Although checkpoint-blocking antibodies dominate the anticancer immunotherapy field today, researchers are increasingly exploring the potential to leverage growing knowledge about tumor-infiltrating lymphocytes to develop new drugs.
Steven A. Rosenberg, MD, PhD
Although checkpoint-blocking antibodies dominate the anticancer immunotherapy field today, researchers are increasingly exploring the potential to leverage growing knowledge about tumor-infiltrating lymphocytes (TILs) to develop new drugs.
TIL therapies are a form of adoptive cell transfer (ACT) immunotherapy in which T cells are grown and expanded from resected metastatic tumor deposits.1,2 In ACT, autologous or allogenic effector lymphocytes, generally obtained from peripheral blood or resected tumors, are expanded and activated ex vivo, then administered to lymphodepleted patients to promote tumor regression and the tumoricidal activities of the effector cells.
The development of novel therapies that employ these strategies is in the early stages. One company, Iovance Biotherapeutics, based in San Carlos, California, is leading the field with platform technology that is being tested in multiple tumor types, including in phase II trials. Academic and research collaboratives in the United States and Europe also are pursuing TIL immunotherapies.Reports of the association between cancerous tissues and lymphoid immune cells predate our understanding of the role of the immune system in limiting oncogenesis and determining the response to anticancer treatments; indeed, the presence of lymphoid cells in cancerous tissue appeared in a report dating back to 1863.3
Components of both the innate and adaptive immune systems have important anticancer roles; for instance, innate immune cells can stimulate T- and B-cell responses by releasing signaling molecules, while cells that mediate adaptive immunity, such as CD8-positive cytotoxic T cells, can infiltrate and attack tumor cells via tumor necrosis factor (TNF)-related pathways.
Immune cells also interact with tumor cells to determine tumor fate through the 3 so-called E-phases of elimination, equilibrium, and escape, first named following seminal studies by Schreiber and colleagues.4,5 Their work also showed increased tumor growth and aggressiveness in immune-deficient mouse models of cancer, further cementing the key role of immune responses in tumor suppression.5
In 1988, pioneering work from Steven A. Rosenberg, MD, PhD, a 2013 Giants of Cancer Care® award winner, and colleagues showed that lymphocytes extracted from freshly resected melanomas could be expanded in vitro and that these TILs, in conjunction with interleukin (IL)-2, could be used as ACT to induce higher response rates in patients with metastatic melanoma.6
Solid tumors are commonly infiltrated by many immune cells, including T and B lymphocytes, natural killer (NK) cells, dendritic cells, macrophages, neutrophils, eosinophils, and mast cells; however, the presence of a high number of T cells, rather than inflammation-associated immune cells, has been identified as an indicator of favorable prognosis in several tumor types, including melanoma and ovarian, colorectal, and breast cancers.7-12
In colorectal cancer (CRC), for example, the presence of memory T cells and CD8-positive T lymphocytes are predictive indicators of progression-free survival (PFS) and overall survival (OS).13,14 More recent studies have shown that TILs can differentiate prognosis within each tumor, node, and metastasis (TNM) stage in CRC and lung cancers, suggesting that the type and density of TILs may be powerful factors that can improve prognostication based on pathological criteria alone.15,16 The presence of CD3-positive and CD8-positive T lymphocytes within colon cancer cell nests and of CD3-positive T cells in CRC stroma was correlated with improved OS.17
In fact, the heightened recognition of the significance of TILs and their subtypes in CRC has resulted in the establishment of a worldwide taskforce committed to classification of CRC tumors based on their lymphocyte profiles.18 Studies and meta-analyses also confirmed an association between TILs, especially intraepithelial CD8-positive TILs, and improved survival outcomes in ovarian cancer.19,20
TILs have also been detected in breast cancers, especially in highly proliferative subtypes such as triple-negative breast cancer (TNBC) and HER2-positive tumors, and their presence before treatment has been associated with pathologic response to anthracycline-based chemotherapy, disease-free survival, and OS after adjuvant chemotherapy.21-23 Moreover, in TNBC, an increase in stromal TILs was correlated with significantly lower risk of recurrence or death, distant recurrence, and overall mortality.24 The examples cited here, along with other data, demonstrate a clear prognostic role for TILs in various cancers.
In addition to serving as a prognostic indicator and predictive biomarker, TILs also have been translated into a therapeutic agent in ACT-based treatments of cancer, mostly of solid malignancies.1,2,7,34-36
The fundamental protocol for ACT-TIL therapy, developed initially by Rosenberg and colleagues for metastatic melanoma,37 consists of metastasectomy followed by fragmenting of the resected lesions and microculturing in the presence of IL-2 to expand the TIL population (Figure). The melanoma-reactive subset of TILs is expanded by stimulation with a soluble anti-CD3 monoclonal antibody, high-concentration IL-2, and irradiated allogeneic or autologous feeder cells. The TILs are then concentrated by suspension and readied for infusion into a lymphodepleted patient.
Although this initial protocol was shown to be functionally effective, yielding objective response rates (ORRs) in the range of 34% to 72%, with some patients developing long-lasting complete remission, significant challenges were also noted.7,37 These challenges include the presence of an appropriate autologous cell line for successful selection of reactive TILs, the lengthy time required for expansion, and the higher likelihood of terminal differentiation of the TILs upon prolonged ex vivo culture, along with the difficulty of obtaining culture-competent TILs from resected cancer tissues.
A significant modification that helped abbreviate the expansion cycle was the exclusion of the tumor-reactivity selection step, resulting in shorter protocol called the “young TIL protocol”7,38; this amended protocol was used in conjunction with a CD8-enrichment step in patients with metastatic melanoma, with an ORR of 58% for the TIL-treated patients.38
Another critical observation is the importance of lymphodepletion, conditioning by depletion of lymphocytes and NK cells and removal of cellular sinks for homeostatic cytokines IL-5 and IL-7, although the role of a more stringent conditioning regimen has not yet been resolved in ACT-TIL therapy.39-43 Additional specifics regarding the optimal protocol that are still under investigation include the IL-2 dosing schedule, the specificity and differentiation status of TILs, and the significance of neoantigen-specific TIL reactivity in improving the efficacy of ACT-TIL therapy.44-49Groundbreaking research from Rosenberg and colleagues at the Surgery Branch of the Center for Cancer Research at the National Cancer Institute (NCI) has spearheaded the translation of TILs and ACT into effective immunotherapies for cancer. Rosenberg was also at the forefront of chimeric antigen receptor (CAR) T-cell therapy, in which T cells isolated from a patient with cancer are engineered to express tumor antigen—specific receptors that facilitate elimination of tumor cells upon reintroduction. Two CAR T-cell therapies, tisagenlecleucel (Kymriah) and axicabtagene ciloleucel (Yescarta), have been FDA approved in hematological malignancies.
The successful signals from ACT-TIL therapy that Rosenberg and others developed in metastatic melanoma are thought to be due, in part, to the high acquired/somatic mutational load and the highly immunogenic nature of this cancer.50 The improvements in high-throughput genetic sequencing have added another layer in the TIL therapy paradigm: identification of TIL-targetable mutations in epithelial cancers, such as breast, esophageal, and ovarian cancers, which carry a lower somatic mutational load than skin cancers or may not be as immunogenic as advanced melanomas.50
The impressive efficacy of ACT-TIL therapy in metastatic melanoma is highlighted not only by higher ORRs (around 50%), but also durable and complete response (CR) rates (13%), which in some studies exceed those of other immunotherapies, such as checkpoint-blockade agents.1
The potential of ACT with TILs as a therapeutic option in breast cancer also is exciting, especially given the veritable explosion of data over the past decade from clinical trials and pooled analyses showing that TILs and their subtypes are independent prognostic indicators of clinical outcomes in 2 of the particularly aggressive and difficult-to-treat forms of breast cancer, TNBC and HER2-positive breast cancers.22,23,26,51-56 In fact, the significance of TILs in breast cancer resulted in the establishment of an International TILs Working Group for standardizing the methods for evaluating TILs.57 There are several reports of the efficacy of ACT-TIL therapy targeting epithelial cancer—specific somatic mutations in cholangiocarcinoma,58 lung metastases from CRC,59 and cervical cancer.60
Notably, a recent report from Rosenberg and colleagues is the first example of a clinical trial of ACT-TIL therapy in breast cancer.61 In this study, a patient with chemorefractory hormone-positive breast cancer was treated with ACT composed of TILs reactive against mutant versions of 4 proteins, SLC3A2, KIAA0368, CADPS2 and CTSB, in conjunction with IL-2 and checkpoint blockade; the patient has achieved complete durable regression of the metastatic breast cancer, currently ongoing for more than 22 months.61 These developments represent another frontier in the utilization of ACT-TIL therapy and may serve as a novel paradigm for treatment of intractable, multirefractory, or advanced epithelial cancers.
The main challenges to the optimization and wider use of ACT-TIL therapy include ameliorating treatment-related toxicities stemming from the use of high-dose IL-2, improving the quality and specificity of the infused cells while maintaining a reasonable expansion period, and devising strategies to minimize the immunosuppressive effects of the tumor microenvironment.1,62Currently, dozens of clinical trials of ACT-TIL therapy for treating various cancers, including melanoma, breast cancer, head and neck squamous cell carcinoma (HNSCC), non—small cell lung cancer (NSCLC), and hepatocellular carcinoma, appear on the ClinicalTrials.gov registry.
Iovance Biotherapeutics has emerged as a leader in the TIL therapy space with 2 drugs that are in phase II development. The company works in conjunction with investigators from the NCI, The University of Texas MD Anderson Cancer Center in Houston, Moffitt Cancer Center in Tampa, Florida, and other academic and industry partners.
The company’s top prospect is lifileucel (LN-144), which is under study in metastatic melanoma in a phase II trial that will be conducted at up to 60 investigational sites in the United States and Europe (NCT02360579). The trial seeks to enroll approximately 85 patients with stage IIIC/ IV melanoma that has progressed after prior anti— PD-1 therapy and, if appropriate, a BRAF inhibitor.
Participants will receive a nonmyeloablative (NMA) lymphodepletion preparative regimen consisting of cyclophosphamide (60 mg/kg for 2 days) and fludarabine (25 mg/m2 for 5 days), followed by lifileucel infusion and up to 6 doses of intravenous IL-2 (600,000 IU/kg).63 Participants may be eligible for TIL retreatment. One of the study’s goals will be to evaluate cryopreserved lifileucel, which can be manufactured in 22 days, a shorter timeframe than for noncryopreserved drugs. Historically, TIL manufacturing takes 5 to 6 weeks and yields a noncryopreserved drug, according to the company.
In interim findings from the study, the company reported a 40% ORR among 10 evaluable patients, all partial responses. Four other participants had stable disease. The most frequently reported grade 3/4 adverse events included anemia (58.8%) and febrile neutropenia (47.1%). Decreases in neutrophil and platelet counts of any grade also were reported in nearly 60% of patients, as was fatigue.64 Another of Iovance’s TIL therapies, LN-145, is being evaluated in 4 phase II clinical trials: HNSCC (NCT03083873)—An NMA lymphodepletion preparative regimen will be administered, followed by LN-145 and IL-2 in an estimated 47 patients with previously treated recurrent and/or metastatic disease.
Cervical cancer (NCT03108495)—NMA lymphodepletion followed by LN-145 and IL-2 in an estimated 47 patients with recurrent, metastatic, or persistent cervical cancer who were unresponsive to or failing prior therapy.
NSCLC (NCT03419559)—LN-145 alone or with the PD-L1 inhibitor durvalumab (Imfinzi) in patients with stage III/IV NSCLC who are checkpoint therapy-naïve and who have received ≥ 1 line of prior systemic therapy in the locally advanced or metastatic setting. Investigators are seeking 12 patients for each cohort. All patients would start with NMA lymphodepletion therapy and also would receive IL-2.
Multiple tumor types (NCT03645928)—LN-145 in combination with the PD-1 inhibitor pembrolizumab (Keytruda) in patients with advanced HNSCC who have not received prior checkpoint blockade therapy and as a single agent in patients with NSCLC who have received prior anti—PD-1/PD-L1 drugs. All patients would start with NMA lymphodepletion therapy and also would receive IL-2.
In metastatic cervical cancer, LN-145 therapy has the potential to significantly improve the prospects for a patient population with few options, according to Amir A. Jazaeri, MD, an associate professor at The University of Texas MD Anderson Cancer Center who is leading the study at his institution. He said the groundwork for testing TIL-ACT therapy in the malignancy was established in an NCI study in which TILs selected for reactivity to human papillomavirus E6 and E7 demonstrated responses in 3 of 9 patients, including 2 CRs.46 Jazaeri said there have been durable responses of more than 40 months in patients treated with the TILs.
“This type of therapy has the potential for long-term remission, which would be a revolutionary finding for patients with recurrent cervix cancer,” Jazaeri, who also is director of the Gynecologic Cancer Immunotherapy Program in the Department of Gynecologic Oncology and Reproductive Medicine, said in an interview with OncologyLive®. “It’s truly a potentially significant and revolutionary change, but of course we have to wait on the results of the trial. It’s exciting because we’re now trying it in other patient populations.”
The promise of TIL in oncology is being recognized more widely, as evidenced by the newly established biopharmaceutical company Optera Therapeutics Corp, a combined venture of The University of Texas MD Anderson Cancer Center in Houston and Berkeley Lights, Inc.65
Another company, TILT Biotherapeutics, is exploring the potential of oncolytic viruses in ACT-TIL therapy.66 TILT-123, which is in the preclinical stage, is a TNFα/IL2—armed oncolytic adenovirus that will be given in combination with TILs for patients with metastatic melanoma.
In developments outside of the United States, researchers from The Netherlands Cancer Institute and colleagues have launched a phase III study that will compare ipilimumab (Yervoy) with TIL therapy in an estimated 168 patients with stage IV metastatic melanoma (NCT02278887). Participants in the TIL arm also will receive NMA therapy plus IL-2.
Although recent technological advancements in immune checkpoint inhibition and engineering of tumor-derived lymphocytes have helped circumnavigate the immune escape mechanisms of tumors, many questions remain to be answered. The full curative potential of ACT with TILs requires a better understanding of the TIL subtypes associated with tumors in individual patients, ideal tumor-specific antigens, and strategies to manage the balance between immune suppression/induction-mediated toxicities and to maximize the cytotoxic effects of TILs.
Treatment response is another crucial factor influenced by TILs in melanoma and breast, thyroid, lung, and bladder cancers, among others.25-33 For instance, the expression of PD-L1 and TILs correlated with pathological complete response to neoadjuvant chemotherapy in breast cancer.25 The correlation between immune checkpoint factors, such as PD-L1/2, and TILs has emerged as a potential predictor of response to immune checkpoint blockade therapy, in particular, in various cancers.25,28-30,33 For instance, a recent study found that the relative abundance of CD8-positive TILs predicted response to anti— PD-1 therapy in melanoma.30