Checkpoint blockade is a transformative therapeutic approach to a broad spectrum of malignancies because it increases the power of antitumor immunity to obtain durable responses. Cytotoxic T-lymphocyte–associated antigen 4 (CTLA-4) is the prototypical inhibitory checkpoint receptor. Since US Food and Drug Administration approval of the anti–CTLA-4 antibody ipilimumab for use in patients with melanoma, there has been ever-increasing excitement among oncologists about new ways to use this method of releasing the “brakes” on patients’ endogenous immune systems. This review will summarize the preclinical and clinical development of CTLA-4–blocking antibodies, discuss recent insights into the biology of CTLA-4 blockade, review the use of these antibodies in combination with established and novel therapeutic modalities, and comment on ongoing questions regarding their administration.
The journey from bench to bedside
Cytotoxic T-lymphocyte–associated antigen 4 (CTLA-4; also known as CD152) is expressed on the surface of T cells, where it primarily suppresses their early stages of activation by inducing inhibitory downstream T-cell receptor (TCR) signaling and counteracting activity of the T-cell costimulatory receptor, CD28.[1,2] CTLA-4 is thought to outcompete CD28 for B7 ligands (CD80 and CD86) on the surface of antigen-presenting cells by binding them with higher affinity and avidity. In preclinical studies, blockade of CTLA-4 led to a 1.5-fold to 2-fold increase in T-cell proliferation and a 6-fold increase in interleukin-2 production.
The physiologic role of CTLA-4 is not only to suppress effector T cells (Teffs), but also to increase the function of immunosuppressive CD4+FoxP3+ regulatory T cells (Tregs). Treg-specific CTLA-4 deficiency was shown to diminish the suppressive capacity of Tregs in cell culture, resulting in upregulation of CD80 and CD86 expression on dendritic cells (DCs). CTLA-4 blockade has been shown to promote T-cell activation, and in preclinical models, to deplete intratumoral Tregs in a process dependent on the presence of Fcγ receptor-expressing macrophages within the tumor microenvironment.[6,7]
CTLA-4 was shown to play a critical role in maintaining immunologic homeostasis when mice genetically deficient in CTLA-4 developed a rapidly progressive, fatal lymphoproliferative disease, characterized by multiorgan T-cell infiltration and death by 3 to 4 weeks of age.[8,9] However, Leach et al subsequently demonstrated in a mouse model that blockade of CTLA-4 with antibodies did not cause lethal systemic autoimmunity. Moreover, anti–CTLA-4 treatment in this preclinical study not only resulted in rejection of pre-established tumors but also in immunity to a secondary exposure to tumor cells without additional CTLA-4 blockade, thereby establishing the development of immune memory.
Based on these preclinical findings, clinical testing of two antibodies that block CTLA-4 in humans, ipilimumab and tremelimumab, was begun. Ipilimumab belongs to the immunoglobulin G (IgG) 1 class of fully human monoclonal antibodies (mAbs) and has a half-life of 12 to 14 days. Tremelimumab belongs to the IgG2 class, which causes less antibody-dependent cellular toxicity than IgG1, and has a half-life of 22 days. Lessons learned from the initial phase I/II studies, which have been summarized in prior reviews, include the emergence of a unique toxicity profile, composed of immune-related adverse events (irAEs), and new response patterns in which new lesions are viewed as part of the total tumor burden and not regarded immediately as progressive disease.[11,12]
US Food and Drug Administration (FDA) approval of ipilimumab was ultimately based on the results of a randomized phase III trial for patients with previously treated, unresectable stage III or IV melanoma who received ipilimumab 3 mg/kg with or without glycoprotein (gp)100 peptide vaccine vs gp100 peptide vaccine alone. Median overall survival (OS) in the ipilimumab and ipilimumab + gp100 cohorts was 10.1 and 10.0 months, respectively, vs 6.4 months for the gp100 control arm (hazard ratio [HR], 0.68; P < .001). More importantly, ipilimumab had an effect on long-term survival, with 18% of the ipilimumab-treated patients surviving beyond 2 years compared with 5% of patients who received the gp100 peptide vaccine alone.
Tremelimumab was tested in a randomized phase III trial in patients with advanced melanoma who received either 15 mg/kg every 3 months as a single agent or dacarbazine/temozolomide. The endpoint of improved OS was not reached despite a proportion of subjects experiencing a durable response after treatment with tremelimumab. The lack of an OS benefit may have been due to crossover to an expanded-access ipilimumab program by patients who received chemotherapy in the control arm. Further, pharmacokinetic simulations subsequently indicated that, despite tremelimumab’s longer half-life, a 15-mg/kg every-3-month dosing strategy resulted in only 50% of subjects reaching the drug’s target concentration. Approximately 90% of subjects reached target levels when treated with tremelimumab 10 mg/kg every 4 weeks for 6 months. Tremelimumab recently showed encouraging clinical activity with the 15-mg/kg every-3-month dosing strategy in previously treated patients with advanced malignant mesothelioma in a phase II trial. For these reasons, tremelimumab continues to be investigated at different doses and in a variety of malignancies and combination regimens, which will be discussed later in this article.
Back to the bench: next-generation sequencing and further insights into the biology of CTLA-4 blockade
Only a subset of patients and tumor types benefits from CTLA-4 blockade. Therefore, immense effort has been expended to understand tumor and host characteristics that contribute to response. Next-generation sequencing may prove to be a valuable tool in helping to achieve this goal.
Approximately 40% of cutaneous metastatic melanomas have an activating mutation that results in the substitution of glutamic acid for valine at codon 600 (BRAF V600E) and leads to constitutive activation of downstream signaling through the mitogen-activated protein kinase (MAPK) pathway. [17,18] Although this mutation is not thought to correlate with response to anti–CTLA-4 therapy, recent data suggest that a mutation in RAS (rat sarcoma) may correlate with response to ipilimumab. In this retrospective study, patients with metastatic melanoma who harbored a mutation in NRAS (neuroblastoma RAS) had a clinical benefit rate of 41% from ipilimumab therapy vs 22% for wild-type patients (P = .018; N = 137).
Further, a preclinical study found that phosphatase and tensin homolog (PTEN) represses the expression of immunosuppressive cytokines by blocking the phosphatidylinositol 3-kinase (PI3K) pathway, which is a downstream target of RAS. Indeed, PTEN loss in malignant melanoma samples was associated with a host response that was not brisk, which could, in theory, predict a poor response to CTLA-4 blockade. The immunologic consequences of these signaling pathways are an area of active research; whether they influence immunotherapy treatment outcomes requires additional investigation.
Another hypothesis implicates the role of immunogenic neoantigens. Different types of malignancies vary with respect to the number of cancer-causing mutations that may encode proteins foreign to the immune system and directly correlate with response to T cell–based immunotherapy.[22,23] However, there does seem to be checkpoint inhibitor activity in tumors with lower median mutational loads, and there also appears to be a lack of activity in certain subjects with tumors classically associated with a high mutational burden. This discrepancy may be due to interindividual differences in the mutational load across a given tumor type, or there may be specific mutations that are more likely to promote an immune response.
To help shed light on this issue, two recent studies have taken a closer look at cancer genome data to determine whether there are mutations that contribute to a T-cell response. The first used bioinformatics and in vitro strategies in a patient with stage IV melanoma to show that a peptide resulting from a mutation in the ATR (ataxia telangiectasia and Rad3 related) gene generated specific T-cell reactivity that increased strongly after successful treatment with ipilimumab. The second performed whole-exome sequencing of tumor DNA from 11 patients who had long-term benefit and 14 who had minimal or no benefit from ipilimumab treatment for advanced melanoma. A preliminary association between certain neoantigens and clinical outcomes was seen.
Whole-exome sequencing and TCR quantitative sequencing have recently been applied to identify patient germline and immune characteristics that may predict clinical benefit from CTLA-4 blockade. In patients with metastatic melanoma treated with ipilimumab, whole-exome sequencing of germline DNA from 30 objective responders and 30 nonresponders identified several single nucleotide polymorphisms that cosegregated with clinical outcomes. Although these results need to be confirmed in functional and larger prospective studies, the genes identified represent chemokine receptors and thus support the biologic plausibility of this result. The effects of anti–CTLA-4 therapy on the T-cell repertoire were recently studied using next-generation sequencing of the TCRβ gene from T cells isolated from samples of peripheral blood mononuclear cells; 25 metastatic castration-resistant prostate cancer (CRPC) patients treated with ipilimumab and granulocyte macrophage colony-stimulating factor (GM-CSF), 21 metastatic melanoma patients treated with tremelimumab, and 9 untreated healthy control subjects were included. Although blockade of CTLA-4 caused global turnover of the T-cell repertoire and an increase in TCR diversity, improved OS was associated only with maintenance of high-frequency TCR clonotypes throughout treatment. This result suggests that high-avidity, pre-existing T cells may be important to the antitumor response seen with CTLA-4 blockade.
These advances also suggest that a more personalized strategy may ultimately be feasible for the application of CTLA-4 blockade. If expeditious and reproducible prescreening methods could be developed, those patients and tumor types most likely to benefit could be identified. Further, these techniques give important mechanistic insights that could aid in the design of combination approaches for the treatment of patients who are unlikely to respond to anti–CTLA-4 monotherapy.
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