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.

 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.

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.

 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.

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.

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.

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).

 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.

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.

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.

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.

Moreover, in TNBC, an increase in stromal TILs was correlated with significantly lower risk of recurrence or death, distant recurrence, and overall mortality.

 The examples cited here, along with other data, demonstrate a clear prognostic role for TILs in various cancers.

From Prognostic Indicator to Therapeutic Agent

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.

The fundamental protocol for ACT-TIL therapy, developed initially by Rosenberg and colleagues for metastatic melanoma,

 consists of metastasectomy followed by fragmenting of the resected lesions and microculturing in the presence of IL-2 to expand the TIL population (

). 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.

 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”

; 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.

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.

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.

Groundbreaking 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.

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.

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.

 There are several reports of the efficacy of ACT-TIL therapy targeting epithelial cancer—specific somatic mutations in cholangiocarcinoma,

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.

 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.

Currently, 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/m

for 5 days), followed by lifileucel infusion and up to 6 doses of intravenous IL-2 (600,000 IU/kg).

 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.

 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 

. “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.

Another company, TILT Biotherapeutics, is exploring the potential of oncolytic viruses in ACT-TIL therapy.

 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.

Verdegaal EME. Adoptive cell therapy: a highly successful individualized therapy for melanoma with great potential for other malignancies. 

 2016;39:90-95. doi: 10.1016/j.coi.2016.01.004.

Hinrichs CS, Rosenberg SA. Exploiting the curative potential of adoptive T-cell therapy for cancer. 

2014;257(1):56-71. doi:10.1111/imr.12132.

Zhang H, Chen J. Current status and future directions of cancer immunotherapy.

 2018;9(10):1773-1781. doi: 10.7150/jca.24577.

Schreiber RD, Old LJ, Smyth MJ. Cancer immunoediting: integrating immunity’s roles in cancer suppression and promotion.

2011;331(6024):1565-1570. doi: 10.1126/science.1203486.

Dunn GP, Old LJ, Schreiber RD. The three Es of cancer immunoediting. 

 2004;22:329-360. doi: 10.1146/annurev.immunol.22.012703.104803.

Rosenberg SA, Packard BS, Aebersold PM, et al. Use of tumor-infiltrating lymphocytes and interleukin-2 in the immunotherapy of patients with metastatic melanoma. a preliminary report. 

 1988;319(25):1676-1680. ncbi.nlm.nih.gov/pubmed/3264384.

Geukes Foppen MH, Donia M, Svane IM, Haanen JB. Tumor-infiltrating lymphocytes for the treatment of metastatic cancer. 

 2015;9(10):1918-1935. doi: 10.1016/j.molonc.2015.10.018.

Clemente CG, Mihm MC Jr, Bufalino R, Zurrida S, Collini P, Cascinelli N. Prognostic value of tumor infiltrating lymphocytes in the vertical growth phase of primary cutaneous melanoma. 

Santoiemma PP, Powell DJ Jr. Tumor infiltrating lymphocytes in ovarian cancer. 

 2015;16(6):807-820. doi: 10.1080/15384047.2015.1040960.

Tuthill RJ, Unger JM, Liu PY, Flaherty LE, Sondak VK; Southwest Oncology Group. Risk assessment in localized primary cutaneous melanoma: a Southwest Oncology Group study evaluating nine factors and a test of the Clark logistic regression prediction model. 

 2002;118(4):504-511. doi: 10.1309/WBF7-N8KH-71KT-RVQ9.

Zhang L, Conejo-Garcia JR, Katsaros D, et al. Intratumoral T cells, recurrence, and survival in epithelial ovarian cancer. 

2003;348(3):203-213. doi: 10.1056/NEJMoa020177.

Pagès F, Galon J, Dieu-Nosjean MC, Tartour E, Sautès-Fridman C, Fridman WH. Immune infiltration in human tumors: a prognostic factor that should not be ignored. 

 2010;29(8):1093-1102. doi: 10.1038/onc.2009.416.

Naito Y, Saito K, Shiiba K, et al. CD8+ T cells infiltrated within cancer cell nests as a prognostic factor in human colorectal cancer. 

Pagès F, Berger A, Camus M, et al. Effector memory T cells, early metastasis, and survival in colorectal cancer. 

 2005;353(25):2654-2666. doi: 10.1056/NEJMoa051424.

Donnem T, Kilvaer TK, Andersen S, et al. Strategies for clinical implementation of TNM-Immunoscore in resected nonsmall-cell lung cancer. 

 2016;27(2):225-232. doi: 10.1093/annonc/mdv560.

Mlecnik B, Tosolini M, Kirilovsky A, et al. Histopathologic-based prognostic factors of colorectal cancers are associated with the state of the local immune reaction.

 2011;29(6):610-618. doi: 10.1200/JCO.2010.30.5425.

Deschoolmeester V, Baay M, Van Marck E, et al. Tumor infiltrating lymphocytes: an intriguing player in the survival of colorectal cancer patients. 

 2010;11:19. doi: 10.1186/1471-2172-11-19.

Galon J, Pagès F, Marincola FM, et al. Cancer classification using the Immunoscore: a worldwide task force. 

2012;10:205. doi: 10.1186/1479-5876-10-205.

Hwang WT, Adams SF, Tahirovic E, Hagemann IS, Coukos G. Prognostic significance of tumor-infiltrating T cells in ovarian cancer: a meta-analysis. 

 2012;124(2):192-198. doi: 10.1016/j.ygyno.2011.09.039.

Sato E, Olson SH, Ahn J, et al. Intraepithelial CD8+ tumor-infiltrating lymphocytes and a high CD8+/regulatory T cell ratio are associated with favorable prognosis in ovarian cancer. 

. 2005;102(51):18538-18543. doi: 10.1073/pnas.0509182102.

West NR, Milne K, Truong PT, Macpherson N, Nelson BH, Watson PH. Tumor-infiltrating lymphocytes predict response to anthracycline-based chemotherapy in estrogen receptor-negative breast cancer. 

Denkert C, Loibl S, Noske A, et al. Tumor-associated lymphocytes as an independent predictor of response to neoadjuvant chemotherapy in breast cancer. 

 2010;28(1):105-113. doi: 10.1200/JCO.2009.23.7370.

Loi S, Sirtaine N, Piette F, et al. Prognostic and predictive value of tumor-infiltrating lymphocytes in a phase III randomized adjuvant breast cancer trial in node-positive breast cancer comparing the addition of docetaxel to doxorubicin with doxorubicin-based chemotherapy: BIG 02-98. 

 2013;31(7):860-867. doi: 10.1200/JCO.2011.41.0902.

Adams S, Gray RJ, Demaria S, et al. Prognostic value of tumor-infiltrating lymphocytes in triple-negative breast cancers from two phase III randomized adjuvant breast cancer trials: ECOG 2197 and ECOG 1199.

2014;32(27):2959-2966. doi: 10.1200/JCO.2013.55.0491.

Wimberly H, Brown JR, Schalper K, et al. PD-L1 expression correlates with tumor-infiltrating lymphocytes and response to neoadjuvant chemotherapy in breast cancer. 

 2015;3(4):326-332. doi: 10.1158/2326-6066.CIR-14-0133.

Chen TH, Zhang YC, Tan YT, et al. Tumor-infiltrating lymphocytes predict prognosis of breast cancer patients treated with anti-HER-2 therapy. 

2017;8(3):5219-5232. doi: doi: 10.18632/oncotarget.14124.

Ingold Heppner B, Untch M, Denkert C, et al. Tumor-infiltrating lymphocytes: a predictive and prognostic biomarker in neoadjuvant-treated HER2-positive breast cancer. 

. 2016;22(23):5747-5754. doi: 10.1158/1078-0432.CCR-15-2338.

Bastman JJ, Serracino HS, Zhu Y, et al. Tumor-infiltrating T cells and the PD-1 checkpoint pathway in advanced differentiated and anaplastic thyroid cancer. 

 2016;101(7):2863-2873. doi: 10.1210/jc.2015-4227.

Sridharan V, Gjini E, Liao X, et al. Immune profiling of adenoid cystic carcinoma: PD-L2 expression and associations with tumor-infiltrating lymphocytes. 

. 2016;4(8):679-687. doi: 10.1158/2326-6066.CIR-16-0031.

Daud AI, Loo K, Pauli ML, et al. Tumor immune profiling predicts response to anti-PD-1 therapy in human melanoma. 

. 2016;126(9):3447-3452. doi: 10.1172/JCI87324.

Pichler R, Fritz J, Zavadil C, Schäfer G, Culig Z, Brunner A. Tumor-infiltrating immune cell subpopulations influence the oncologic outcome after intravesical Bacillus Calmette-Guérin therapy in bladder cancer. 

2016;7(26):39916-39930. doi: 10.18632/oncotarget.9537.

Yu X, Zhang Z, Wang Z, Wu P, Qiu F, Huang J. Prognostic and predictive value of tumor-infiltrating lymphocytes in breast cancer: a systematic review and meta-analysis. 

 2016;18(5):497-506. doi: 10.1007/s12094-015-1391-y.

Jacquelot N, Roberti MP, Enot DP, et al. Predictors of responses to immune checkpoint blockade in advanced melanoma.

 2017;8(1):592. doi: 10.1038/s41467-017-00608-2.

Fournier C, Martin F, Zitvogel L, Kroemer G, Galluzzi L, Apetoh L. Trial watch: adoptively transferred cells for anticancer immunotherapy. 

2017;6(11):e1363139. doi: 10.1080/2162402X.2017.1363139.

Chandran SS, Somerville RPT, Yang JC, et al. Treatment of metastatic uveal melanoma with adoptive transfer of tumour-infiltrating lymphocytes: a single-centre, two-stage, single-arm, phase 2 study. 

 2017;18(6):792-802. doi: 10.1016/S1470-2045(17)30251-6.

Khammari A, Knol AC, Nguyen JM, et al. Adoptive TIL transfer in the adjuvant setting for melanoma: long-term patient survival. 

 2014;2014:186212. doi: 10.1155/2014/186212.

Rosenberg SA, Yannelli JR, Yang JC, et al. Treatment of patients with metastatic melanoma with autologous tumor-infiltrating lymphocytes and interleukin 2. 

Dudley ME, Gross CA, Langhan MM, et al. CD8+ enriched “young” tumor infiltrating lymphocytes can mediate regression of metastatic melanoma. 

 2010;16(24):6122-6131. ncbi.nlm.nih.gov/pmc/articles/PMC2978753/pdf/nihms224664.pdf.

Gattinoni L, Finkelstein SE, Klebanoff CA, et al. Removal of homeostatic cytokine sinks by lymphodepletion enhances the efficacy of adoptively transferred tumor-specific CD8+ T cells. 

. 2005;202(7):907-912. doi: 10.1158/1078-0432.CCR-10-1297.

Wallen H, Thompson JA, Reilly JZ, Rodmyre RM, Cao J, Yee C. Fludarabine modulates immune response and extends in vivo survival of adoptively transferred CD8 T cells in patients with metastatic melanoma. 

 2009;4(3):e4749. doi: 10.1371/journal.pone.0004749.

Dudley ME, Wunderlich JR, Yang JC, et al. Adoptive cell transfer therapy following non-myeloablative but lymphodepleting chemotherapy for the treatment of patients with refractory metastatic melanoma.

2005;23(10):2346-2357. doi: 10.1200/JCO.2005.00.240.

Dudley ME, Yang JC, Sherry R, et al. Adoptive cell therapy for patients with metastatic melanoma: evaluation of intensive myeloablative chemoradiation preparative regimens.

2008;26(32):5233-5239. doi: 10.1200/JCO.2008.16.5449.

Goff SL, Dudley ME, Citrin DE, et al. Randomized, prospective evaluation comparing intensity of lymphodepletion before adoptive transfer of tumor-infiltrating lymphocytes for patients with metastatic melanoma. 

 2016;34(20):2389-2397. doi: 10.1200/JCO.2016.66.7220.

Yee C, Thompson JA, Byrd D, et al. Adoptive T cell therapy using antigen-specific CD8+ T cell clones for the treatment of patients with metastatic melanoma: in vivo persistence, migration, and antitumor effect of transferred T cells. 

2002;99(25):16168-16173. doi: 10.1073/pnas.242600099.

Chacon JA, Sarnaik AA, Chen JQ, et al. Manipulating the tumor microenvironment ex vivo for enhanced expansion of tumor-infiltrating lymphocytes for adoptive cell therapy. 

. 2015;21(3):611-621. doi: 10.1158/1078-0432.CCR-14-1934.

Stevanovi&#263; S, Draper LM, Langhan MM, et al. Complete regression of metastatic cervical cancer after treatment with human papillomavirus-targeted tumor-infiltrating T cells. 

Andersen R, Donia M, Westergaard MC, Pedersen M, Hansen M, Svane IM. Tumor infiltrating lymphocyte therapy for ovarian cancer and renal cell carcinoma. 

 2015;11(12):2790-2795. doi: 10.1080/21645515.2015.1075106.

Hall M, Liu H, Malafa M, et al. Expansion of tumor-infiltrating lymphocytes (TIL) from human pancreatic tumors. 

. 2016;4:61. doi: 10.1186/s40425-016-0164-7.

Fernandez-Poma SM, Salas-Benito D, Lozano T, et al. Expansion of tumor-infiltrating CD8+ T cells expressing PD-1 improves the efficacy of adoptive T-cell therapy. 

 2017;77(13):3672-3684. doi: 10.1158/0008-5472.CAN-17-0236.

Chalmers ZR, Connelly CF, Fabrizio D, et al. Analysis of 100,000 human cancer genomes reveals the landscape of tumor mutational burden. 

. 2017;9(1):34. doi: 10.1186/s13073-017-0424-2.

Dushyanthen S, Beavis PA, Savas P, et al. Relevance of tumor-infiltrating lymphocytes in breast cancer. 

2015;13:202. doi: 10.1186/s12916-015-0431-3.

Lee HJ, Kim JY, Park IA, et al. Prognostic significance of tumor-infiltrating lymphocytes and the tertiary lymphoid structures in HER2-positive breast cancer treated with adjuvant trastuzumab. 

 2015;144(2):278-288. doi: 10.1309/AJCPIXUYDVZ0RZ3G.

Miyashita M, Sasano H, Tamaki K, et al. Prognostic significance of tumor-infiltrating CD8+ and FOXP3+ lymphocytes in residual tumors and alterations in these parameters after neoadjuvant chemotherapy in triple-negative breast cancer: a retrospective multicenter study. 

 2015;17:124. doi: 10.1186/s13058-015-0632-x.

Kotoula V, Chatzopoulos K, Lakis S, et al. Tumors with high-density tumor infiltrating lymphocytes constitute a favorable entity in breast cancer: a pooled analysis of four prospective adjuvant trials. 

2016;7(4):5074-5087. doi: 10.18632/oncotarget.6231.

Perez EA, Ballman KV, Tenner KS, et al. Association of stromal tumor-infiltrating lymphocytes with recurrence-free survival in the N9831 adjuvant trial in patients with early-stage HER2-positive breast cancer. 

2016;2(1):56-64. doi: 10.1001/jamaoncol.2015.3239.

Pruneri G, Gray KP, Vingiani A, et al. Tumor-infiltrating lymphocytes (TILs) are a powerful prognostic marker in patients with triple-negative breast cancer enrolled in the IBCSG phase III randomized clinical trial 22-00. 

. 2016;158(2):323-331.  doi: 10.1007/s10549-016-3863-3.

Articles

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.

TILs Show Growing Potential as Novel Immunotherapy

Less than 5 years after Bruton tyrosine kinase (BTK) inhibition was introduced in hematologic malignancies, the need for strategies to address primary and secondary mechanisms of resistance has emerged.

The robust potency of ibrutinib (Imbruvica), which, in December 2013, became the first BTK inhibitor to gain FDA approval, has paved the way for second-generation agents with improved specificity profiles and expanded the potential for adding the drug to novel combination therapies. Ibrutinib use in a wider range of patients has also revealed the significance of resistance mechanisms and the need for options to manage ibrutinib-resistant cancers.

Figure. BCR Signaling and Key Factors Involved in Ibrutinib Resistance

In October, the FDA approved acalabrutinib (Calquence), another BTK inhibitor, as second-line therapy in mantle cell lymphoma (MCL). Several other BTK-targeting drugs, including agents designed to address resistance mutations, are being investigated in clinical studies (

The identification of BTK as a target for anticancer therapy stems from its involvement in aberrant B-cell receptor (BCR) signaling, which plays a central role in B-cell malignancies. BCR signaling is initiated by antigen binding, resulting in receptor aggregation and subsequent phosphorylation of the cytoplasmic tyrosine-based activation motifs in BCR by the SRC family kinases SYK and LYN.

This helps drive BTK to amplify and transmit BCR signaling, through phosphorylation of phospholipase C gamma 2 (PLC-gamma-2), mobilization of calcium secondary messenger, and activation of transcriptional programs driven by the nuclear factor &#1082;-B (NF—

B), AKT, RAS, mitogen-activated protein kinase, and nuclear factor of activated T cells pathways, ultimately promoting B-cell proliferation and survival (

BTK is a member of the highly conserved TEC kinase family.

 loss-of-function mutations result in X-linked agammaglobulinemia, characterized by absence of B cells, low serum immunoglobulin levels, and recurring infections, all consequences of impaired B-cell development. In addition to its role in BCR signaling, BTK is involved in chemokine-receptor, toll-like receptor, and Fc-receptor signaling in B cells.

The expression of BTK in B-cell malignancies and its pivotal role in the BCR signaling cascade, B-cell development, and lymphomagenesis mark BTK as a unique druggable target, providing a compelling rationale for use of BTK inhibitors in hematological malignancies.

Ibrutinib was initially approved for patients with MCL as a secondline monotherapy and has since gained indications in chronic lymphocytic leukemia (CLL)/small lymphocytic leukemia (SLL), and Waldenström macroglobulinemia (WM) as first-line therapy, as well as in marginal zone lymphoma (MZL) and chronic graft versus host disease after at least 1 prior therapy.

Ibrutinib inactivates BTK by binding covalently to cysteine 481 (C481) within the ATP-binding pocket in the kinase domain, acting as an irreversible inhibitor.

 Ibrutinib—BTK binding inhibits phosphorylation of BTK and its downstream targets and abrogates downstream BCR signaling. The potent activity of ibrutinib, first in MCL and subsequently in CLL, paired with the drugs relative tolerability underscores a paradigm shift in the treatment of aggressive B-cell malignancies.

In CLL, ibrutinib has been a transformative therapy. The FDA approval of ibrutinib in CLL, for patients who had received at least 1 prior therapy and for previously untreated patients, was based on significantly improved overall survival (OS) compared with standard-of-care therapies.

In the first-line setting in CLL, the OS rate at 24 months among participants in the phase III RESONATE-2 study was 98% for patients treated with ibrutinib versus 85% among those treated with standard-of-care chlorambucil, which translated to an 84% reduction in the risk of death (HR, 0.16; 95% CI, 0.05-0.56; 

In, improvements were noted in progression- free survival (PFS) and overall response rates (ORRs) in refractory/relapsed B-cell malignancies, including CLL and MCL.

In previously treated patients with WM, ibrutinib was found to be highly active (ORR, 90.5%) and was associated with durable responses.

 Ibrutinib induced durable responses (ORR, around 50%) with a favorable benefit—risk profile in patients with previously treated MZL.

Adverse events (AEs) with ibrutinib in clinical trials have mostly been limited to grade 1 or 2 toxicities; however, ibrutinib is associated with high bleeding risk, with major bleeding events reported in around 3% of patients receiving the drug. Careful monitoring and concomitant anticoagulant and antiplatelet agents have been used to mitigate these AEs. Additionally, atrial fibrillation has been observed in up to 16% of patients.

Investigators hypothesize that ibrutinib’s toxicity profile stems from off-target inhibition of other kinases, including those of TEC, EGFR, and TXK.

 Another notable characteristic in CLL with ibrutinib treatment is the development of lymphocytosis, because CLL cells mobilize from lymph nodes and the spleen. Although ibrutinib-associated lymphocytosis typically resolves within 8 months, it may persist past 12 months in some patients, concomitant with a continued response to ibrutinib.

Two mechanisms account for ibrutinib’s clinical efficacy: inhibition of intrinsic B-cell signaling pathways that impede proliferation and survival and disruption of tumor—microenvironment interactions.

In CD40- or BCR-activated CLL cells, ibrutinib inhibits the ERK, PI3K, and NF-&#1082;B pathways, thereby suppressing cell survival.

 Ibrutinib also impedes migration of CLL cells toward chemokines and suppresses BCR-dependent chemokine secretion, suggesting that its clinical activity involves interfering with relocation and retention of malignant cells in their survival niches.

In pivotal clinical trials across multiple indications, ibrutinib has demonstrated high response rates. These range from an ORR of 42.6% as a single agent in the phase III RESONATE study in patients with relapsed/refractory CLL/SLL to 82.7% in combination with bendamustine and rituximab (Rituxan) in the phase III HELIOS study in a similar patient population.

Despite the clinical efficacy of ibrutinib, primary and secondary resistance has been reported.

 As a larger proportion of patients undergo treatment with this drug, the mechanisms of resistance and salvage or combination therapies for ibrutinib- resistant cancers have taken center stage in aggressive leukemia/lymphoma management. One-third of patients with MCL exhibit primary resistance to ibrutinib; moreover, further resistance develops with continued therapy, despite substantial response rates.

To date, bypass mutations in or sustained activation of downstream factors, rather than mutations in 

, have been identified as drivers of primary resistance to ibrutinib.

 For instance, sustained downstream PI3K/AKT activity, rather than BTK activity, correlated with resistance to ibrutinib in MCL.

 Gene-sequencing analysis of ibrutinibresistant MCL cells identified mutations in effectors of the BTK-independent nonclassical NF-&#1082;B pathway.

 A study evaluating ibrutinib resistance in ABC-subtype diffuse large B-cell lymphoma (DLBCL) found that aberrant activation of the NF-&#1082;B pathway, driven by mutations in myeloid differentiation primary response 88 (MYD88), was associated with primary ibrutinib resistance.

Acquired mutations, arising after malignant clone persistence, have been described in secondary resistance to reversible kinase inhibitors such as BCR-ABL kinase—directed imatinib (Gleevec) and EGFR-directed erlotinib (Tarceva).

 Recent studies highlight the importance of mutations in 

 and its downstream targets in secondary resistance to ibrutinib.

Less than 5 years after BTK inhibition was introduced in hematologic malignancies, the need for strategies to address primary and secondary mechanisms of resistance has emerged.

Krithika Subramanian, PhD

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