-γ gene signatures (55). Unexpectedly, in this hyperprogression cohort, IFN-γ promotes fibroblast growth factor 2 (FGF2) signaling, which decreases NAD⁺, resulting in reduced β-catenin deacetylation and enhanced β-catenin acetylation, reprogramming for tumor stemness, and driving an aggressive tumor phenotype (55). There are currently no reliable biomarkers to quickly distinguish between hyperprogressive disease, pseudoprogression, and natural progression with immunotherapy resistance. There is continuing clinical discussion about the benefits of continuing treatment in stable patients until the next imaging assessment within 4–8 weeks (38).

Evidence-based approaches for treating patients after the development of secondary resistance are few, as the patient population is small and difficult to study.Retrospective analysis has revealed that patients treated with single-agent salvage chemotherapy after immunotherapy have an increased response compared with historical data on patients with a variety of cancer types whose disease progressed on second-line immunotherapy (56). Case reports have sporadically revealed significant responses to some chemotherapy agents after immunotherapy failure, but larger studies are essential to confirm and standardize these results, as current observations are largely mixed (57, 58). There are no standard guidelines for treatment after progression under immunotherapy. Clinical studies that explore different responses to chemotherapy after immunotherapy and basic science investigation into why immunotherapy fails to reprogram the TME are essential to improve treatment strategies.

In addition to defining the overall response to immunotherapy, it is important to differentiate between primary and acquired resistance. Primary resistance is defined as a patient displaying no clinical benefit after at least two cycles of therapy and radiographic assessment (2). Primary resistance occurs due to the absence of tumor antigens, alterations in the antigen presentation machinery, loss of human leukocyte antigen (HLA) expression, mutation of immunogenic signaling pathways to circumvent immune response, or constitutive PD-L1 expression, and new mechanisms are continually being discovered.

Acquired resistance is when the cancer initially responds to immunotherapy but relapse eventually occurs. Acquired resistance is seen in one-fourth of patients with metastatic melanoma treated with ICB (59).Acquired resistance can occur due to novel genomic and epigenetic changes as tumors continue to grow.Multiple mechanisms such as β2-microglobulin (β2M) mutations, loss of HLA, loss of target antigen expression, somatic escape mutations, and alterations in interferon signaling can occur in the tumor during or after the treatment period. Acquired resistance can also occur due to increased infiltration of regulatory T cells or myeloid-derived suppressor cells (MDSCs) or upregulation of alternative immune inhibitory checkpoints. Understanding the best combinations of immunotherapy, chemotherapy, and targeted therapies and the optimal timing strategy is critical to circumvent primary resistance and to prevent or treat the development of acquired resistance. Many studies are using serial tumor biopsies to develop a dynamic model of response to immunotherapy for the development of a potential biomarker, as baseline biomarker studies have been largely ineffective. Overcoming both primary and acquired therapeutic resistance is an area of focus of the cancer immunology community and will likely require personalized approaches. We discuss approaches to enhance endogenous T cell function, prevent cancer cell immune escape, and develop combination targeted therapies in the sections below.

4. TUMOR-INTRINSIC RESISTANCE MECHANISMS

4.1. Tumor Genetic Factors

A variety of tumor-intrinsic mechanisms contribute to immunotherapy resistance (Figure 2). Mutational load, neoantigen burden, and DNA repair pathway mutations were associated with increased ICB efficacy reported in two early landmark papers (60, 61). Loss of tumor neoantigen expression can lead to acquired resistance to ICB. This process can be driven by immunoediting, which selects for clones without neoantigens (62). Therefore, increased intratumor heterogeneity leads to an increased likelihood that a nonimmunogenic subclone will proliferate unchecked, leading to resistance (63). In fact, matched pretreatment and postacquired resistance biopsies of NSCLC show the loss of 7 to 18 putative mutation-associated neoantigens in postresistance specimens of metastatic melanoma (64). In both ovarian cancer and melanoma, breast cancer gene 1 (BRCA1)- and BRCA2-mutated tumors correlate with high mutational burden and better response to ICB (65,66). In general, tumors with defects in DNA repair machinery have high microsatellite instability (MSI-high), may express more neoantigens, and typically have a higher tumor mutational burden and therefore are more sensitive to ICB, as evidenced by the favorable response of patients with MSI-high colorectal cancer (67, 68). PD-L1 expression is another possible mediator of ICB response. PD-L1 expression in melanocytes is strongly associated with the presence of tumor-infiltrating lymphocytes in human melanoma tissues and benign nevi, suggesting that T cells may trigger their own inhibition by secreting cytokines such as IFN-γ that drive the expression of tumoral PD-L1 (69). Hence, induction of the PD-L1/PD-1 pathway is a plausible adaptive immune resistance mechanism in response to immune stress from endogenous T cell immunity and ICB-induced T cell activation. Pretreatment with one form of immunotherapy can also alter the molecular characteristics of response to other ICB agents.For example, patients with advanced melanoma treated with prior anti-CTLA4 therapy who respond to later PD-1 blockade exhibit higher tumor mutational burden and increased inflammatory signatures than do patients who do not respond to PD-1 therapy (70). This finding suggests that the initial immunotherapy treatment has an impact on the tumor’s response profile to future immunotherapy agents.Further research will identify the wide range of targetable factors to preemptively determine resistance patterns and explore optimal clinical strategies for layering and combining ICB with different therapeutic approaches (71).

Figure 2.

Tumor-intrinsic mechanisms of immunotherapy response. Tumors can downregulate tumor antigen expression to avoid immune surveillance, or they can upregulate novel immune inhibitory checkpoints to inactivate or exclude T cells. JAK1/2 loss also often leads to resistance mediated by the downregulation of PD-L1 expression. Antigen presentation failure prevents immune cell recognition of the tumor cell in question and can be driven by β2M mutations, loss of optineurin, or the downregulation of MHC. Oncogenic pathways can also drive resistance to immunotherapy. Loss of PTEN leads to the activation of PI3K, which leads to T cell exclusion and increased apoptosis resistance. The Wnt/β-catenin signaling pathway can lead to T cell exclusion and increased activation of regulatory T cells, sheltering the cancer from immune attack. Genetic and epigenetic mutations, such as the SWI/SNF chromatin remodeling complex, can affect ICB resistance via altering neoantigens and T cell tumor trafficking. Finally, tumor heterogeneity can drive selection for immune-resistant clones. Abbreviations: β2M, β2-microglobulin; ARID1A, AT-rich interactive domain-containing protein 1A; ICB, immune checkpoint blockade; PD-L1, programmed cell death ligand 1; PI3K, phosphatidylinositol 3-kinase; PTEN, phosphatase and tensin homolog; STAT, signal transducer and activator of transcription; SWI/SNF, switch/sucrose non-fermentable; TME, tumor microenvironment; WNT, Wingless/Integrated.

4.2. Oncogenic Signaling Pathway Mutations and Activation

Alterations in classical oncogenic pathways modulate the sensitivity of tumor cells to T cell killing and actively reprogram the TME to become more immunosuppressive. For example, mutations in the phosphatase and tensin homolog (PTEN) pathway correlate to decreased CD8⁺ T cell infiltration and increased resistance to T cell–induced apoptosis (72). The mitogen-activated protein kinase (MAPK) pathway can support the expression of IL-6 and IL-10. IL-6 drives protumor activity of signal transducer and activator of transcription (STAT) signaling, and IL-10 inhibits antitumor effector T cell function. Activation of the β-catenin pathway prevents tumor infiltration by DCs (73). V-raf murine sarcoma viral oncogene homolog B1 (BRAF) mutations are particularly prevalent in melanoma. Treatment with a BRAF inhibitor, vemurafenib, leads to an increase in tumor antigens and infiltrating T cells, suggesting a clinical impetus to combine BRAF inhibition and ICB (74). Tumors with increased Wingless/Integrated (WNT) signaling are less likely to respond to ICB (75). Thus, inhibiting WNT signaling could reverse immune exclusion in nonresponsive tumors. Tumor cell cycle genes are also involved in the regulation of tumor immunogenicity. Inhibiting CDK4 or CDK6 can increase the production of doublestranded RNA and synergize with anti-PD-L1 therapy in a breast cancer model (76). Moreover, serine/threonine kinase 11 (STK11) mutations drive ICB resistance in Kirsten rat sarcoma viral oncogene (KRAS) mutant lung adenocarcinomas clinically. In murine models, loss of STK11 promoted ICB resistance, highlighting the utility of genomic profiling to predict ICB responders to develop personalized immunotherapy (77).

4.3. IFN-γ Signaling

IFN-γ signaling is critical for MHC expression, antigen presentation, T cell priming and activation, and tumor cell ferroptosis (78, 79). T cell– and natural killer (NK) cell–derived IFN-γ activates the JAK/STAT signaling pathway, playing a key role in ICB-induced tumor immunity (80). Loss of the key IFN pathway genes, such as JAK1, JAK2, and β2M, is an ICB resistance mechanism (81). JAK1 and JAK2 mutations result in a lack of response to IFN-γ receptor signaling, while β2M mutations lead to loss of MHC-I expression.Several groups have utilized an in vivo CRISPR/Cas9 genome-editing approach in mice to identify genes critical for ICB response (82, 83).Manguso et al.(82) confirmed that defects in IFN-γ receptor signaling led to resistance to immunotherapy in mice with melanoma. Deletion of the protein tyrosine phosphatase nonreceptor type 2 (Ptpn2) potentiated the immunotherapy response by enhancing IFN-γ mediated effects on antigen presentation and growth suppression (82). In addition to genetic mutations in the IFN-γ signaling pathway, ICB-activated IFN-γ stimulates tumor FGF2 signaling via inhibiting pyruvate kinase M2 (PKM2), a glycolytic enzyme. This diminishes sirtuin-mediated deacetylation of β-catenin via NAD⁺ reduction, which eventually leads to the activation of the c-myelocytomatosis (c-Myc) oncogene and ICB resistance (55). In line with this, IFN-γ-driven metabolic editing modulates c-Myc expression in tumor cells, enabling tumor immune evasion in mouse models (84). IFN-γ signaling may play a dual role in tumor immunity and ICB resistance. How to effectively target the IFN-γ signaling pathway in cancer immunotherapy is clinically challenging (80).

4.4. Antigen Presentation Failure

Downregulating MHC expression to evade T cell killing, particularly in the class I pathway, is a straightforward pathway of tumor cell–intrinsic resistance. β2M was identified early on as part of the MHC-I molecule. Loss of β2M eliminates antigen recognition. Mutations in β2M are found in 29.4% of metastatic melanoma patients with progressing disease on ICB, while β2M loss of heterozygosity is threefold higher in nonresponders than in responders (85). Recent studies demonstrate that the autophagy pathway targets expression of MHC in tumors, contributing to tumor immune evasion in colorectal cancer (86, 87). For example, the autophagy receptor optineurin is lost early on in colorectal cancer (86). Loss of optineurin leads to low IFN-γ and MHC-I expression and reduced immunotherapy efficacy in murine cancer models and cancer patients. Optineurin prevented degradation of IFN-γR1 in lysosomes, maintaining MHC-I signaling integrity. Thus, stabilizing MHC signaling may be a viable approach to increase sensitivity to immunotherapy.

4.5. Alternative Immune Checkpoints

Apart from PD-L1/PD-1 and CTLA-4, there has been a growing recognition of the potential of alternative immune checkpoints in cancer immunotherapy. One of these novel immune checkpoints is LAG-3, which limits the expansion of activated T cells and the size of the memory T cell pool (1, 88). Ieramilimab, the first anti-LAG-3 agent, has been approved by the US Food and Drug Administration to be given in combination with nivolumab to patients with untreated unresectable or metastatic melanoma (89). Siglec-15, a member of the Siglec gene family, has a sialic acid–binding immunoglobulin-type lectin structure. Siglec-15 is upregulated on human cancer cells and myeloid cells and exhibits mutual exclusivity with PD-L1 (B7-H1), as it is downregulated by IFN-γ (90). Siglec-15 suppresses antigen-specific T cell responses, thereby attenuating antitumor immunity. Treatment with blocking antibody against Siglec-15 leads to amplified antitumor immune responses and reduced tumor progression in animal models (90).Clinical study will determine whether Siglec-15 is an effective target for reenergizing immunotherapy in resistant patients with high levels of Siglec-15 expression. Another immune-related target of interest is tumoral stanniocalcin 1 (STC1).STC1 expression is negatively associated with patient survival across various cancer types(91).STC1 has been implicated in promoting tumor progression and resistance to ICB through its interaction with calreticulin, an eat-me signal. STC1 prevents calreticulin membrane exposure, thereby inhibiting APC-mediated phagocytosis and antigen presentation (91). Targeting STC1 may represent an approach to sensitize previously resistant patients to cancer immunotherapy. Continuing effort is being applied to identify additional checkpoints associated with ICB resistance and determine their potential for cancer immunotherapy.

4.6. Epigenetic Factors

Epigenetic modification alters the expression of immune-related genes, shaping the TME composition and modulating ICB efficacy. Epigenetic histone modifications by polycomb repressive complex 2 (PRC2) and switch/sucrose non-fermentable (SWI/SNF) complexes are relatively well studied in the context of tumor immune response and immunotherapy. Enhancer of zeste homologue 2 (EZH2), a PRC2 component, mediates histone H3 lysine 27 trimethylation (H3K27me3) and represses tumor production of Th1-type chemokines, CXCL9 and CXCL10 (92, 93). Conversely, AT-rich interactive domain-containing protein 1A (ARID1A), a core member of the SWI/SNF complex, supports expression of CXCL9 and CXCL10 in tumors, resulting in an increase in T cell tumor trafficking (94). ARID1A mutations result in impaired IFN-γ signaling, reduced T cell infiltration, and shortened survival in both human patients and murine cancer models (94). ARID1A interacts with mismatch repair machinery to promote mismatch repair. Thus, loss of ARID1A may lead to increased MSI and potential tumor immunogenicity (95). Tumors formed by ARID1A-deficient ovarian cancer cells had increased tumor-infiltrating lymphocytes and increased response to anti-PD-L1 antibody (95). Similar results are observed in mice bearing genetic loss of Polybromo-associated Brahma-related gene associated factor (PBAF), another component of the SWI/SNF complex (83). The chromatin effector pygopus family plant homeodomain finger 2 (Pygo2) was found to promote immunotherapy resistance by mediating the expression of the kinase Kit and activating indoleamine 2, 3-dioxygenase 1 (96). Inhibiting Pygo2 sensitized prostate cancer, a historically unresponsive, immunologically cold tumor, to ICB (96). These findings highlight the complicated relationship between epigenetic modifications and Tcell infiltration and ICB response, suggesting that the immune effect of epigenetic mutations is largely context dependent.

In addition to histone modifications, DNA methylation by DNA methyltransferases (DNMTs) and demethylation by ten-eleven translocation family of protein 2 (TET2) can also regulate the immune responses in the TME, thereby affecting ICB efficacy. DNMT1 suppresses the tumor production of CXCL9 and CXCL10 and subsequently reduces T cell migration into tumors and dampens ICB efficacy (92). While IFN-γ stimulation results in phosphorylation and nuclear translocation of STAT1, leading to STAT1–TET2 association, many IFN-γ-responsive genes are silenced via DNA methylation (97). TET2-mediated DNA demethylation causes an increase in 5-hydroxymethylcytosine levels on the promoters of IFN-γ-responsive genes, thereby reducing potential ICB resistance and promoting antitumor immunity (98). Further research is needed to better understand the mechanisms by which epigenetic alterations contribute to ICB resistance and to identify potential therapeutic strategies.

5. TUMOR MICROENVIRONMENT RESISTANCE MECHANISMS

5.1. Immune Infiltrate

The composition of the tumor immune microenvironment plays a critical role in determining the strength of the initial immune response to the tumor and the response to immunotherapy (Figure 3). Improved ICB efficacy is associated with increased infiltrating effector T cells and decreased regulatory T cells, MDSCs, and tumor-associated macrophages (TAMs) (99). In fact, early signatures of adaptive immunity after treatment with ICB are highly predictive of response to ICB (100). MDSCs correlate with reduced antigen-specific T cells in melanoma (101). Monocytic MDSCs are more prevalent in ICB-resistant melanoma patients than in ICB responders (102). Inhibiting MDSC tumor trafficking by CXCR2 antibody treatment resensitized murine rhabdomyosarcoma tumors to anti-PD-1 therapy (103). Selective inhibition of the gamma isoform of phosphatidylinositol 3-kinase (PI3K), which is highly expressed in myeloid cells, restores sensitivity to ICB and promotes T cell–mediated tumor regression without directly targeting cancer cells (104). It appears that PI3Kγ signals through the Akt and mammalian target of rapamycin (mTOR) pathways to inhibit NF-κB activation and to stimulate C/EBPB activation, which induces an immunosuppressive transcriptional program for MDSC development (105, 106). Inactivation of PI3Kγ synergizes with checkpoint therapy to promote tumor regression. Eganelisib, a selective inhibitor for PI3Kγ, is currently in phase I clinical trials (107). Regulatory T cells traffic into the TME via CCL22 and CCR4 and inhibit the antitumor immune response (108, 109). Regulatory T cells are a major component of immunosuppressive networks that limit the antitumor function of effector T cells and APCs in the TME (110). Targeting regulatory T cells is potentially instrumental to improving antitumor immunity and ICB efficacy in patients with cancer.

Figure 3.

Mechanisms of resistance in the TME. (a) Tumors secrete chemokines and cytokines that lead to the generation of an immunosuppressive TME with an increased proportion of MDSCs and regulatory T cells. Inhibiting the recruitment of these cell types is a viable approach to reduce tumor resistance. (b) Tumors secrete a variety of molecules that modulate immune cell function, including cholesterol, lactate, TGF-β, and CSF1. Blocking the production of these molecules metabolically or protecting T cells from their binding using antibodies could reactivate immunotherapy in resistant tumors. (c) Tumors also undergo DNA methylation changes that trigger T cell exclusion, and avidly consume nutrients in the TME, leading to the starvation of infiltrating immune cells. Abbreviations: CSF1, colony-stimulating factor 1; EZH2, enhancer of zeste homologue 2; MDSC, myeloid-derived suppressor cell; PD-L1, programmed cell death ligand 1; TAM, tumor-associated macrophages; TGF-β, transforming growth factor beta; TME, tumor microenvironment.

TAMs regulate extracellular matrix remodeling, immunosuppression, metastasis, and resistance to ICB (111). However, macrophages can also phagocytose and kill cancer cells and stimulate both the adaptive and the innate immune systems (112, 113). Inhibiting the recruitment and polarization of tumor-promoting macrophages while activating their antitumor functions is an active area of research. Macrophage-centered therapeutics are lagging T cell–driven drugs but provide a plethora of modifying metabolites, cytokines, and signaling pathways that could synergize with ICB.

Immunologically cold tumors, where little to no adaptive immune response is initiated and tumor-infiltrating lymphocytes are rare, provide a significant challenge to successful immunotherapy. Melanoma response to ICB correlates with an increase in tumor-infiltrating lymphocytes between baseline and 3 weeks of treatment (114). One mechanism used to prevent T cell infiltration by tumors is secreted galectin, which reprograms the endothelium to upregulate PD-L1 (115). Galectin blockade increases T cell infiltration, which naturally leads to a better response to anti-PD1 therapy (115). In patients with ovarian cancer and colorectal cancer, EZH2-mediated H3K27me3 and DNMT1-mediated DNA methylation repress the production of Th-1-type chemokines (CXCL9 and CXCL10) and reduce T cell infiltration (92).Exploring how to improve T cell infiltration is critical for expanding the reach of immunotherapy. Metastatic melanoma secretes extracellular vesicles with high levels of PD-L1, which suppresses and excludes CD8⁺ T cells from the tumor (116). Tumor-released exosomes also block the differentiation of myeloid precursors into functional DCs, thereby preventing T cell activation and trafficking into the TME (117). How to reshape the immune composition of the TME for a favorable response to ICB is a promising therapeutic strategy.

5.2. Metabolic Reprogramming of the Tumor Microenvironment

Tumors maintain an alkaline intracellular pH and an acidic microenvironment through the Na/H exchanger isoform 1 (NHE1) (118). Inhibition of NHE1 reduced tumor growth and increased survival while increasing cytotoxic T cell infiltration. Combining NHE1 blockade and anti-PD1 antibody treatment with temozolomide led to longer survival in a mouse model of glioma, suggesting that modulating the TME pH is an approach to increase T cell function (118). Lactate is released by many cells in the TME, including fibroblasts and tumor cells, and acidifies the TME. As a result, lactate inhibits the transcription of the FAK family interacting protein of 200 kDa (FIP200) by downregulating the levels of NAD (119). Loss of FIP200 in T cells triggers naive T cells to undergo apoptosis. Therefore, lactate released by tumors metabolically targets naive T cells to prevent antitumor immunity. Furthermore, lactate can lead to sirtuin 1 (SIRT1)-mediated degradation of T-bet and can increase regulatory T cells, leading to increased aggression in prostate cancer (120).

In addition to lactate, an expanding range of tumor metabolites can drive T cell dysfunction and ICB resistance in the TME. Succinate dehydrogenase mutations drive succinate accumulation in tumors (121). Succinate suppresses T cell IFN-γ secretion by inducing impaired glucose flux through the tricarboxylic acid cycle. Thus, pharmacologically reducing succinate levels may reactivate an antitumor immune response. Colorectal cancer shows an increase in ammonia levels, which leads to T cell metabolic reprogramming and T cell exhaustion (122). In line with this, cancer patients have increased serum ammonia, and ammonia gene signature levels correlate with T cell response and response to ICB (122). In mouse models of metastatic and primary colorectal cancer, enhancing ammonia clearance results in T cell activation, reduced tumor growth, and sensitized tumor response to ICB (122).

Hypoxia is implicated in tumor immune escape in the TME.Hypoxia-induced factor 1α (HIF-1α) interacts with histone deacetylase 1 (HDAC1), leading to T cell dysfunction via chromatin remodeling and to epigenetic repression of T cell effector genes. Inhibiting HIF-1α reversed T cell dysfunction and overcame resistance to ICB in syngeneic and humanized mouse models of triple-negative breast cancer (123). Administering a hypoxia-specific chemotherapeutic pro-drug alongside ICB sensitized prostate cancer to ICB by reducing myeloid cell suppression and enhancing the infiltration of effector T cells, leading to a durable remission (124, 125). Thus, disrupting hypoxia has the potential to sensitize tumor response to ICB.

Another way the TME metabolic environment influences ICB resistance is through nutrient consumption. Key nutrients, such as glucose and amino acids, are essential metabolic elements in immune cells and tumor cells in the TME. To meet their bioenergetic, biosynthetic, and redox demands, tumors compete with immune cells for these nutrients, and nutrient deprivation can impair antitumor immunity and promote ICB resistance. For example, tumor cells disrupt methionine metabolism in CD8⁺ T cells, which reduces levels of S-adenosylmethionine, resulting in loss of demethylation at lysine 79 of histone H3 (H3K79). This leads to Stat5 signaling, damaged T cell function, and potential ICB resistance (126). Methionine supplementation increased T cell immunity in both tumor-bearing mice and patients with colorectal cancer (126). Arginine is also critical for T cell function in the TME. Increasing arginine levels switches T cells to oxidative phosphorylation and promotes their increased antitumor activity (127). Hence, reducing arginine in the TME may trigger T cell dysfunction and induce ICB resistance.

Glutamine antagonist 6-diazo-5-oxo-l, a glutamine antagonist, suppresses the glycolytic metabolism of cancer cells while upregulating oxidative metabolism in T cells (128). These divergent responses to glutamine blockade between cancer cells and T cells expose a critical difference in cell-type-related metabolic patterns and provide a potentially potent intervention node in patients with cancer (127). It is unknown how cystine levels are regulated in the TME in patients with cancer. It has been reported that T cell–secreted IFN-γ downregulates the expression of the cystine-glutamate transporters SLC3A2 and SLC7A11, reducing intracellular cystine, cysteine, and glutathione, and promotes tumor cell lipid peroxidation and ferroptosis. Depletion of cystine by cystinase enhances tumor immunity and ICB efficacy in tumor-bearing mouse models (78).

Tumor-secreted cholesterol negatively affects tumor immunity. TME cholesterol induces CD36 in tumor-infiltrating CD8⁺ T cells (129). Fatty acid uptake through CD36 leads to lipid peroxidation and ferroptosis in tumor-infiltrating T cells and eventually leads to impaired antitumor immunity(129).Cholesterol induces expression of immune checkpoints and increases exhaustion in tumor-infiltrating CD8⁺ T cells by inducing endoplasmic reticulum stress, which regulates the expression of PD-1 (130). Inhibiting cholesterol esterification in T cells potentiates CD8⁺ T cell proliferation and T cell effector function (131). However, as plasma membrane cholesterol is critical for T cell receptor clustering and signaling, modifying cholesterol metabolism to increase antitumor immune response to ICB will most likely be clinically challenging (131).Other lipids such as arachidonic acid, palmitoleic acid, and oleic acid can promote tumor ferroptosis in response to IFN-γ and synergize with ICB therapy in murine models (79). Nonetheless, there are limited studies of the contribution of different lipids to tumor immunity and ICB.

Apart from amino acids and lipids, glycolysis reprogramming also affects tumor immunity and ICB efficacy. Activated T cells are dependent on aerobic glycolysis for successful antitumor function (105, 132). Tumors and myeloid cells compete with T cells for glucose in the TME, restricting T cell activity by inhibiting Notch signaling and reducing their mTOR activity and IFN-γ production, allowing tumor progression (133, 134). Simply enhancing glycolysis in a tumor overrides the ability of T cells to control tumor growth. Phosphoenolpyruvate, a glycolytic metabolite, is important for sustaining T cell NFAT signaling and effector functions (135). Increasing phosphoenolpyruvate in the TME increases T cell effector function (135). Thus, tumors reprogram core metabolic pathways in immune cells, thereby evading anti-immunity and enabling resistance to ICB.

5.3. Cytokines

Both tumor- and immune system–derived cytokines modulate the TME to alter response to immunotherapy. Transforming growth factor beta (TGF-β) is the most researched cytokine contributing to ICB resistance. A TGF-β signature is associated with lack of response to anti-PD-L1 in metastatic urothelial cancer (136). TGF-β leads to immune exclusion from the tumor stroma. Administering both a TGF-β- and a PD-L1/PD-1-blocking antibody facilitates T cell penetration into tumors and induces tumor regression (136, 137). Increased TGF-β in the TME is one of the primary mechanisms of ICB resistance. Unfortunately, TGF-β inhibitors have failed to show significant benefit in combination with ICB in clinical trials, highlighting the difficulties of translating preclinical findings to therapeutic impact (138). This reality is potentially due to the lack of a biomarker(s) for patient selection and disease indication.

High levels of serum IL-8 are associated with poor outcome in patients with multiple cancer types treated with ICB. IL-8 is a potential biomarker for ICB response (139). Similarly, the levels of serum colony-stimulating factor 1 (CSF1) are higher in patients with metastatic melanoma than in healthy subjects and are associated with resistance to PD-1 blockade (140). Both IL-8 and CSF1 can be produced by TAMs. In a murine melanoma model, blocking both PD-1 and CSF1 with antibodies induces tumor regression in a macrophage-dependent manner (140). In pancreatic adenocarcinoma models, inhibiting CSF1 receptor (CSF1R) signaling functionally reprograms macrophages, enhancing antigen presentation and T cell activation, and combining CSF1R blockade and ICB led to tumor regression in large, established tumors (141). However, a combination clinical study of PLX3397, a CSF1R inhibitor, with pembrolizumab to treat advanced melanoma and other solid tumors has been terminated for insufficient evidence of clinical efficacy (142).

IL-10 is generally thought to support tumor growth via immune suppression. However, ablation of IL-10 increases tumor progression and metastasis due to increased levels of both MDSCs and regulatory T cells, which lead to a tumor-promoting immune microenvironment (143).IL-10 may contribute to the development of antigen-specific effector T cells, and IL-10-deficient mice develop spontaneous tumors (144).Treatment with engineered IL-10 with drugs such as pegilodecakin induced an increase in T cell immunity in tumor-bearing mice (145, 146). However, despite evidence of biological effect in peripheral blood of the combination of pegilodecakin and pembrolizumab, the CYPRESS 1 and CYPRESS 2 trials failed to improve progression-free survival or overall survival in NSCLC and induced significantly higher overall toxicity (147).

Hence, despite the immunological roles of different cytokines in the TME, directly targeting the signaling pathways of CSF-1, IL-10, and TGF-β has so far failed to have a major therapeutic impact.

6. PATIENT FACTORS

6.1. Baseline Factors

ICB resistance may be affected by several patient factors. Counterintuitively, immunotherapy is associated with a larger survival benefit in older patients with melanoma (148,149).Older mice are more sensitive to immunotherapy compared with younger mice.Younger mice have increased regulatory T cells compared with old mice (149).Younger patients with melanoma also have increased regulatory T cells, potentially explaining this phenotype. Women in general respond better to ICB, with longer survival after treatment compared with men (150, 151). The Eastern Cooperative Oncology Group (ECOG) performance status score is utilized to assess the fitness of patients. Patients with a higher ECOG status have less response to immunotherapy (152, 153). As more than 40% of cancer patients have an ECOG score of 2, studying resistance in this population is important (154).

Peripheral lab values can have a role in identifying ICB response. Even patients who clinically fail ICB reveal a reduction in peripheral exhausted CD8⁺ T cells (155). Low lactate dehydrogenase levels, low monocyte counts, low C-reactive protein, high eosinophils, and high relative lymphocyte counts are associated with improved survival after ICB treatment (156, 157).

6.2. Diet and Obesity

A high-fiber diet or a Mediterranean diet is associated with significantly improved progression-free survival in ICB-treated patients (158).Obesity shapes ICB resistance. In a pan-cancer survival analysis, obese patients showed increased survival after ICB after a multivariable regression analysis (159). Although obesity leads to faster immune aging, increased tumor progression, and PD-1-mediated T cell dysfunction driven by leptin, it appears that obesity is associated with increased ICB efficacy in both murine models and cancer patients (160).This is the so-called obesity immunotherapy paradox (161, 162). Given that ferroptosis is caused by increased lipid peroxidation, this paradox may be partially explained by recent findings on the ability of ICB-activated immune cells to promote ferroptosis in cancer cells (78, 79).

6.3. Microbiome

Mice treated with different commensal bacteria had different responses to immunotherapy that were eliminated after fecal transfer (163). For example, administering Bifidobacterium is enough to inhibit tumor outgrowth when combined with anti-PD-L1 antibodies (163). The effect was mediated through increased T cell accumulation and functionality in the TME. Another early study showed that CTLA-4 blockade is dependent on Bacteroides and that tumors in antibiotic-treated mice are completely resistant (164). In addition, primary resistance to ICB can be attributed in part to dysbiosis of the gut (165). The commensal bacteria composition affects the response to ICB in metastatic melanoma patients, with colonization with Bifidobacterium longum, Collinsella aerofaciens, and Enterococcus faecium significantly associated with improved tumor control and a better T cell response (166). Mostly translationally, clinical trials showed that fecal microbiota transplantation can induce clinical responses with favorable changes in immune infiltrate in the tumor and overcome tumor immune resistance (167, 168). How to durably and effectively modulate the microbiome to amplify ICB efficacy is an active area of research development (169, 170).

7. THERAPEUTIC APPROACHES AND FUTURE DIRECTIONS

Considerable efforts have been devoted to establishing reliable and consistent biomarkers to predict ICB response, with mixed results (100). It is generally thought that enhancing T cell tumor infiltration, augmenting T cell function, and counteracting the immunosuppressive networks in the TME are critical for overcoming ICB resistance. Recent translational research and clinical trials have focused on combinatorial therapies, but no clear, optimal treatment plan has emerged (Table 1). One predominant strategy is to combine current ICB with different immunebased treatments, such as different checkpoints, epigenetic modifiers, adoptive T cell therapy, and vaccines (Figure 4). The second is to combine current ICB with traditional therapies, such as chemotherapy, targeted therapy, and radiation therapy.

Figure 4.

Combination therapy. The future of immunotherapy lies in personalized medicine to design rational combination therapies. To reach this goal, researchers must make significant effort to identify baseline and response biomarkers using peripheral blood composition, analysis of immune populations, TME factors, and fecal microbiome profiling. Clinical research studies should also be expanded to populations historically excluded, such as those with poor functional status and older adults. In the future, profiling will identify fundamental characteristics of a patient’s tumor, and then drugs will be used to proactively combat this resistance, whether through metabolic manipulation or targeted drug therapy. Abbreviations: BRAF, V-raf murine sarcoma viral oncogene homolog B1; ICB, immune checkpoint blockade; IDO, indoleamine 2, 3-dioxygenase; MAPK, mitogen-activated protein kinase; MDSC, myeloid-derived suppressor cell; PI3K, phosphatidylinositol 3-kinase; PTEN, phosphatase and tensin homolog; TME, tumor microenvironment; Treg, regulatory T cell. Adapted from figure created with BioRender.com.

7.1. Different Checkpoint Combinations

Combining blocking antibodies that target CTLA-4 and PD-1/PD-L1 has resulted in increased response rates in many types of cancer (171). Many studies have identified overlapping and unique effects of each blockade on T cells and the TME. However, combined ICB has been associated with a significant increase in adverse events. It is worth exploring whether developing alternative checkpoint inhibitors would minimize this problem. Targeting TIM-3, TIGIT, LAG-3, Siglec-10, and CD47 is effective in preclinical models, as is the blockade of CSF1 or STC1 (91). Further human studies will determine whether and how targeting these different checkpoints are therapeutically effective in patients with cancer.

7.2. Combination with Microbiome Manipulation

A series of recent papers have highlighted how the commensal microbiome composition modulates the effect of ICB in a variety of murine models (172). Perhaps most encouragingly, clinical trials have assessed the feasibility of fecal microbiota transplantation and retreatment with anti-PD-1 therapy in refractory metastatic melanoma patients and revealed clinical responses in 3 of 10 treated patients (168, 173). The fecal microbiota transplant induced favorable changes in immune cell infiltration, suggesting that microbiome modulation could be effective in cancer patients. How to scale this approach, select the most appropriate fecal donor, and maintain the new microbiome composition long term remain active research questions.

7.3. Combination with Epigenetic Modifiers

DNMTs and HDACs affect cancer immunity. Combining epigenetic modulation with immunotherapy is an emerging area of exploration. In preclinical models, low-doseHDACinhibition reduces the suppressive activity of TAMs and reduces the recruitment of MDSCs (174). The DNMT inhibitor decitabine increases T cell function. Combining decitabine with anti-PD-1 therapy in patients with Hodgkin lymphoma has shown a striking increase in complete response rate, suggesting that decitabine may ameliorate anti-PD-1 resistance (175). Nevertheless, determining optimal timing, dosing, and patient populations for combination strategies will need to be clinically evaluated for different cancer types.

7.4. Combination with Chimeric Antigen Receptor T Cell Therapy

Chimeric antigen receptor (CAR) T cell therapy has provided durable and meaningful responses, specifically in treating hematological malignancies. CAR is an engineered chimeric T cell receptor that is designed to have an antibody-binding domain and a T cell receptor signaling domain (176, 177). However, solid tumors often have an immunosuppressive TME that impairs and excludes CAR-T cells, reducing their tumor-killing efficacy. Therefore, combining CAR T cell therapy and ICB may produce a synergistic effect, where CAR T cells provide the initial tumor infiltrate and then ICB maintains the function of these CAR T cells (178). Findings from mouse models have suggested that promise in this combination therapy. In an orthotopic mouse model of pleural mesothelioma, CAR T cells eradicated tumors at high doses but rapidly lost cytotoxic function in the tumors at lower doses (179). Treatment with anti-PD-1 therapy restored the function of exhausted CAR T cells (179). In a squamous cell carcinoma model, adoptive T cell transfer and infusion of an anti-PD-L1-blocking antibody cured 60% of the animals, supporting that coadministration of ICB and CAR T cells could improve therapeutic efficacy of single therapy (180). Thus, CAR T cell therapy may be considered an effective approach to be combined with ICB.

7.5. Combination with Cancer Vaccine

Cancer vaccine in combination with ICB has been tested in animal models and patients with cancer (181). Treatment of a murine model of pancreatic cancer with a granulocyte-macrophage colony-stimulating factor (GM-CSF) vaccine upregulates PD-L1 expression (182). Combining this vaccine with PD-1 antibody increases effector T cells in the TME and improves survival of tumor-bearing mice. A cancer-testis antigen SSX2 (synovial sarcoma X 2) vaccine was also investigated. This vaccine increased expression of PD-L1 on tumor cells, suggesting that combining the vaccine with ICB might improve vaccine immunogenicity (183). Potentially, combining cancer vaccines with ICB can combat the vaccine-induced upregulation of PD-L1 and could lead to improved tumor response. Recent clinical trials have evaluated neoantigen vaccines in a variety of tumor types, including melanoma. In melanoma, a long-peptide vaccine that targets up to 20 predicted personal tumor neoantigens induced long-term neoantigen-specific polyfunctional effector T cells that in some cases recognized autologous tumor, with some patients experiencing no tumor recurrence 25 months after vaccination (184, 185). This multiepitope, personalized approach is also feasible in a low-mutation-load tumor such as glioblastoma. Patients given a personalized neoantigen vaccine who did not receive dexamethasone generated polyfunctional neoantigen specific T cells and an increase in tumor-infiltrating T cells (186). Mutated IDH1 describes a subtype of glioma.A mutant-IDH1-specific peptide vaccine induces therapeutic T helper cell responses that are effective against mutant glioma in syngeneic MHC-humanized mice (187). In patients with astrocytomas, the trial met its safety endpoint and induced an immune response in 93.3% of patients, with patients with an immune response having a longer progression-free survival rate. Together, these data provide a strong rationale for further development of this approach in combination with ICB to overcome primary and secondary resistance.

7.6. Combination with Chemotherapy

Immunotherapy and chemotherapy cotreatment of tumors is perhaps the most common and well-studied combination therapy. Metastatic nonsquamous NSCLC patients showed longer survival withapembrolizumabandchemotherapycombinationthandidpatientswhoreceivedplaceboand chemotherapy (188). These results were echoed in the IMpower130 trial, which showed a significant and clinically meaningful improvement in survival when atezolizumab was combined with platinum-based chemotherapy (189). In both small-cell lung cancer and squamous NSCLC, combining chemotherapy with ICB significantly improved survival (49, 190). Even in inoperable or metastatic triple-negative breast cancer, a notoriously aggressive malignancy with poor outcomes, combining ICB with chemotherapy significantly extended progression-free survival (191,192).Of note, there is some evidence in NSCLC and Hodgkin lymphoma that in heavily pretreated patients who subsequently fail treatment with ICB, the ICB treatment resensitizes patients to salvage chemotherapy (193, 194).

7.7. Combination with Targeted Therapy

Targeted therapy, aimed at specific mutations or metabolic abnormalities such as hypoxia or altered amino acid levels, has the potential to enhance T cell infiltration and facilitate a more robust immune response to ICB. Specifically, combining BRAF inhibition with ICB in melanoma patients is promising. BRAF inhibition leads to a decrease in immunosuppressive cytokines, an increase in tumor antigen expression, and T cell infiltration (74). Adding a mitogen-activated extracellular signal-related kinase (MEK) inhibitor to the combination of anti-PD-L1 and the BRAF inhibitor dabrafenib improved survival even further in patients with melanoma (195). Clinically, the phase 3 trial IMspire150 revealed that adding atezolizumab to treatment with the BRAF inhibitor vemurafenib and the MEK inhibitor cobimetinib significantly increased progression-free survival in patients with BRAF-mutated advanced melanoma (196). Combining PI3K inhibition with ICB improves antitumor effects in mice and humans (197). Cotreatment selectively decreased intratumoral regulatory T cells and enhanced the generation of antigen-specific memory T cells. Inhibition of IDO1 is thought to restore T cell function in the TME (198). However, despite early encouraging results, no significant difference was found in progression-free survival or overall survival in the combination pembrolizumab and epacadostat (IDO1 inhibitor) compared with pembrolizumab alone (199). Therefore, the usefulness of IDO1 inhibition to enhance the activity of ICB remains unclear. This trial highlights the difficulties in expanding preclinical findings to clinical application. CD73 is the cell surface enzyme that generates adenosine, which inhibits T cell effector function. Combining CD73 and PD-L1 blockade led to an increase in tumor-infiltrating T cells with increased IFN-γ and tumor necrosis factor alpha (TNF-α) in a murine model of NSCLC (200). Optimizing dosing and timing of these combination treatments, expanding preclinical metabolic manipulation to clinical trials, and identifying biomarkers for response remain active areas of interest.

7.8. Combination with Radiation Therapy

Immunotherapy sensitizes tumors to radiotherapy, as both therapies induce tumor cell ferroptosis (201). IFN-γ from CD8⁺ T cells and ataxia telangiectasia mutated (ATM) activated by radiotherapy both suppressed the glutamate-cystine antiporter SLC7A11, resulting in increased lipid oxidation and ferroptosis, and improved tumor control (201). Clinically, the combination of radiation and ICB has been quite promising. For melanoma brain metastases, stereotactic radiation is well tolerated in patients under nivolumab treatment (202). In patients with advanced NSCLC, progression-free survival and overall survival with pembrolizumab were significantly longer in patients who had received radiotherapy than in patients who had not (203). However, patients who had previously received thoracic radiotherapy had an increased rate of pulmonary toxicity (203). The picture for prostate cancer is more complicated. In patients with castration-resistant, bone metastatic prostate cancer who had progressed after docetaxel treatment did not show any significant difference between bone-directed radiation and ipilimumab and only radiation (204). However, the proportional hazards assumption changed over time, indicating that the hazard ratio between the two treatment groups changed over time and that further optimization is needed. Radiation therapy and CTLA-4 blockade induce systemic antitumor T cells and an objective response in 18% of patients with chemorefractory metastatic NSCLC, where anti-CTLA4 antibodies have previously not demonstrated significant efficacy alone or with chemotherapy (205). However, radiation also shapes the TME with TAM and MDSC infiltration (206).Understanding how radiation and ICB modulate the TME in different types of cancer and different treatment schemata is critical to leveraging this combination therapy, as is basic science research that dissects the contributions of ferroptosis and neoantigens.

In summary, ICB can induce durable clinical responses in patients with cancers of a wide range of histologies. However, ICB resistance is a significant hurdle that must be overcome to expand therapeutic benefit to most cancer patients. One strategy is to design novel clinical trials based on the current mechanistic understanding of ICB resistance. Another is to continuously explore novel actionable immune targets as the next generation of immune checkpoints. Additionally, standardized guidance on optimal diet, nutrient supplementation, and microbiota transplantation would be beneficial in improving ICB efficacy. Finally, given the extensive array of studies outlining metabolic impact on the TME and immune cell function in preclinical models and patients with cancer, selectively targeting metabolic pathways may provide an option to optimize immunotherapy response and potentiate combination treatment with ICB (207).