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Engineered T cell therapy has shown remarkable efficacy in hematologic malignancies and has the potential for application to common epithelial cancers. Diverse T cell therapy strategies including adoptive transfer of tumor-infiltrating lymphocytes, chimeric antigen receptor (CAR)-T cells, and T cell receptor (TCR)-T cells have been studied in clinical trials. Recent research has established treatment of human papillomavirus (HPV)-associated cancers with TCR-T cells as a model for proof-of-principle studies in epithelial cancers. These studies and others have provided critical insight into mechanisms of tumor regression, therapeutic targets, treatment safety, treatment design, and barriers to curative cell therapies for common types of cancer. This perspective will review and consolidate understanding gained from clinical trials to treat viral and non-viral epithelial cancers with cell and gene therapy and will examine how past experience may guide future strategy in treatment and biomarker discovery.

BackgroundCell therapy has shown promise in the treatment of certain solid tumors, but its efficacy may be limited by inhibition of therapeutic T cells by the programmed cell death protein-1 (PD-1) receptor. Clinical trials are testing cell therapy in combination with PDCD1 disruption or PD-1-axis blockade. However, preclinical data to support these approaches and to guide the treatment design are lacking.MethodsMechanisms of tumor regression and interaction between cell therapy and PD-1 blockade were investigated in congenic murine tumor models based on targeting established, solid tumors with T-cell receptor T cells directed against tumor-restricted, non-self antigens (ie, tumor neoantigens).ResultsIn solid tumor models of cell therapy, PD-1 blockade mediated a reproducible but non-synergistic increase in tumor regression following adoptive T-cell transfer. Tumor regression was associated with increased tumor infiltration by endogenous T cells but not by transferred T cells. The effect was independent of PD-1 receptor expression by transferred T cells and was dependent on the endogenous T-cell repertoire and on tumor antigenicity. PD-1 blockade primarily induced cell state changes in endogenous tumor-antigen-specific T cells rather than transferred T cells.ConclusionsTogether, these findings support the concept that PD-1 blockade acts primarily through endogenous rather than transferred T cells to mediate a non-synergistic antitumor effect in solid tumor cell therapy. These findings have important implications for strategies to leverage PD-1 receptor disruption or blockade to enhance the efficacy of cell therapy.

PURPOSE Genetically engineered T-cell therapy is an emerging treatment of hematologic cancers with potential utility in epithelial cancers. We investigated T-cell therapy for the treatment of metastatic human papillomavirus (HPV)–associated epithelial cancers. METHODS This phase I/II, single-center trial enrolled patients with metastatic HPV16-positive cancer from any primary tumor site who had received prior platinum-based therapy. Treatment consisted of autologous genetically engineered T cells expressing a T-cell receptor directed against HPV16 E6 (E6 T-cell receptor T cells), a conditioning regimen, and systemic aldesleukin. RESULTS Twelve patients were treated in the study. No dose-limiting toxicities were observed in the phase I portion. Two patients, both in the highest-dose cohort, experienced objective tumor responses. A patient with three lung metastases experienced complete regression of one tumor and partial regression of two tumors, which were subsequently resected; she has no evidence of disease 3 years after treatment. All patients demonstrated high levels of peripheral blood engraftment with E6 T-cell receptor T cells 1 month after treatment (median, 30%; range, 4% to 53%). One patient’s resistant tumor demonstrated a frameshift deletion in interferon gamma receptor 1, which mediates response to interferon gamma, an essential molecule for T-cell–mediated antitumor activity. Another patient’s resistant tumor demonstrated loss of HLA-A*02:01, the antigen presentation molecule required for this therapy. A tumor from a patient who responded to treatment did not demonstrate genetic defects in interferon gamma response or antigen presentation. CONCLUSION Engineered T cells can induce regression of epithelial cancer. Tumor resistance was observed in the context of T-cell programmed death-1 expression and defects in interferon gamma and antigen presentation pathway components. These findings have important implications for development of cellular therapy in epithelial cancers.

T cell receptor (TCR) gene-engineered T cells have shown promise in the treatment of melanoma and synovial cell sarcoma, but their application to epithelial cancers has been limited. The identification of novel therapeutic TCRs for the targeting of these tumors is important for the development of new treatments. Here, we describe the preclinical characterization of a TCR directed against Kita-Kyushu Lung Cancer Antigen-1 (KK-LC-1, encoded by CT83), a cancer germline antigen with frequent expression in human epithelial malignancies including gastric cancer, breast cancer, and lung cancer. Gene-engineered T cells expressing the KK-LC-1 TCR (KK-LC-1 TCR-Ts) demonstrated recognition of CT83+ tumor lines in vitro and mediated regression of established CT83+ xenograft tumors in immunodeficient mouse models. Cross-reactivity studies based on experimental determination of the recognition motifs for the target epitope did not demonstrate cross-reactivity against other human proteins. CT83 gene expression studies in 51 non-neural tissues and 24 neural tissues showed expression restricted exclusively to germ cells. CT83 was however expressed by a range of epithelial cancers, with the highest expression noted in gastric cancer. Collectively, these findings support the further investigation and clinical testing of KK-LC-1 TCR-Ts for gastric cancer and possibly other malignancies. Electronic supplementary material The online version of this article (10.1186/s40425-019-0678-x) contains supplementary material, which is available to authorized users.

Background The objective of this study was to characterize the clinicopathologic features of sicca syndrome associated with immune checkpoint inhibitor (ICI) therapy. Subjects, Materials, and Methods Consecutive patients with new or worsening xerostomia in the setting of ICI treatment for benign or malignant neoplastic disease were evaluated, including labial salivary gland biopsy (LSGB). Results Twenty patients (14 male; median age 57 years) had metastatic melanoma (n = 10), metastatic carcinoma (n = 6), or recurrent respiratory papillomatosis (n = 4) and were being treated with avelumab (n = 8), nivolumab (n = 5), pembrolizumab (n = 4), nivolumab/ipilimumab (n = 2), and M7824, a biologic targeting programmed cell death ligand 1 (PD-L1) and transforming growth factor ß (n = 1). Four had pre-existing autoimmune disease. Nineteen had very low whole unstimulated saliva flow; six had new dry eye symptoms. The median interval between ICI initiation and dry mouth onset was 70 days. Rheumatoid factor and anti-Sjögren's Syndrome-related Antigen A (Anti-SSA) were both positive in two subjects. LSGB showed mild-to-severe sialadenitis with diffuse lymphocytic infiltration and architectural distortion. There were lymphocytic aggregates in eight patients, composed mainly of CD3+ T cells with a slight predominance of CD4+ over CD8+ T cells. ICI targets (e.g., programmed cell death 1 and PD-L1) were variably positive. In direct response to the advent of the sicca immune-related adverse event, the ICI was held in 12 patients and corticosteroids were initiated in 10. Subjective improvement in symptoms was achieved in the majority; however, salivary secretion remained very low. Conclusion ICI therapy is associated with an autoimmune-induced sicca syndrome distinct from Sjögren's syndrome, often abrupt in onset, usually developing within the first 3 months of treatment, and associated with sialadenitis and glandular injury. Improvement can be achieved with a graded approach depending on severity, including withholding the ICI and initiating corticosteroids. However, profound salivary flow deficits may be long term. Implications for Practice Sicca syndrome has been reported as an immune-related adverse event (irAE) of immune checkpoint inhibitor therapy (ICI) for neoplastic diseases. Severe dry mouth (interfering with eating or sleeping) developed abruptly, typically within 90 days, after initiation of ICI therapy. Salivary gland biopsies demonstrated mild-to-severe sialadenitis distinct from Sjögren's syndrome, with diffuse T-cell lymphocytic infiltration and acinar injury. Recognition of the cardinal features of ICI-induced sicca will spur appropriate clinical evaluation and management, including withholding of the ICI and corticosteroid, initiation. This characterization should help oncologists, rheumatologists, and oral medicine specialists better identify patients that develop ICI-induced sicca to initiate appropriate clinical evaluation and therapy to reduce the likelihood of permanent salivary gland dysfunction.

TPS2678 Background: T cell receptor (TCR)-T cell therapy is an emerging cancer treatment strategy. Thus far, demonstration of clinical activity has been limited to a subset of solid tumors including melanoma, synovial cell sarcoma and HPV-associated cancer. It is estimated that metastatic epithelial cancers are responsible for approximately 90% of cancer deaths in the United States. The estimated 600,000 cancer deaths each year is driven largely by lung adeno- and squamous cell carcinoma and invasive breast cancer, which account for approximately 30% of all cancer-related deaths. Kita-Kyushu Lung Cancer Antigen 1 (KK-LC-1) is a cancer germline antigen with expression restricted to germ cells in adults and certain epithelial cancers including lung, breast, gastric and cervical. We identified a KK-LC-1 TCR from the tumor-infiltrating lymphocytes (TIL) of a patient with cervical cancer who had a complete tumor response to TIL therapy. The KK-LC-1 TCR was the most dominate clonotype in the infused TIL product and persisted in the peripheral blood following infusion, suggesting that it may have contributed to cancer regression in this patient. Methods: We are conducting a phase I cell dose escalation trial to test the safety and efficacy of KK-LC-1 TCR-T cell therapy in patients with metastatic KK-LC-1 positive epithelial cancer. Patients receive a lymphocyte depleting conditioning regimen followed by a one-time infusion of genetically engineered T cells expressing the KK-LC-1 TCR (KK-LC-1 TCR-T cells) and high-dose systemic aldesleukin. KK-LC-1 positivity is determined by RNAscope assay measuring the percentage of cancer cells expressing CT83 (gene encoding KK-LC-1) with a percentage of 25 or greater considered positive. Main inclusion criteria include HLA-A*01:01 allele, prior first-line therapy, ECOG of 0 or 1 and adequate organ and hematologic function. Main exclusion criteria include active major medical illness of the cardiovascular, respiratory or immune system, primary or secondary immunodeficiency and autoimmune disease. Participants will be entered in sequential dose levels and receive escalating doses of cells beginning at 1x108 and ending with 6x1010. Dose-limiting toxicities will be assessed during the first 30 days of cell infusion. The primary objective is to determine the maximally tolerated dose of KK-LC-1 TCR-T cells. Exploratory objectives include assessing the safety and efficacy of KK-LC-1 TCR-T cells and to conduct immunologic studies to understand and improve the administered treatment. Clinical trial information: NCT05035407.

Purpose: Circulating cell-free (ccf) human papillomavirus (HPV) DNA may serve as a unique tumor marker for HPV-associated malignancies, including cervical cancer. We developed a method to genotype and quantify circulating HPV DNA in patients with HPV16- or HPV18-positive metastatic cervical cancer for potential disease monitoring and treatment-related decision making. Experimental Design: In this retrospective study, HPV ccfDNA was measured in serum samples from 19 metastatic cervical cancer patients by duplex digital droplet PCR (ddPCR). Nine patients had received tumor-infiltrating lymphocyte (TIL) immunotherapy. ccfDNA data were aligned with the tumor HPV genotype, drug treatment, and clinical outcome. Results: In blinded tests, HPV ccfDNA was detected in 19 of 19 (100%) patients with HPV-positive metastatic cervical cancer but not in any of the 45 healthy blood donors. The HPV genotype harbored in the patients' tumors was correctly identified in 87 of 87 (100%) sequential patient serum samples from 9 patients who received TIL immunotherapy. In three patients who experienced objective cancer regression after TIL treatment, a transient HPV ccfDNA peak was detected 2–3 days after TIL infusion. Furthermore, persistent clearance of HPV ccfDNA was only observed in two patients who experienced complete response (CR) after TIL immunotherapy. Conclusions: HPV ccfDNA represents a promising tumor marker for noninvasive HPV genotyping and may be used in selecting patients for HPV type–specific T-cell-based immunotherapies. It may also have value in detecting antitumor activity of therapeutic agents and in the long-term follow-up of cervical cancer patients in remission. Clin Cancer Res; 23(22); 6856–62. ©2017 AACR.

Background Recurrent respiratory papillomatosis (RRP) is a human papillomavirus (HPV)-driven disorder that causes substantial morbidity and can lead to fatal distal airway obstruction and post-obstructive pneumonias. Patients require frequent surgical debridement of disease, and no approved systemic adjuvant therapies exist. Methods A phase II study was conducted to investigate the clinical activity and safety of programmed death-ligand 1 (PD-L1) blockade with avelumab in patients with RRP. Results Twelve patients were treated. All patients with laryngeal RRP displayed improvement in disease burden, and 5 of 9 (56%) displayed partial responses. None of 4 patients with pulmonary RRP displayed a response. Using each patient’s surgical history as their own control, patients required fewer surgical interventions after avelumab treatment (p = 0.008). A subset of partial responders developed HPV-specific reactivity in papilloma-infiltrating T-cells that correlated with reduced HPV viral load and an increased Tissue Inflammation Signature. Conclusions Avelumab demonstrated safety and clinical activity in patients with laryngeal RRP. Further study of immune checkpoint blockade for RRP, possibly with longer treatment duration or in combination with other immunotherapies aimed at activating antiviral immunity, is warranted. Trial registration NCT, number NCT02859454, registered August 9, 2016. Electronic supplementary material The online version of this article (10.1186/s40425-019-0603-3) contains supplementary material, which is available to authorized users.

BackgroundRecurrent respiratory papillomatosis (RRP) is a human papillomavirus (HPV) driven neoplastic disorder of the upper aerodigestive tract that causes significant morbidity and can lead to fatal airway obstruction. Prior clinical study demonstrated clinical benefit with the programmed death-ligand 1 (PD-L1) monoclonal antibody avelumab. Bintrafusp alfa is a bifunctional inhibitor of PD-L1 and transforming growth factor-beta (TGF-b) that has shown clinical activity in several cancer types.MethodsWe conducted a phase II clinical trial evaluating bintrafusp alfa in adults with RRP. Papilloma samples before and after treatment with bintrafusp alfa were assessed for correlates of response with multiplex immunofluorescence as well as immunological and genomic analyses. Post hoc analyses of papilloma samples before and after treatment with avelumab were assessed for comparison.ResultsDual PD-L1/TGF-b inhibition failed to abrogate papilloma growth in most subjects and increased the frequency of clinically indicated interventions after treatment in four of eight subjects based on each subject’s own historical control. TGF-b neutralization consistently decreased pSMAD3 and p21 and increased Ki67 expression within the basal layers of papillomas, indicating that TGF-b restrained proliferation. These alterations were not observed in papillomas treated with PD-L1 blockade alone. Dual PD-L1/TGF-b inhibition did not enhance anti-HPV immunity within papillomas beyond that observed with PD-L1 blockade. Genomic alterations in TGF-b superfamily genes were infrequent in papillomas and normal mucosa but present in a significant fraction of head and neck carcinomas.ConclusionsIntact TGF-b signaling restrains proliferation within papillomas, and the use of clinical agents that abrogate this pathway should be avoided in patients with RRP.Trial registration numbersNCT03707587 and NCT02859454.

Introduction: As T cell receptor (TCR) therapy emerges as a powerful therapy in cancer treatment, the field is still limited by a relatively small number of targets that are expressed widely in tumor cells but not expressed healthy normal tissues. Kita-Kyushu lung cancer antigen 1 (KK-LC-1) is a cancer-testis antigen that is expressed only in the testis of normal adult tissues. KK-LC-1 is widely expressed across a large percentage of solid tumors. Transcriptional profiling reveals expression in triple-negative breast (TNBC, 53% of tumors), non-small cell lung (NSCLC, 40%), gastric (81%), and cervical cancers (40%). T-Cure is developing a TCR targeting KK-LC-1 (820TCR) that is restricted to HLA-A*01, which is expressed in approximately 25-30% of the U.S. population. The 820TCR was identified to be specific for KK-LC-152-60 peptide NTDNNLAVY from a responder cervical carcinoma patient. Methods: KK-LC-1 gene expression was measured by qPCR in donor total RNA. Jurkat cells and PBMCs from 5 healthy donors were transduced with a retroviral construct containing the 820TCR, and transduction efficiency was measured by flow staining of the mouse TCR-B constant region. Cell-free binding assays were performed using A*01 tetramers loaded with KK-LC-152-60 peptide labeled with fluorescent markers assayed by flow. TCR specificity was measured by co-culture of transduced Jurkat and T cells with peptide pulsed APCs and on/off-target tumor cell panels. Off-target reactivity was assayed by co-culture of transduced T cells with a panel of normal primary cells across multiple organ systems. All co-cultures measured cytokine release by ELISA and multi-cytokine Luminex panels. Results: Gene expression analysis of RNA from a panel of normal primary donor tissues shows that KK-LC-1 expression is restricted to testis. Cell lines derived from affected histologies also maintain high KK-LC-1 expression in vitro. 820TCR transduced Jurkat and donor T cells showed over 85% of transduced T cells exhibited A*01-KK-LC-152-60 tetramer binding. 820TCR-transduced donor T cells displayed high specificity in co-culture with a panel of cell lines of mixed A*01 and KK-CL-1 status. Reactivity was observed in all naturally expressing KK-LC-1 lines that are A*01+. Reactivity was observed across a wide range of expression levels, and high expression was not required for tumor reactivity. No reactivity was observed in any non-A*01 HLA type or in A*01+/KK-LC-1- tumors. Reactivity could be induced in these cells by exogenous expression of A*01 in non-A*01/KK-LC1+ cells and KK-LC-1 in A*01/KK-LC-1- cells. Off target analysis revealed no reactivity of 820TCR with any normal primary cells. Conclusion: KK-LC-1 is a promising target for adoptive TCR cell therapy, and the 820TCR targeting KK-LC-1 is a highly potent and specific TCR with no observed off-target effects. A phase I clinical trial with 820TCR is being planned for patients with cervical, NSCLC, TNBC, and gastric cancers. Citation Format: Nicholas S. Davis, Catherine Leites, Helicia Paz, Leslie Ryan, Nathaniel Magilnick, Christian Hinrichs, Gang Zeng, Erika von Euw. KK-LC-1 targeting T cell receptor for adoptive T cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1496.

2501 Background: There are more than 630,000 cases of HPV associated malignancies including cervical, oropharyngeal and anal cancer worldwide annually. HPV 16 is responsible for the majority of these cases. About 15-20% of HPV associated malignancies respond to PD-(L)1 inhibitors, but for the overwhelming majority of patients who progress on these immunotherapies there is no effective standard of care therapy. Preclinical studies have shown that the triple combination of PDS0101 (Versamune-HPV), a liposomal multipeptide therapeutic vaccine targeting HPV 16 E6/E7, M9241, a tumor-targeting immunocytokine composed of IL-12 heterodimers fused to a monoclonal antibody targeting free DNA in necrotic tumor regions, and bintrafusp alfa, a bifunctional fusion protein targeting TGF-β and PD-L1, resulted in maximum HPV-specific T cell responses, T cell tumor infiltration and tumor reduction as compared to any one or two of these agents alone. Methods: Fourteen pts with HPV 16+ relapsed or refractory advanced cancer were enrolled to the triple combination of PDS0101, M9241 and bintrafusp alfa (NCT04287868). Pts received bintrafusp alfa at 1200 mg flat dose i.v. every 2 weeks, M9241 at 16.8 mcg/kg s.c. every 4 weeks and PDS0101 given as two separate 0.5 ml s.c. injections every 4 weeks. Dose reductions of M9241 to 8 mcg/kg were allowed as well as skipped doses of any agent for ongoing toxicities. Results: Fourteen pts with advanced HPV 16+ cancers (5 cervical, 2 vaginal/vulvar, 4 anal, 3 oropharyngeal) were treated. 4/14 (28.6%) pts had a grade 3 treatment related toxicity including grade 3 hematuria in 2 pts with cervical ca and prior pelvic radiation and grade 3 AST/ALT elevation in 2 pts, one with anal ca and one with vaginal ca. For one patient with grade 3 AST/ALT elevation dose reduction of M9241 from 16.8 to 8 mcg/kg allowed for continued treatment with AST/ALT remaining at grade 1 or less. One additional patient had transient asymptomatic grade 4 neutropenia. No other treatment related grade 3 or greater toxicities were noted. 10/14 (71%) pts have had objective responses: 1 CR (anal ca) and 9 PRs (3 cervical, 2 vulvar/vaginal, 2 anal, 2 oropharyngeal) with 9/10 of these responses ongoing after a median 5 month of follow up. Of the 14 pts, 6 pts have checkpoint naïve disease and 8 pts have checkpoint refractory disease. 5/6 (83%) pts with checkpoint naïve disease and 5/8 (63%) pts with checkpoint refractory disease have had objective responses. Analyses of immune responses and other immune correlates are ongoing. Conclusions: Triple combination of PDS0101, M9241 and bintrafusp alfa appears to have a manageable safety profile along with early evidence of notable clinical activity for pts with both checkpoint naïve as well as checkpoint refractory HPV 16+ advanced malignancies. Clinical trial information: NCT04287868.

BackgroundAs heterogeneous tumors develop in the face of intact immunity, tumor cells harboring genomic or expression defects that favor evasion from T-cell detection or elimination are selected. For patients with such tumors, T cell-based immunotherapy alone infrequently results in durable tumor control.MethodsHere, we developed experimental models to study mechanisms of T-cell escape and demonstrated that resistance to T-cell killing can be overcome by the addition of natural killer (NK) cells engineered to express a chimeric antigen receptor (CAR) targeting programmed death ligand-1 (PD-L1).ResultsIn engineered models of tumor heterogeneity, PD-L1 CAR-engineered NK cells (PD-L1 t-haNKs) prevented the clonal selection of T cell-resistant tumor cells observed with T-cell treatment alone in multiple models. Treatment of heterogenous cancer cell populations with T cells resulted in interferon gamma (IFN-γ) release and subsequent upregulation of PD-L1 on tumor cells that escaped T-cell killing through defects in antigen processing and presentation, priming escape cell populations for PD-L1 dependent killing by PD-L1 t-haNKs in vitro and in vivo.ConclusionsThese results describe the underlying mechanisms governing synergistic antitumor activity between T cell-based immunotherapy that results in IFN-γ production, upregulation of PD-L1 on T-cell escape cells, and the use of PD-L1 CAR-engineered NK cells to target and eliminate resistant tumor cell populations.

BackgroundBintrafusp alfa is a first-in-class bifunctional fusion protein composed of the extracellular domain of transforming growth factor (TGF)-βRII (a TGF-β ‘trap’) fused to a human IgG1 mAb blocking programmed cell death ligand 1. This is the largest analysis of patients with advanced, pretreated human papillomavirus (HPV)-associated malignancies treated with bintrafusp alfa.MethodsIn these phase 1 (NCT02517398) and phase 2 trials (NCT03427411), 59 patients with advanced, pretreated, checkpoint inhibitor-naive HPV-associated cancers received bintrafusp alfa intravenously every 2 weeks until progressive disease, unacceptable toxicity, or withdrawal. Primary endpoint was best overall response per Response Evaluation Criteria in Solid Tumors (RECIST) V.1.1; other endpoints included safety.ResultsAs of April 17, 2019 (phase 1), and October 4, 2019 (phase 2), the confirmed objective response rate per RECIST V.1.1 in the checkpoint inhibitor-naive, full-analysis population was 30.5% (95% CI, 19.2% to 43.9%; five complete responses); eight patients had stable disease (disease control rate, 44.1% (95% CI, 31.2% to 57.6%)). In addition, three patients experienced a delayed partial response after initial disease progression, for a total clinical response rate of 35.6% (95% CI, 23.6% to 49.1%). An additional patient with vulvar cancer had an unconfirmed response. Forty-nine patients (83.1%) experienced treatment-related adverse events, which were grade 3/4 in 16 patients (27.1%). No treatment-related deaths occurred.ConclusionBintrafusp alfa showed clinical activity and manageable safety and is a promising treatment in HPV-associated cancers. These findings support further investigation of bintrafusp alfa in patients with advanced, pretreated HPV-associated cancers.

101 Background: Genetically engineered T-cell therapy has shown remarkable clinical activity in hematologic malignancies. It is not known if this type of treatment can be applied effectively to epithelial cancers, which account for 80% to 90% of human malignancies. Methods: We conducted a phase I clinical trial with a 3 + 3 dose escalation in which patients with metastatic HPV-16+ epithelial cancers were treated with a one-time infusion of genetically engineered T cells expressing a T-cell receptor targeting an HLA-A*02:01-restricted epitope of HPV-16 E7 (E7 TCR-T cells). A lymphocyte-depleting conditioning regimen was administered before cell infusion, and high-dose systemic aldesleukin was administered after cell infusion. Results: Twelve patients, previously treated with a median of 4 (range, 3 to 7) anticancer agents, were treated. The cell dose was not limited by toxicity. Six patients demonstrated objective clinical responses, which included regression of bulky tumors and complete elimination of some tumors. Responses occurred in patients with vulvar, anal, head and neck, and cervical cancer. Four patients who previously received PD-1-based therapy responded. Response duration ranged from 3 to 9 months. Sustained, high-level engraftment of E7 TCR-T cells in peripheral blood was observed (median after approximately 6 weeks, 66% of total T cells, range 1% to 88%) and correlated with cell dose but not with clinical response. Infused T cell characteristics did not correlate strongly with response. Of the 4 resistant tumors that were studied, 3 demonstrated genetic defects in HLA-A*02:01 or B2M (necessary components of the target complex) and 1 demonstrated copy loss with decreased expression of antigen presentation and interferon response molecules (i.e. TAP1, TAP2, IFNGR1, IFNGR2). Of the 3 sensitive tumors studied, 0 showed genetic defects in these molecules. Conclusions: E7 TCR-T cells demonstrated safety and clinical activity in the treatment of highly refractory metastatic HPV-16+ cancers. Treatment resistance was linked to definitive genetic defects in the targeted peptide-HLA complex and to manifold defects in antigen processing and interferon response. Clinical trial information: NCT02858310.

Key Points Question Does the cross-reactivity of T cells against the human papillomavirus (HPV) oncoproteins in HPV-positive cancers support current immunotherapeutic vaccine clinical trial designs in which the HPV type of the tumor is not matched with the HPV type of the vaccine? Findings In an analysis of 16 samples, HPV-reactive tumor-infiltrating lymphocytes from patients with HPV-positive cancers displayed HPV-16/HPV-18 oncoprotein cross-reactivity in 1 of 16 samples. None of the 10 HPV-reactive T-cell receptors from the researchers’ library exhibited HPV-16/HPV-18 oncoprotein cross-reactivity. Meaning The low frequency of HPV-16/HPV-18 T-cell cross-reactivity supports clinical trial designs that match the HPV type of therapeutic vaccines to that of patient tumors., This study characterizes the prevalence of human papillomavirus 16 (HPV-16) and HPV-18 metastatic cervical cancers among National Cancer Institute (NCI) trial participants and the cross-reactivity of archived T cells between the 2 HPV types to determine whether treating patients with an HPV-16–targeted vaccine would be effective for women with HPV-18 cancers., Importance Clinical trials are testing vaccines that target human papillomavirus 16 (HPV-16) oncoproteins for the treatment of cervical cancer regardless of the HPV type of the tumor. For patients with HPV-18–positive cancers, this strategy relies on cross-reactivity of HPV-16–reactive T cells against the HPV-18 oncoproteins. Objectives To determine the prevalence of HPV-16 and HPV-18 metastatic cervical cancers in women enrolling in clinical trials at a US medical center and to assess whether HPV oncoprotein–targeting tumor-infiltrating lymphocytes (TILs) and T-cell receptors (TCRs) possess HPV-16/HPV-18 oncoprotein cross-reactivity. Design, Setting, and Participants This study was conducted at the National Institutes of Health Clinical Center, a tertiary care research hospital in the United States. The HPV type of the tumors from 65 consecutive patients with cervical cancer who were evaluated for participation in clinical trials was determined by retrospective medical record review. Immunological assays testing HPV cross-reactivity were conducted on all available archived samples of oncoprotein-reactive TILs from HPV-positive tumors (n = 16) and on a library of previously identified TCRs (n = 10). Interventions The HPV genotype of each patient’s tumor was determined. The cross-reactivity of archived TILs and a library of TCRs was assessed. Main Outcomes and Measures The main outcomes were the prevalence of each HPV genotype and the frequency of TILs or TCRs with HPV oncoprotein–T-cell cross-reactivity. Cross-reactivity was assessed by enzyme-linked immunospot assays and interferon-γ production assays. Results The median (range) age of 65 referred patients was 44 (24-64) years. Ethnicity was recorded for 39 of 65 patients; 35 (89.7%) were white, 3 (7.7%) were Asian, and 1 (2.6%) was American Indian/Alaskan Native. Histologic tumor subtype was recorded for 41 of 65 patients; 25 (61.0%) were squamous cell carcinomas, 12 (29.3%) were adenocarcinomas, 2 (4.9%) were adenosquamous cell carcinomas, and 2 (4.9%) were neuroendocrine tumors. Thirty-nine of 65 patients (60.0%) had HPV-16–positive tumors and 21 patients (32.3%) had HPV-18–positive tumors. In the analysis of cross-reactivity, 1 of 16 oncoprotein-reactive archived TILs (9 from cervical cancers and 7 from other cancers) displayed HPV-16/HPV-18 cross-reactivity. None of the 10 oncoprotein-reactive TCRs displayed HPV-16/HPV-18 cross-reactivity. Conclusions and Relevance Cervical cancers that tested positive for HPV-18 were common in this study and may be common in other US clinical trial populations. Results showed that HPV-16/HPV-18 intergenotype T-cell cross-reactivity of T cells from HPV-16–positive and HPV-18–positive cancers was uncommon. These findings support clinical trial designs in which the HPV type targeted by a therapeutic vaccine is matched with the HPV type of a cancer and suggest a change is necessary in the design of active clinical trials.

Purpose: Cellular therapy is an emerging cancer treatment modality, but its application to epithelial cancers has been limited. This clinical trial evaluated tumor-infiltrating lymphocyte (TIL) therapy for the treatment of patients with metastatic human papillomavirus (HPV)-associated carcinomas. Patients and Methods: The trial was a phase II design with two cohorts, cervical cancers and noncervical cancers. Cell infusion was preceded by a lymphocyte-depleting conditioning regimen and followed by systemic high-dose aldesleukin. Results: Objective tumor responses occurred in 5 of 18 (28%) patients in the cervical cancer cohort and 2 of 11 (18%) patients in the noncervical cancer cohort. Two of the responses in cervical cancer were complete and are ongoing 67 and 53 months after treatment. Responses in the noncervical cancer cohort were in anal cancer and oropharyngeal cancer. The HPV reactivity of the infused T cells correlated with clinical response. Peripheral blood repopulation with HPV-reactive T cells also correlated with clinical response. Conclusions: These findings support the concept that cellular therapy can mediate the regression of epithelial cancers, and they suggest the importance of predictive biomarkers and novel treatment platforms for more effective therapies.

T cell receptor (TCR) T cell therapy is a promising cancer treatment modality. However, its successful development for epithelial cancers may depend on the identification of high-avidity TCRs directed against tumor-restricted target antigens. The human papillomavirus (HPV) E7 antigen is an attractive therapeutic target that is constitutively expressed by HPV+ cancers but not by healthy tissues. It is unknown if genetically engineered TCR T cells that target E7 can mediate regression of HPV+ cancers. We identified an HPV-16 E7-specific, HLA-A*02:01-restricted TCR from a uterine cervix biopsy from a woman with cervical intraepithelial neoplasia. This TCR demonstrated high functional avidity, with CD8 coreceptor-independent tumor targeting. Human T cells transduced to express the TCR specifically recognized and killed HPV-16+ cervical and oropharyngeal cancer cell lines and mediated regression of established HPV-16+ human cervical cancer tumors in a mouse model. These findings support the therapeutic potential of this approach and established the basis for an E7 TCR gene therapy clinical trial in patients with metastatic HPV+ cancers (NCT02858310).

Adoptive transfer of T cells genetically engineered to express a tumor-targeting chimeric antigen receptor (CAR) or T-cell receptor (TCR) can mediate cancer regression in some patients. CARs are synthetic single-chain proteins that use antibody domains to target cell surface antigens. TCRs are natural heterodimeric proteins that can target intracellular antigens through recognition of peptides bound to human leukocyte antigens. CARs have shown promise in B-cell malignancies and TCRs in melanoma, but neither approach has achieved clear success in an epithelial cancer. Treatment of epithelial cancers may be particularly challenging because of a paucity of target antigens expressed by carcinomas and not by important healthy tissues. In addition, epithelial cancers may be protected by inhibitory ligands and soluble factors in the tumor microenvironment. One strategy to overcome these negative regulators is to modulate expression of T-cell genes to enhance intrinsic T-cell function. Programmable nucleases, which can suppress inhibitory genes, and inducible gene expression systems, which can enhance stimulatory genes, are entering clinical testing. Other work is delineating whether control of genes for immune checkpoint receptors (e.g., PDCD1, CTLA4) and cytokine and TCR signaling regulators (e.g., CBLB, CISH, IL12, IL15) can increase the antitumor activity of therapeutic T cells. Clin Cancer Res; 22(7); 1559–64. ©2016 AACR.

Purpose: The E6 and E7 oncoproteins of HPV-associated epithelial cancers are in principle ideal immunotherapeutic targets, but evidence that T cells specific for these antigens can recognize and kill HPV+ tumor cells is limited. We sought to determine whether TCR gene engineered T cells directed against an HPV oncoprotein can successfully target HPV+ tumor cells. Experimental Design: T-cell responses against the HPV-16 oncoproteins were investigated in a patient with an ongoing 22-month disease-free interval after her second resection of distant metastatic anal cancer. T cells genetically engineered to express an oncoprotein-specific TCR from this patient's tumor-infiltrating T cells were tested for specific reactivity against HPV+ epithelial tumor cells. Results: We identified, from an excised metastatic anal cancer tumor, T cells that recognized an HLA-A*02:01–restricted epitope of HPV-16 E6. The frequency of the dominant T-cell clonotype from these cells was approximately 400-fold greater in the patient's tumor than in her peripheral blood. T cells genetically engineered to express the TCR from this clonotype displayed high avidity for an HLA-A*02:01–restricted epitope of HPV-16, and they showed specific recognition and killing of HPV-16+ cervical, and head and neck cancer cell lines. Conclusions: These findings demonstrate that HPV-16+ tumors can be targeted by E6-specific TCR gene engineered T cells, and they provide the foundation for a novel cellular therapy directed against HPV-16+ malignancies, including cervical, oropharyngeal, anal, vulvar, vaginal, and penile cancers. Clin Cancer Res; 21(19); 4431–9. ©2015 AACR.

BackgroundInterleukin-12 (IL-12) is a potent, proinflammatory cytokine that holds promise for cancer immunotherapy, but its clinical use has been limited by its toxicity. To minimize systemic exposure and potential toxicity while maintaining the beneficial effects of IL-12, we developed a novel IL-12-based therapeutic system that combines tumor-specific T-cell-mediated delivery of IL-12 with membrane-restricted IL-12 localization and inducible IL-12 expression.MethodsTherapeutic T cells targeting a tumor antigen were genetically engineered to express membrane-anchored IL-12 (aIL-12). Expression, function, and shedding of the aIL-12 molecule was assessed in vitro. Tumor treatment efficacy was assessed in vivo with T cell receptor (TCR) transgenic murine tumor models and a tumor xenograft model. Key outcomes were change in tumor size, circulating levels of IL-12 and other cytokines, and survival. Toxicity was assessed via change in body weight. Tumor growth curve measurements were compared using repeated-measures two-way analyses of variance.ResultsRetroviral gene transfer resulted in cell membrane expression of aIL-12 by transduced T cells. In each of two transgenic murine tumor models, tumor-specific T cells constitutively expressing aIL-12 demonstrated increased antitumor efficacy, low circulating IL-12 and interferon-γ, and no weight loss. Expression of aIL-12 via aNFAT-inducible promoter resulted in coordinate expression of aIL-12 with T cell activation. In an OT-I TCR transgenic murine tumor model, theNFAT-inducible aIL-12 molecule improved tumor treatment and did not result in detectable levels of IL-12 in serum or in weight loss. In a human tumor xenograft model, theNFAT-inducible aIL-12 molecule improved antitumor responses by human T cells coexpressing a tumor-specific engineered TCR. Serum IL-12 levels were undetectable with theNFAT-inducible construct in both models.ConclusionExpression of aIL-12 by tumor-targeting therapeutic T cells demonstrated low systemic exposure and improved efficacy. This treatment strategy may have broad applications to cellular therapy with tumor-infiltrating lymphocytes, chimeric antigen receptor T cells, and TCR T cells.

Background: TGF-β pathway dysregulation may play a critical role in carcinogenesis and immune evasion in HPV-associated cancers (HACs), including anal, cervical, and p16+ squamous cell carcinoma of the head and neck (SCCHN). Targeting this pathway may also enhance clinical response to PD-(L)1 antibodies. M7824 is an innovative first-in-class bifunctional fusion protein composed of two extracellular domains of TGF-βRII (a TGF-β “trap”) fused with a human IgG1 monoclonal antibody against PD-L1. In the dose-escalation cohort of the ongoing Phase I trial of M7824 (NCT02517398), patients with HACs who developed an HPV-specific immune response on M7824 had improved clinical responses. Here we report efficacy and safety data from patients with HPV-positive malignancies from the dose-escalation cohort pooled with data from HAC expansion cohorts for patients with HPV-positive disease. Methods: Patients received M7824 Q2W at 1-30 mg/kg in the dose-escalation phase or at the recommended phase 2 dose of 1200 mg in the expansion cohorts. Treatment continued until progressive disease (PD), unacceptable toxicity, or trial withdrawal; treatment past PD was allowed if clinically justified. Safety and best overall response by RECIST v1.1 were the primary endpoints of the dose-escalation and expansion phases, respectively. Results: As of August 24, 2018, 36 patients with pretreated HPV-positive HACs (4 anal; 18 cervical; 14 SCCHN) had received M7824 for a median duration of 19.0 weeks (range, 2.0-96.0). 10 patients had a response per investigator (2 complete response [CR]; 8 partial response [PR]; ORR 27.8%). 1 additional patient developed a PR after data cutoff; 3 additional patients had a delayed PR after initial PD (total clinical response rate 38.9%). Both patients with CR and 5 patients with PR have an ongoing response. The median duration of response was not reached (range, 4-22+ months). 9 patients (25%) had grade 3 treatment-related adverse events (TRAE): anemia, cystitis, diabetic ketoacidosis, increased γ-glutamyltransferase, gastroparesis, hyperkeratosis, hypokalemia, keratoacanthoma, macular rash, pleural effusion, pneumonitis, and skin SCC. The patient with gastroparesis had asymptomatic grade 3→4 hypokalemia (1 grade 4 TRAE, 2.7%). No treatment-related deaths occurred. 6 patients (16.7%) permanently discontinued M7824 due to TRAEs (gastroparesis, infusion-related reaction, cystitis, pneumonitis, and acneiform and psoriasiform dermatitis). Conclusions: M7824 has a manageable safety profile and shows encouraging clinical efficacy in patients with HPV-positive HACs (total clinical response rate 38.9%). M7824 is a promising therapeutic agent for these patients and is under further evaluation in phase 1b and 2 trials. Updated survival results will be reported. Citation Format: Julius Strauss, Margaret E. Gatti-Mays, Byoung Cho, Sébastien Salas, Edward McClay, Jason Redman, Houssein Abdul Sater, Renee N. Donahue, Elizabeth Lamping, Andrea Burmeister, Jennifer L. Marté, Lisa Cordes, Laureen S. Ojalvo, Christoph Helwig, Alex Rolfe, Christian S. Hinrichs, Ravi A. Madan, Jeffrey Schlom, James Gulley. Phase I evaluation of M7824, a bifunctional fusion protein targeting TGF-β and PD-L1, in patients with human papillomavirus (HPV)-associated malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT075.

T cell receptor (TCR) gene therapy can mediate tumor regression in patients; however, successful treatment of epithelial cancers has been limited. We studied the targeting of Kita-Kyushu lung cancer antigen 1 (KK-LC-1), a cancer germline antigen expressed in multiple epithelial cancers, with TCR gene-engineered T cells. We thoroughly vetted KK-LC-1 expression in both cancerous and healthy tissue by pulling data from BioGPS and cBioPortal and analyzing a broad range of patient samples, primary cancer lines, and healthy tissue by both qRT-PCR and fluorescence in situ hybridization. Our findings show that KK-LC-1 is expressed in numerous epithelial cancers including breast, lung, cervical, ovarian, melanoma, and prostate whereas expression in healthy tissue is limited to immune-privileged sites including the epididymis and testis. We identified an HLA-A*01:01-restricted KK-LC-152-60-specific TCR isolated from the tumor infiltrating lymphocytes of a patient with metastatic cervical adenocarcinoma. T cells from two healthy donors were transduced with the KK-LC-1 TCR and tested for in vitro targeting of tumor cell lines. The KK-LC-1 TCR recognized the HLA-A*01:01+, KK-LC-1+ cell lines 4156, EKVX, and PC-3, as determined by IFN-γ release. The HLA-A*01:01- cell lines, HeLA, DU-145, and MDA-MD-468, were initially not recognized by the KK-LC-1 TCR; however, following stable transduction with HLA-A*01:01, these cell lines were targeted. The HLA-A*01:01+, KK-LC-1- cell lines, PC-3 and 3748, were not recognized by the KK-LC-1 TCR. To assess the TCR’s potential for off-target activity, we used amino acid scanning to determine the recognition motif, allowing for subsequent targeted in silico searches to identify peptides containing the same recognition motif. Using NCBI Blast and the ScanProsite database, 10 peptides with homologous essential but different non-essential residues were identified. A retroviral vector encoding the KK-LC-152-60 TCR was used to genetically engineer peripheral blood T cells. No peptide recognition by the TCR was observed when dendritic cells were pulsed with the individual peptides and co-cultured with KK-LC-1 TCR-transduced T cells. Finally, we assessed if human genetically engineered T cells expressing the KK-LC-1 TCR could mediate in vivo tumor regression in an NSG mouse model. Genetically engineered T cells expressing the KK-LC-1 TCR induced tumor regression in both a melanoma and cervical cancer line in a dose-dependent manner. Collectively, these findings indicate that KK-LC-1 is a suitable target for TCR gene therapy and they provide preclinical support for testing of the KK-LC-1 TCR in a phase I clinical trial. Citation Format: Bridget Marcinkowski, Carylinda Serna, Benjamin Jin, Scott Norberg, Christian Hinrichs. Preclinical characterization of a KK-LC-1-specific T cell receptor for the treatment of epithelial cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1429.

Adoptive cell transfer of tumor-infiltrating lymphocytes (TILs) can mediate cancer regression in patients with metastatic melanoma, but whether this approach can be applied to common epithelial malignancies remains unclear. In this study, we compared the phenotype and function of TILs derived from liver and lung metastases from patients with gastrointestinal (GI) cancers (n = 14) or melanoma (n = 42). Fewer CD3+ T cells were found to infiltrate GI compared with melanoma metastases, but the proportions of CD8+ cells, T cell differentiation stage, and expression of costimulatory molecules were similar for both tumor types. Clinical-scale expansion up to ∼50 × 109 T cells on average was obtained for all patients with GI cancer and melanoma. From GI tumors, however, TIL outgrowth in high-dose IL-2 yielded 22 ± 1.4% CD3+CD8+ cells compared with 63 ± 2.4% from melanoma (p < 0.001). IFN-γ ELISA demonstrated MHC class I–mediated reactivity of TIL against autologous tumor in 5 of 7 GI cancer patients tested (9% of 188 distinct TIL cultures) and in 9 of 10 melanoma patients (43% of 246 distinct TIL cultures). In these assays, MHC class I–mediated up-regulation of CD137 (4-1BB) expression on CD8+ cells suggested that 0–3% of TILs expanded from GI cancer metastases were tumor-reactive. This study implies that the main challenge to the development of TIL adoptive cell transfer for metastatic GI cancers may not be the in vitro expansion of bulk TILs, but the ability to select and enrich for tumor-reactive T cells.

Purpose: Adoptive cell transfer (ACT) of tumor infiltrating or genetically engineered T cells can cause durable responses in patients with metastatic cancer. Multiple clinically modifiable parameters can comprise this therapy, including cell dose and phenotype, in vivo antigen restimulation, and common gamma-chain (γc) cytokine support. However, the relative contributions of each these individual components to the magnitude of the antitumor response have yet to be quantified. Experimental Design: To systematically and quantitatively appraise each of these variables, we employed the Pmel-1 mouse model treating large, established B16 melanoma tumors. In addition to cell dose and magnitude of in vivo antigen restimulation, we also evaluated the relative efficacy of central memory (TCM), effector memory (TEM), and stem cell memory (TSCM) subsets on the strength of tumor regression as well as the dose and type of clinically available γc cytokines, including IL-2, IL-7, IL-15, and IL-21. Results: We found that cell dose, T-cell differentiation status, and viral vaccine titer each were correlated strongly and significantly with the magnitude of tumor regression. Surprisingly, although the total number of IL-2 doses was correlated with tumor regression, no significant benefit to prolonged (≥6 doses) administration was observed. Moreover, the specific type and dose of γc cytokine only moderately correlated with response. Conclusion: Collectively, these findings elucidate some of the key determinants of successful ACT immunotherapy for the treatment of cancer in mice and further show that γc cytokines offer a similar ability to effectively drive antitumor T-cell function in vivo. Clin Cancer Res; 17(16); 5343–52. ©2011 AACR.

Background: Adoptive T cell therapy with gene-engineered T cells is an emerging cancer treatment strategy that has been applied successfully to the treatment of hematological cancers. We conducted a clinical trial to test proof of principle for this type of treatment in an epithelial cancer. Patients with human papillomavirus (HPV) 16-associated cancers were treated with gene-engineered T cells targeting HPV16 E7. Methods: The clinical trial was a phase I study with a 3 + 3 design and three dose levels (DL) of gene-engineered T cells (DL1: 1 x 109, DL2: 1 x 1010, DL3: 1 x 1011). Patients had metastatic HPV-16+ cancers from any primary tumor site. Treatment consisted of a one-time infusion of autologous T cells that were gene-engineered to express an HLA-A*02:01-restricted T-cell receptor (TCR) that targets HPV-16 E7 (E7 T cells). A lymphocyte-depleting conditioning regimen was administered before treatment. E7 T cell infusion was followed by high-dose systemic aldesleukin. Results: Twelve patients were treated (DL1, n=3; DL2, n=3; DL3, n=6). The age range was 31 to 59 years. The site of the primary cancer was vulva (n=1), head and neck (n=4), uterine cervix (n=5), and anus (n=2). Each patient had multiple metastases and had previously received 3 to 7 anti-cancer agents. The conditioning regimen consisted of cyclophosphamide 30 mg/Kg (n=6) or 60 mg/Kg (n=6) iv daily for 2 days overlapping with fludarabine 25 mg/m2 iv daily for 5 days. The E7 TCR was expressed by 90-99% of the infused T cells for each patient. E7 T cell cross-reactivity against healthy tissues was not identified. Cytokine-release syndrome was not observed. A single patient, at DL3, experience dose-limiting toxicity. Four patients experienced confirmed responses, and two patients experienced unconfirmed responses (i.e. met criteria for response at only one assessment) (Figure 1). Confirmed responses occurred in patients with cervical cancer, oropharyngeal cancer, vulvar cancer, and anal cancer. The duration of responses was 3 months (ongoing), 4 months, 8 months, and 9 months, respectively. These patients had previously received 7, 4, 7 and 3 prior anti-cancer agents, respectively. Three patients with confirmed responses had previously received PD-1 or PD-L1 checkpoint blockade. Four patients whose cancer progressed after E7 T cells received PD-1 or PD-L1 checkpoint blockade; none responded. Conclusions: Tumor regression can occur following treatment of an epithelial cancer with gene-engineered T cells. These findings support continued study of E7 T cells and possibly other types of gene-engineered T cells in epithelial cancers. Disclosures Adhikary: Kite Pharma: Employment. Schweitzer:Kite Pharma: Employment. Astrow:Kite Pharma: Employment. Hinrichs:Kite Pharma: Research Funding; NIH: Patents & Royalties: NIH patents related to cell therapy.

Purpose: Circulating cell-free (ccf) human papillomavirus (HPV) DNA is a unique tumor marker for metastatic cervical cancers. We developed a method to genotype and quantify circulating HPV DNA in patients with HPV16- or HPV18-positive metastatic cervical cancer for disease monitoring and treatment-related decision making. Patients and Methods: In this retrospective study, HPV ccfDNA was measured in serum samples from metastatic cervical cancer patients by duplex digital droplet (dd) PCR. Nine patients had received tumor-infiltrating lymphocyte (TIL) immunotherapy. ccfDNA data were aligned with the tumor HPV data, drug treatment, and clinical outcome. Results: In blinded tests, HPV ccfDNA was detected in 19/19 (100%) patients with HPV-positive metastatic cervical cancer but not in any of the 45 healthy blood donors. The HPV genotype harbored in the patients' tumors was correctly identified in 87/87 (100%) sequential patient serum samples. In three patients who experienced objective responses after TIL immunotherapy, a transient HPV ccfDNA peak was detected 2-3 days after TIL infusion. Furthermore, a persistent clearance of HPV ccfDNA was observed in only two patients with a complete response (CR) after TIL immunotherapy. Conclusions: HPV ccfDNA represents a promising tumor marker for non-invasive HPV genotyping and may be used in selecting patients for HPV type-specific T-cell-based immunotherapies. It may also have value in detecting the anti-tumor activities of therapeutic agents and in the long-term follow-up of cervical cancer patients in remission. Citation Format: Zhigang Kang, Sanja Stevanović, Christian Hinrichs, Laing Cao. Circulating cell-free DNA for metastatic cervical cancer detection, genotyping and monitoring [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1581.

3043Background: Adoptive T-cell therapy with gene-engineered T cells is an emerging cancer treatment strategy. Study of this approach in epithelial cancers, the most common types of malignancies, h...

3019Background: Engineered T-cell therapy is an emerging treatment for hematological cancers. In epithelial cancers, its study has been limited. Human papillomavirus (HPV)-16+ epithelial cancers co...

3004Background: Adoptive T-cell therapy (ACT) is a promising cancer treatment modality. However, its study in epithelial cancers has been limited. Human papillomavirus (HPV)-associated cancers are ...

Immunotherapy, particularly the adoptive cell transfer (ACT) of tumor-infiltrating lymphocytes (TIL), is a very promising therapy for metastatic melanoma. Some patients unable to receive TIL have been successfully treated with autologous peripheral blood lymphocytes (PBL), genetically modified to express human leukocyte antigen (HLA) class I antigen–restricted, melanoma antigen–reactive T-cell receptors; however, substantial numbers of patients remain ineligible due to the lack of expression of the restricting HLA class I allele. We sought to overcome this limitation by designing a non–MHC-restricted, chimeric antigen receptor (CAR) targeting the high molecular weight melanoma-associated antigen (HMW-MAA), which is highly expressed on more than 90% of human melanomas but has a restricted distribution in normal tissues. HMW-MAA–specific CARs containing an antigen recognition domain based on variations of the HMW-MAA–specific monoclonal antibody 225.28S and a T-cell activation domain based on combinations of CD28, 4-1BB, and CD3ζ activation motifs were constructed within a retroviral vector to allow stable gene transfer into cells and their progeny. Following optimization of the HMW-MAA–specific CAR for expression and function in human PBL, these gene-modified T cells secreted cytokines, were cytolytic, and proliferated in response to HMW-MAA–expressing cell lines. Furthermore, the receptor functioned in both CD4+ and CD8+ cells, was non–MHC restricted, and reacted against explanted human melanomas. To evaluate this HMW-MAA–specific CAR in patients with metastatic melanoma, we developed a clinical-grade retroviral packaging line. This may represent a novel means to treat the majority of patients with advanced melanoma, most notably those unable to receive current ACT therapies. Cancer Res; 70(8); 3027–33. ©2010 AACR.

Purpose Metastatic cervical cancer is a prototypical chemotherapy-refractory epithelial malignancy for which better treatments are needed. Adoptive T-cell therapy (ACT) is emerging as a promising cancer treatment, but its study in epithelial malignancies has been limited. This study was conducted to determine if ACT could mediate regression of metastatic cervical cancer. Patients and Methods Patients enrolled onto this protocol were diagnosed with metastatic cervical cancer and had previously received platinum-based chemotherapy or chemoradiotherapy. Patients were treated with a single infusion of tumor-infiltrating T cells selected when possible for human papillomavirus (HPV) E6 and E7 reactivity (HPV-TILs). Cell infusion was preceded by lymphocyte-depleting chemotherapy and was followed by administration of aldesleukin. Results Three of nine patients experienced objective tumor responses (two complete responses and one partial response). The two complete responses were ongoing 22 and 15 months after treatment, respectively. One partial response was 3 months in duration. The HPV reactivity of T cells in the infusion product (as measured by interferon gamma production, enzyme-linked immunospot, and CD137 upregulation assays) correlated positively with clinical response (P = .0238 for all three assays). In addition, the frequency of HPV-reactive T cells in peripheral blood 1 month after treatment was positively associated with clinical response (P = .0238). Conclusion Durable, complete regression of metastatic cervical cancer can occur after a single infusion of HPV-TILs. Exploratory studies suggest a correlation between HPV reactivity of the infusion product and clinical response. Continued investigation of this therapy is warranted.

3009 Background: Engineered T-cell therapy has shown promise in B-cell malignancies and melanoma, but clinical investigation in epithelial cancers has been limited. Methods: We conducted a phase I/II clinical trial of T cells genetically engineered to express a T-cell receptor that targets an HLA-A*02:01-restricted epitope of E6 (E6 TCR T Cells) for patients with metastatic HPV-16+ carcinoma. The cell dose was escalated in cohorts of single patients (1 x 109, 1 x 1010, and 1-2 x 1011cells). Patients received a nonmyeloablative conditioning regimen of cyclophosphamide and fludarabine, a single infusion of E6 TCR T Cells, and systemic high-dose aldesleukin. Results: Twelve patients were treated, 9 at the highest cell dose, plus one retreatment. The cancer types were 6 cervical, 4 anal, 1 oropharyngeal, and 1 vaginal. No dose-limiting toxicity, autoimmune adverse events, or cytokine storm were observed. Two patients with anal cancer treated at the highest cell dose experienced partial tumor responses lasting 6 and 3 months after treatment. The patient with a 6-month response had complete regression of one tumor and partial regression of two tumors that were resected upon progression; she has no evidence of disease 22 months after treatment. T-cell receptor gene transfer efficiency was 45 and 51% in the responding patients, and 47-76% (median 61%) in the non-responding patients. Responding patients showed robust levels of E6 TCR T cell memory (30 and 46% of circulating T cells 1-month after treatment). Non-responding patients showed wide-ranging levels of E6 TCR T cell memory (range 4-53%, median 29%). Expression of programmed cell death protein 1 (PD-1) by circulating E6 TCR T Cells 1-month after treatment was low in all patients ( < 5%). The patient with a 6-month response had 7% E6 TCR T Cells in a resected tumor 10 months after treatment, 25% of which expressed PD-1. A patient with no response had no detectable E6 TCR T Cells in a resected tumor 3 months after treatment. Conclusions: E6 TCR T-cell therapy was safe at doses up to 2 x 1011 cells. Regression of metastatic HPV+ carcinoma occurred in two patients following treatment, suggesting that TCR T-cell therapy can mediate epithelial cancer regression. Clinical trial information: NCT02280811.

Adoptive T-cell therapies for cancer can induce tumor responses in patients with metastatic melanoma, synovial sarcoma, and B-cell malignancies. However, no cellular therapy has demonstrated activity in a common epithelial tumor. Adoptive transfer of tumor infiltrating lymphocytes (TIL) has high response rates and can induce complete and durable tumor regression in metastatic melanoma. We sought to determine the objective tumor response rate of TIL therapy for human papillomavirus (HPV)-associated malignancies. These common epithelial cancers occur at varied sites but universally express the immunogenic E6 and E7 viral oncoproteins making them rational targets for T cell-based therapeutic approaches. Herein we report the results of treatment of the first eight subjects (seven evaluable) from the cohort of patients with cervical cancer (which is necessarily associated with high-risk HPV infection) treated with TIL therapy. Tumors were HPV genotyped and TIL were generated from individual tumor fragment cultures then tested for reactivity against autologous dendritic cells loaded with pools of overlapping peptides spanning type-specific HPV E6 or E7 proteins. Cultures were selected for rapid expansion and administration to patients based on oncoprotein reactivity, T-cell growth rate, and CD8+ T cell frequency. Patients received non-myeloablative lymphoconditioning chemotherapy before cell infusion and high-dose bolus IL-2 after cell infusion. TIL from 6/8 patients displayed reactivity against E6 or E7. Objective tumor responses by RECIST criteria were observed in 3/7 evaluable patients; two have complete and ongoing regression of measurable disease at three and nine months after treatment. Adverse events were the expected combined toxicities of the preparative chemotherapy regimen and interleukin-2, and were consistent with those of TIL therapy for metastatic melanoma. No autoimmunity was observed. These early clinical trial results indicate that tumor responses can occur following administration of TIL for metastatic cervical cancer.

Background: Adoptive transfer of genetically-modified T cells is being explored as a salvage treatment for patients with selected metastatic cancers. Most of the current strategies utilize MHC class I-restricted T cell receptor (TCR) or chimeric antigen receptor (CAR) technologies to genetically modify CD8+ T cells or bulk T cells for patient treatment. Evidence indicates that CD4+ T cells can induce tumor regression, similar to CD8+ T cells. To test this hypothesis, an HLA-DPB1*0401-restricted TCR recognizing MAGE-A3 was isolated from a patient's peripheral blood after MAGE-A3 peptide vaccination. Because HLA-DPB1*0401 is present in ∼60% of the Caucasian population and MAGE-A3 is expressed in up to one third of tumor specimens from a variety of cancer types, this TCR immunotherapy could potentially be applicable for a significant portion of cancer patients. Trial Design: Eligible patients were HLA-DPB1*0401 positive with MAGE-A3 positive tumor specimens, and had not responded or had recurred following at least one standard first line therapy for their disease. Patients received a lymphodepleting preparative regimen, followed by adoptive transfer of purified CD4+ T cells transduced with the HLA-DPB1*0401-restricted MAGE-A3 TCR plus systemic high-dose interleukin-2 (IL-2). A cell dose-escalation was conducted, treating 1 patient at each cohort (0.01, 0.03, 0.1, up to 30 billion cells), followed by 6 patients at the highest dose level (up to 100 billion cells). Clinical trial information: NCT02111850. Results: Fourteen patients were treated in this phase I study, including the last 6 patients at the highest dose level (78∼100 billion cells). Objective partial responses (RECIST) were observed in a patient with metastatic cervical cancer (ongoing at 11+ months), esophageal cancer (duration 3 months) and urothelial cancer (ongoing at 4+ months). High levels of IL-6 were detected in all patients’ serum samples after adoptive cell transfer. One month after the treatment, TCR-transduced T cells persisted at high levels in the peripheral blood of 6 patients who received the highest dose level, compared to patients who receive lower dose levels (0.01 ∼ 30 billion cells) (p = 0.0082). These results demonstrate the safety of administering autologous CD4+ T cells genetically-engineered to express an MHC class II-restricted anti-tumor TCR targeting MAGE-A3 and presents evidence for efficacy. We have started the phase II clinical trial to study the efficacy of this TCR therapy in different types of metastatic cancer. This clinical trial extends the reach of TCR gene therapy to patients with metastatic cancer. To our knowledge, this clinical trial represents the first genetically-modified CD4+ T cell immunotherapy against metastatic cancer. Citation Format: Yong-Chen Lu, Linda L. Parker, Tangying Lu, Zhili Zheng, Xin Yao, Paul F. Robbins, Steven A. Feldman, Pierre van der Bruggen, Christopher A. Klebanoff, Christian S. Hinrichs, Stephanie L. Goff, Richard M. Sherry, Udai S. Kammula, James C. Yang, Steven A. Rosenberg. A phase I study of an HLA-DPB1*0401-restricted T-cell receptor targeting MAGE-A3 for patients with metastatic cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT003.

Lymphodepletion with chemotherapeutic agents or total body irradiation (TBI) before adoptive transfer of tumor-specific T cells is a critical advancement in the treatment of patients with melanoma. More than 50% of patients that are refractory to other treatments experience an objective or curative response with this approach. Emerging data indicate that the key mechanisms underlying how TBI augments the functions of adoptively transferred T cells include (a) the depletion of regulatory T cells (Treg) and myeloid-derived suppressor cells that limit the function and proliferation of adoptively transferred cells; (b) the removal of immune cells that act as “sinks” for homeostatic cytokines, whose levels increase after lymphodepletion; and (c) the activation of the innate immune system via Toll-like receptor 4 signaling, which is engaged by microbial lipopolysaccharide that translocated across the radiation-injured gut. Here, we review these mechanisms and focus on the effect of Toll-like receptor agonists in adoptive immunotherapy. We also discuss alternate regimens to chemotherapy or TBI, which might be used to safely treat patients with advanced disease and promote tumor regression.

Postmastectomy radiotherapy (PMRT) has proven benefits for certain patients with breast cancer; however, one of its complications is lymphedema. This study examines the incidence of and risk factors associated with lymphedema secondary to PMRT. The charts of patients treated with mastectomy at Roswell Park Cancer Institute between January 1, 1995, and April 20, 2001, who received PMRT were reviewed. Univariate analysis of patient, disease, and treatment variables was conducted. Multivariate analysis was performed on variables found to be significant in univariate analysis. One hundred five patients received PMRT. The incidence of lymphedema was 27%. Patient age, body mass index, disease stage, positive lymph nodes, nodes resected, postoperative infection, duration of drainage, chemotherapy, and hormonal therapy were not associated with lymphedema. Total dose (P = .032), posterior axillary boost (P = .047), overlap technique (P = .037), radiotherapy before 1999 (P = .028), and radiotherapy at Roswell Park Cancer Institute (P = .028) were significantly associated with lymphedema. Increased lymphedema was noted with supraclavicular, internal mammary, mastectomy scar boost, and chest wall tangential photon beam radiation, but the associations were not statistically significant. The high incidence and debilitating effects of lymphedema must be weighed against the benefits of PMRT. Efforts to prevent lymphedema should be emphasized.

LBA3008 Background: Adoptive T-cell therapy (ACT) is a promising cancer treatment modality with potentially broad application. It is not known if ACT can mediate regression of carcinomas, the most common solid tumors in humans. We studied carcinoma of the uterine cervix, a virally induced malignancy that constitutively expresses the HPV E6 and E7 oncoproteins, as a model cancer to test if ACT can mediate regression of an epithelial malignancy. Methods: We initiated a clinical trial to treat metastatic HPV+ cancers with tumor-infiltrating lymphocytes (TIL) selected for HPV E6- and E7-reactivity (HPV-TIL). Patients from the cervical cancer cohort are reported here. HPV-TIL infusion was preceded b y non-myeloablative conditioning and followed by high-dose bolus aldesleukin. HPV-reactivity was assessed by ELISPOT, IFN-gamma production, and CD137 expression assays. Results: Nine patients were treated on the study. They received a median of 81 x 109 T cells (range 33 to 159 x 109) as a single infusion. The infused cells possessed reactivity against high-risk HPV E6 and/or E7 in 6/8 patients. The two patients with no HPV reactivity did not respond to treatment. 3/6 patients with HPV reactivity demonstrated objective tumor responses by RECIST (1 PR and 2 CR). One patient had a 39% best response. Two patients with widespread metastases had complete tumor responses that are ongoing 18 and 11 months after treatment. One patient with a complete response had a chemotherapy-refractory HPV-16+ squamous cell carcinoma and the other a chemoradiation-refractory HPV-18+ adenocarcinoma. Both patients demonstrated prolonged repopulation with HPV-reactive T cells following treatment. Increased frequencies of HPV-specific T cells were detectable after 13 months in one patient and 6 months in the other. Two patients with HPV-reactive TIL that did not respond to treatment did not display repopulation with HPV-reactive T cells. Conclusions: HPV-TIL can mediate durable, complete regression of metastatic cervical cancer. Continued investigation of HPV-TIL for cervical cancer, and possibly other HPV+ malignancies, is warranted. Cellular therapy can mediate complete regression of an epithelial malignancy. Clinical trial information: NCT01585428.

Meeting abstracts Adoptive transfer of genetically-modified T cells is being explored as a salvage treatment for patients with selected metastatic cancers. Most of the current strategies utilize MHC class I-restricted T cell receptor (TCR) or chimeric antigen receptor (CAR) technologies to