Adoptive Transfer Of Lymphocytes Research Articles

Interleukin-21 receptor signaling promotes metabolic dysfunction-associated steatohepatitis-driven hepatocellular carcinoma by inducing immunosuppressive IgA+ B cells

BackgroundDysregulation of immune surveillance is tightly linked to the development of metabolic dysfunction-associated steatohepatitis (MASH)-driven hepatocellular carcinoma (HCC); however, its underlying mechanisms remain unclear. Herein, we aimed to determine the role of interleukin-21 receptor (IL-21R) in MASH-driven HCC.MethodsThe clinical significance of IL-21R was assessed in human HCC specimens using immunohistochemistry staining. Furthermore, the expression of IL-21R in mice was assessed in the STAM model. Thereafter, two different MASH-driven HCC mouse models were applied between IL-21R-deficient mice and wild type controls to explore the role of IL-21R in MASH-driven HCC. To further elucidate the potential mechanisms by which IL-21R affected MASH-driven HCC, whole transcriptome sequencing, flow cytometry and adoptive lymphocyte transfer were performed. Finally, flow cytometry, enzyme-linked immunosorbent assay, immunofluorescent staining, chromatin immunoprecipitation assay and western blotting were conducted to explore the mechanism by which IL-21R induced IgA+ B cells.ResultsHCC patients with high IL-21R expression exhibited poor relapse-free survival, advanced TNM stage and severe steatosis. Additionally, IL-21R was demonstrated to be upregulated in mouse liver tumors. Particularly, ablation of IL-21R impeded MASH-driven hepatocarcinogenesis with dramatically reduction of lipid accumulation. Moreover, cytotoxic CD8+ T lymphocyte activation was enhanced in the absence of IL-21R due to the reduction of immunosuppressive IgA+ B cells. Mechanistically, the IL-21R-STAT1-c-Jun/c-Fos regulatory axis was activated in MASH-driven HCC and thus promoted the transcription of Igha, resulting in the induction of IgA+ B cells.ConclusionsIL-21R plays a cancer-promoting role by inducing IgA+ B cells in MASH-driven hepatocarcinogenesis. Targeting IL-21R signaling represents a potential therapeutic strategy for cancer therapy.

Abstract LB073: Adoptive transfer of one CD4TCR and one CD8TCR targeting autochthonous neoantigens are essential and sufficient for eradication of naturally heterogeneous solid tumors

Abstract Despite advances in checkpoint blockade therapy or adoptive transfer of tumor-infiltrating lymphocytes (TILs), achieving cure remains challenging, but success of immunotherapy seems to depend on recognition of tumor-specific antigens (neoantigens). We aimed to determine how many neoantigens need to be recognized by how many different TCRs for eradication of solid tumors. Unmanipulated, naturally expressed (autochthonous) neoantigens were targeted with adoptively transferred TCR-engineered autologous T cells (TCR-therapy). Investigated were effects of TCR-engineered CD8+ T cells, TCR-engineered CD4+ T cells or a combination of both. The targeted tumors were established for at least three weeks and derived from primary autochthonous cancer cell cultures, resembling natural solid tumors and their heterogeneity as found in humans. CD8TCR-therapy was only effective against homogenous tumors and relapse was common in heterogeneous tumors, even when targeting multiple different autochthonous neoantigens. By contrast, a combination of CD8TCR-therapy with CD4TCR-therapy, each targeting an independent neoantigen, eradicated large and established solid tumors of natural heterogeneity. CD4TCR-therapy targeted a mutant neoantigen on tumor stroma while direct cancer cell recognition by CD8TCR-therapy was essential for cure. Thus, two cancer-specific TCRs can be essential and sufficient to eradicate heterogeneous solid tumors expressing unmanipulated, autochthonous neoantigens. Citation Format: Steven Wolf, Vasiliki Anastasopoulou, Kimberley Drousch, Markus Diehl, Boris Engels, Poh Yin Yew, Kazuma Kiyotani, Yusuke Nakamura, Karin Schreiber, Hans Schreiber, Matthias Leisegang. Adoptive transfer of one CD4TCR and one CD8TCR targeting autochthonous neoantigens are essential and sufficient for eradication of naturally heterogeneous solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB073.

Abstract 15: Massively parallel base-editing screens to map variant effects on anti-tumor hallmarks of primary human T cells and improve cell-based cancer immunotherapies

Abstract Base editing enables precise generation of single nucleotide variants, but large-scale screening in primary human T cells has been limited by low editing efficiency. Here, we developed a high-throughput approach for highly efficient, massively parallel adenine and cytosine base-editor screening in primary human T cells. We performed multiple large-scale pooled screens editing 108 genes with central functions in T cells and whole-gene tiling mutagenesis of selected genes. All screens were functionally read out to capture the effect of variants on hallmarks of T cell anti-tumor immunity, including activation, proliferation, long-term persistence, and cytokine production. We discovered a broad landscape of gain-of-function and loss-of-function mutations and characterized their impact across multiple axes of T cell functions. Among others, we identified gain-of-function mutations in PIK3CD and its regulatory subunit PIK3R1, LCK, AKT1, and loss-of-function mutants of CTLA-4, driving increased T cell polyfunctionality. We validated several of these mutations by probing downstream signaling and functional outputs. To demonstrate the feasibility of using such screen results for improving cell-based cancer immunotherapies, we used human T cells with engineered T cell receptors recognizing specific epitopes in melanoma models. We base-edited these T cells with mutations identified in our screens and demonstrate that these mutations indeed significantly enhance cytotoxic cytokine production and tumor-lytic capacity with minimal off-target activity. Lastly, we also provide proof-of-feasibility for high-efficiency combinatorial base-editing in T cells. This study presents a framework for large-scale base editing screens with multi-dimensional functional readouts in cells that are typically difficult to molecularly engineer in a precise manner. This work also has important therapeutic implications: T cells are the substrate for clinically active cell-based therapies, such as adoptive transfer of tumor-infiltrating lymphocytes (TILs) or chimeric-receptor antigen T (CAR-T) cells. Our screen-informed base editing to achieve an improved T cell product against melanoma exemplifies the therapeutic power of generating synthetic protein variants that improve a wide range of existing and future T cell-based cancer immunotherapies. Citation Format: Zachary Hudson Walsh, Parin Shah, Neeha Kothapalli, Gergo Nikolenyi, Shivem Shah, Giuseppe Leuzzi, Neil Vasan, Mohammed AlQuarishi, Alberto Ciccia, Johannes Melms, Benjamin Izar. Massively parallel base-editing screens to map variant effects on anti-tumor hallmarks of primary human T cells and improve cell-based cancer immunotherapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 15.

Bacterial ghost cell based bivalent candidate vaccine against Salmonella Typhi and Salmonella Paratyphi A: A prophylactic study in BALB/c mice

Typhoid and emerging paratyphoid fever are a severe enteric disease worldwide with high morbidity and mortality. Licensed typhoid vaccines are in the market, but no paratyphoid vaccine is currently available. In the present study we developed a bivalent vaccine against Salmonella Typhi and Paratyphi A using a bacterial ghost platform. Bacterial ghost cells (BGs) are bacteria-derived cell membranes without cytoplasmic contents that retain their cellular morphology, including all cell surface features. Furthermore, BGs have inherent adjuvant properties that promote an enhanced humoral and cellular immune reaction to the target antigen. Sodium hydroxide was used to prepare ghost cells of Salmonella Typhi and Paratyphi A. The bacterial ghost cells were characterised using electron microscopy. Then BALB/c mice were immunized three times (0th, 14th and 28th day) with the bivalent typhoidal bacterial ghost cells. Haematological study of adult mice throughout immunization period reflected that the immunogen was safe to administer and does not affect the animals’ health. After complete immunization, we found significant serum antibody titter against whole cell lysate, outer membrane protein and lipopolysaccharide of both bacteria, and cell-mediated immunity was observed in an ex-vivo experiment. CD4+, CD8a+ and CD19+ splenic cell populations were increased in immunized animals. Bivalent Typhoidal ghost cell immunized mice showed better survival, less bacterial colonization in systemic organs, and less inflammation and/or destruction of tissue in histopathological analysis than non-immunized control mice.Serum antibodies of immunized animals can significantly inhibit bacterial motility and mucin penetration ability with better killing properties against Salmonella Typhi and Paratyphi A. Furthermore, significant passive protection was observed through the adoptive transfer of serum antibody and lymphocytes of immunized animals to naïve animals after bacterial infection. In summary, Bivalent Typhoidal Bacterial Ghost cells (BTBGs) enhances immunogenic properties and serves as a safe and effective prevention strategy against Salmonella Typhi and Paratyphi A.

Using patient-derived tumor organoids from common epithelial cancers to analyze personalized T-cell responses to neoantigens

Adoptive cell transfer of tumor-infiltrating lymphocytes (TIL) can mediate durable complete responses in some patients with common epithelial cancers but does so infrequently. A better understanding of T-cell responses to neoantigens and tumor-related immune evasion mechanisms requires having the autologous tumor as a reagent. We investigated the ability of patient-derived tumor organoids (PDTO) to fulfill this need and evaluated their utility as a tool for selecting T-cells for adoptive cell therapy. PDTO established from metastases from patients with colorectal, breast, pancreatic, bile duct, esophageal, lung, and kidney cancers underwent whole exomic sequencing (WES), to define mutations. Organoids were then evaluated for recognition by autologous TIL or T-cells transduced with cloned T-cell receptors recognizing defined neoantigens. PDTO were also used to identify and clone TCRs from TIL targeting private neoantigens and define those tumor-specific targets. PDTO were successfully established in 38/47 attempts. 75% were available within 2 months, a timeframe compatible with screening TIL for clinical administration. These lines exhibited good genetic fidelity with their parental tumors, especially for mutations with higher clonality. Immunologic recognition assays demonstrated instances of HLA allelic loss not found by pan-HLA immunohistochemistry and in some cases WES of fresh tumor. PDTO could also be used to show differences between TCRs recognizing the same antigen and to find and clone TCRs recognizing private neoantigens. PDTO can detect tumor-specific defects blocking T-cell recognition and may have a role as a selection tool for TCRs and TIL used in adoptive cell therapy.

Neutrophils infiltrate sensory ganglia and mediate chronic widespread pain in fibromyalgia

Fibromyalgia is a debilitating widespread chronic pain syndrome that occurs in 2 to 4% of the population. The prevailing view that fibromyalgia results from central nervous system dysfunction has recently been challenged with data showing changes in peripheral nervous system activity. Using a mouse model of chronic widespread pain through hyperalgesic priming of muscle, we show that neutrophils invade sensory ganglia and confer mechanical hypersensitivity on recipient mice, while adoptive transfer of immunoglobulin, serum, lymphocytes, or monocytes has no effect on pain behavior. Neutrophil depletion abolishes the establishment of chronic widespread pain in mice. Neutrophils from patients with fibromyalgia also confer pain on mice. A link between neutrophil-derived mediators and peripheral nerve sensitization is already established. Our observations suggest approaches for targeting fibromyalgia pain via mechanisms that cause altered neutrophil activity and interactions with sensory neurons.

Engineered Tcell therapy for viral and non-viral epithelial cancers.

Engineered Tcell therapy has shown remarkable efficacy in hematologic malignancies and has the potential for application to common epithelial cancers. Diverse Tcell therapy strategies including adoptive transfer of tumor-infiltrating lymphocytes, chimeric antigen receptor (CAR)-T cells, and Tcell 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.

Adoptive cell transfer therapy for melanoma.

Adoptive cell transfer (ACT) of tumor-infiltrating lymphocytes (TILs) for melanoma is an example of the most successful cancer immune therapy. It achieves a durable complete response about ~20% of patients, and they might be cured. However, the ratio of patients with durable benefits is not high, and its complicated procedure prevents its diffusion. Therefore, many efforts to enhance the effect and simplify the protocol of TIL therapy have been made so far, resulting in the establishment of a simple and effective current TIL therapy that has been propagated to other institutes and countries. Moreover, TIL therapy and translational research using clinical samples derived from durable responders elucidate the important element for developing more effective cancer immune therapies in the future. This review introduced the brief history, attempts for the improvement and important findings elucidated by translational research of ACT for melanoma.

EXTH-98. ENHANCING T CELL TRAFFICKING OF CD3-ENGAGING IMMUNOTHERAPY TO TUMORS OF THE CENTRAL NERVOUS SYSTEM

Abstract BACKGROUND Effective immunotherapy against tumors of the central nervous system (CNS) requires that the drug the blood brain barrier (BBB) and encounter immune cells. We have previously described a mechanism which may facilitate transport of CD3-engaging therapeutics into the CNS, via carriage on activated T cells. Building on this, we sought to produce specific T cell phenotypes that rapidly enter and accumulate in the CNS. METHODS 8–10-week-old C57/Bl6 mice (n=5-6 per group) were implanted with 30,000 CT2AvIII cells which established over 14 days. Mice received (1) CD45.1 lymphocytes activated with IL-7 and Concanavalin-A (single intravenous (IV) injection, 1 x 107 adoptive lymphocyte transfer (ALT)) or (2) CD45.1 lymphocytes activated with IL-2 and serial Con-A stimulation. Mice were sacrificed 3 hours following ALT and their brains analysed via flow cytometry. A follow-up timepoint of 48 hours was also analysed. Groups were compared using a Mann-Whitney U test. RESULTS Ex vivo culture in group 1 yielded a mixed population of T effector and T central memory cells whereas group 2 resulted in terminally differentiated T effector memory cells only. VLA-4, a migratory integrin involved in T cell entry into the CNS was upregulated in the IL-7 group. Mice in Group 1 demonstrated significantly enhanced entry of CD8+ effector and memory T cells into tumor bearing hemispheres 3- and 48-hours following administration compared to Group 2 (p = 0.005, p = 0.0159 respectively). CONCLUSIONS Varying the cytokine cocktail used for ex vivo activation and expansion of T cells results in markedly different trafficking properties and phenotype composition. Ongoing work will determine how enhanced T cell localization to tumor affects accumulation of immunotherapy via the hitchhiking mechanism. Further, we will evaluate the safety of combinatorial CD3-engaging immunotherapy and ALT in Phase I trials (NCT04903795) using a cGMP Brain Bi-specific T cell engager (BRiTE).

Internal checkpoint regulates T cell neoantigen reactivity and susceptibility to PD1 blockade

Adoptive transfer of tumor-infiltrating lymphocytes (TIL) fails to consistently elicit tumor rejection. Manipulation of intrinsic factors that inhibit Tcell effector function and neoantigen recognition may therefore improve TIL therapy outcomes. We previously identified the cytokine-induced SH2 protein (CISH) as a key regulator of Tcell functional avidity in mice. Here, we investigate the mechanistic role of CISH in regulating human Tcell effector function in solid tumors and demonstrate that CRISPR/Cas9 disruption of CISH enhances TIL neoantigen recognition and response to checkpoint blockade. Single-cell gene expression profiling was used to identify a negative correlation between high CISH expression and TIL activation in patient-derived TIL. A GMP-compliant CRISPR/Cas9 gene editing process was developed to assess the impact of CISH disruption on the molecular and functional phenotype of human peripheral blood Tcells and TIL. Tumor-specific Tcells with disrupted Cish function were adoptively transferred into tumor-bearing mice and evaluated for efficacy with or without checkpoint blockade. CISH expression was associated with Tcell dysfunction. CISH deletion using CRISPR/Cas9 resulted in hyper-activation and improved functional avidity against tumor-derived neoantigens without perturbing Tcell maturation. Cish knockout resulted in increased susceptibility to checkpoint blockade invivo. CISH negatively regulates human Tcell effector function, and its genetic disruption offers a novel avenue to improve the therapeutic efficacy of adoptive TIL therapy. This study was funded by Intima Bioscience, U.S. and in part through the Intramural program CCR at the National Cancer Institute.

Abstract 1378: CD8 T cell mediated killing of MHC class 1 negative tumors requires antigen presenting myeloid cells and interferon gamma

Abstract Major Histocompatibility Complex (MHC) Class I downregulation is a well described mechanism of tumor immune escape, posing a challenge for T cell based immunotherapies including immune checkpoint blockade (ICB). Recent studies, however, have demonstrated mixed roles of MHC Class 1 and the critical component beta-2-microglobulin (β2m) expression in cancer progression and ICB response, with some studies showing inactivation of antigen presentation to be associated with resistance to ICB and others showing low β2m expression to be associated with favorable prognosis. Glioblastoma (GBM) in particular expresses little or no MHC Class 1 and patients remain unresponsive to ICB. We thus sought to evaluate the role of MHC Class 1 in ICB, given that we have previously demonstrated that combination ICB with anti-PD-1 and co-stimulation with 4-1BB agonism has marked efficacy against intracranial murine glioma tumors in a CD8 T cell dependent manner. Surprisingly, in a CT2A murine glioma tumor line expressing the antigen TRP2 and lacking cell surface MHC I (CT2A-TRP2-β2mKO), the efficacy of combination 4-1BB and PD-1 therapy (ICB) was re-demonstrated in a CD8 dependent manner, independent of NK cells, CD4 T cells, and B cells. Furthermore, the efficacy of immunotherapy against intracranial CT2A-TRP2-β2mKO was demonstrated to be antigen dependent, with an adoptive lymphocyte transfer (ALT) of TRP2 TCR transduced T cells (TRP2 T cells) into a CD8KO mouse sufficient to eliminate CT2A-TRP2-β2mKO in the setting of ICB. Additionally, an ALT of TRP2 T cells did not kill CT2A-β2mKO tumors in the setting of ICB, while OT-1 mice whose CD8+ T cells primarily recognize OVA peptide with CT2A-TRP2-β2mKO tumors did not respond to ICB. In vitro studies revealed that TRP2 T cells demonstrated anti-tumor cytotoxicity against MHC Class I negative CT2A-TRP2-β2mKO tumor cells in the presence of TRP2 loaded bone marrow derived macrophages (TRP2 Mφ), but neither cell type was individually sufficient to induce tumor cell death, while the combination of TRP2 T cells and TRP2 Mφ demonstrated no cytotoxicity against CT2A-β2mKO tumors. Transwell experiments in which TRP2 Mφ and CT2A-TRP2-β2mKO tumor cells were separated by a 0.5µm membrane permeable to T cells but not Mφ or tumor cells revealed that contact between TRP2 Mφ and tumor cells was not necessary to induce T cell dependent killing. Indeed, tumor-bearing β2mKO bone marrow chimeras lacking MHC class 1 on hematopoeitically derived cells did not respond to ICB, highlighting the importance of antigen presentation from myeloid cells. The mechanism of killing was found to be dependent on interferon gamma (IFNγ), as IFNγKO mice did not respond to ICB. These findings demonstrate that tumors with low MHC Class 1 expression may still be targeted by T cell dependent immunotherapies such as ICB when antigen presentation can occur from myeloid cells. Citation Format: Emily Lerner, Vincent D'Anniballe, William Tomaszewski, Jonathan Perera, Xiuyu Cui, Jessica Waibl-Polania, Daniel Wilkinson, Michael D. Gunn, Peter E. Fecci, Karolina Woroniecka. CD8 T cell mediated killing of MHC class 1 negative tumors requires antigen presenting myeloid cells and interferon gamma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1378.

First-in-human clinical trial of an oncolytic adenovirus armed with TNFa and IL-2 in patients with advanced melanoma receiving adoptive cell transfer of tumor-infiltrating lymphocytes.

TPS9590 Background: The long-term complete remission experienced by some cancer patients after receiving immunotherapies, such as adoptively transferred tumor-infiltrating lymphocytes (ACT-TIL), represent the ideal outcome to pursue for the development of therapies for oncology. At the same time, those responses are limited to a minority of treated patients, and adverse events resulting from preconditioning chemotherapy and postconditioning IL2 are a rather common scenario. TILT-123 is an oncolytic adenovirus (Ad5/3-E2F-D24-TNFa-IRES-IL2) designed to enable T-cell therapies and checkpoint inhibition against cancer. Ultimately, the aim of this approach is to expand the proportion of patients benefiting from immunotherapies. Extensive preclinical studies with this technology showed that the virus repolarizes the tumor’s immune microenvironment in a way that favors T-cell presence and their activity against tumor cells. When TILT-123 was used together with TILs in preclinical in vivo models, animals had a higher chance to display curative results while showing reduced toxicity as the use of TILT-123 replaces the pre and post conditioning (Havunen R. et al Mol Ther Oncolytics 2016, Santos J.M. Mol Ther 2018). A Phase I clinical trial (NCT04217473) is ongoing to evaluate safety of the approach in advanced melanoma patients. Extensive biological assays of patient aim to characterize viral transduction of tumors through the intravenous and intratumoral routes, and the recruitment of activated lymphocytes to tumors in order to elucidate the immunological impact of the drug. Methods: The primary aim of NCT04217473 is to evaluate safety of TILT-123 in advanced melanoma patients. Refractory or recurrent stage III/IV patients, which cannot be treated with curative intent with available therapies, and are eligible for ACT-TIL therapy, can potentially participate in the study. TILT-123 administration begins while the manufacturing of ACT-TILs takes place and continues after the TIL-therapy is administered to the patient. In contrast with standard ACT-TIL therapy, patients are not conditioned with lymphodepletion or IL-2 in this approach. Patients must present at least one biopsiable/operable tumor for the generation of TILs and another injectable lesion for intratumoral administration of TILT-123. The open label, dose escalation trial has the main endpoint of establishing TILT-123 safety by day 36 (prior to TIL administration), which is based on the incidence of adverse events, severe adverse events, vital signs, ECG, and safety laboratory results. Secondary endpoints include safety and tolerability after TIL therapy has been administered, evaluation of antitumor responses and studies of tumor immune repolarization. Cohorts 1-3 have been completed without DLTs. Enrollment in cohort 4 was initiated in December 2021. Clinical trial information: NCT04217473.

Neoantigen Identification and Response to Adoptive Cell Transfer in Anti-PD-1 Naïve and Experienced Patients with Metastatic Melanoma.

Immune checkpoint blockade (ICB) agents and adoptive cell transfer (ACT) of tumor-infiltrating lymphocytes (TIL) are prominent immunotherapies used for the treatment of advanced melanoma. Both therapies rely on activation of lymphocytes that target shared tumor antigens or neoantigens. Recent analysis of patients with metastatic melanoma who underwent treatment with TIL ACT at the NCI demonstrated decreased responses in patients previously treated with anti-PD-1 agents. We aimed to find a basis for the difference in response rates between anti-PD-1 naïve and experienced patients. We examined the tumor mutational burden (TMB) of resected tumors and the repertoire of neoantigens targeted by autologous TIL in a cohort of 112 anti-PD-1 naïve and 69 anti-PD-1 experienced patients. Anti-PD-1 naïve patients were found to possess tumors with higher TMBs (352.0 vs. 213.5, P = 0.005) and received TIL reactive with more neoantigens (2 vs. 1, P = 0.003) compared with anti-PD-1 experienced patients. Among patients treated with TIL ACT, TMB and number of neoantigens identified were higher in ACT responders than ACT nonresponders in both anti-PD-1 naïve and experienced patients. Among patients with comparable TMBs and predicted neoantigen loads, treatment products administered to anti-PD-1 naïve patients were more likely to contain T cells reactive against neoantigens than treatment products for anti-PD-1 experienced patients (2.5 vs. 1, P = 0.02). These results indicate that decreases in TMB and targeted neoantigens partially account for the difference in response to ACT and that additional factors likely influence responses in these patients. See related commentary by Blass and Ott, p. 2980.

Abstract P2-14-07: Marrow-infiltrating lymphocytes as adoptive immunotherapy for breast cancer

Abstract Background: The bone marrow harbors tumor-antigen specific T cells in patients with breast cancer (1). Marrow-infiltrating lymphocytes (MILs®) are the product of activating and expanding bone-marrow T cells (2). In multiple myeloma, transfer of MILs as adoptive cell therapy has demonstrated anti-tumor activity (3). The use of adoptive transfer of autologous tumor-infiltrating lymphocytes (TILs) has been used successfully for hormone-receptor positive (HR+) metastatic breast cancer (4). Expansion of MILs was previously shown to be feasible in breast cancer, non-small cell lung cancer (NSCLC), prostate, head and neck, glioblastoma and multiple myeloma (5). Using an expanded cohort, we demonstrate that tumor-specific MILs can be expanded from bone marrow in patients with breast cancer. Methods: Bone marrow aspiration and blood samples were collected from 8 patients with metastatic and high-risk early-stage breast cancer, including 3 patients with HR+/HER2-, 2 patients with HER2+, and 3 patients with triple negative subtypes, at Providence Cancer Institute (Portland, OR). MILs and peripheral blood lymphocytes were activated and expanded from patient samples during a 10-day proprietary process. T cell lineage-specific markers CD3, CD4 and CD8 were characterized by flow cytometry pre- and post- expansion. Tumor-specific T cells were quantitated in expanded MILs and peripheral blood lymphocytes using a previously described cytokine-secretion assay (3). Briefly, they were defined as the IFNy-producing population by flow cytometry. Autologous antigen-presenting cells (APCs) were pulsed with lysates from allogeneic cancer cell lines and co-cultured with activated MILs or peripheral blood lymphocytes. APCs pulsed with irrelevant mis-matched cancer cell line lysates or media alone were used as negative controls. Results: In all harvested samples, MILs expansion successfully resulted in a selective increase in CD3+ T cells. Cytokine-producing CD4+ and CD8+ T cells were detected in all expanded MILs samples, but not in any of the matched activated and expanded peripheral blood lymphocytes. MILs were successfully expanded in HR+/HER2-, HER2+, and triple negative subtypes without apparent differences in activity as measured by cytokine production. Conclusion: MILs isolated from this expanded cohort of metastatic and high-risk early stage breast cancer patients demonstrated cytokine production in response to pulsed cancer cell line lysates, consistent with our experience with other solid (5) and hematological (3) malignancies. A phase II trial to evaluate MILs in combination with a checkpoint inhibitor is underway in patients with anti-PD1/PDL1-refractory NSCLC (NCT04069936). These preclinical data demonstrate that expanding MILs is feasible and could be evaluated therapeutically in breast cancer.

Adoptive tumor infiltrating lymphocyte transfer as personalized immunotherapy.

Cancer is a leading cause of death worldwide and, despite new targeted therapies and immunotherapies, a large group of patients fail to respond to therapy or progress after initial response, which brings the need for additional treatment options. Manipulating the immune system using a variety of approaches has been explored for the past years with successful results. Sustained progress has been made to understand the T cell-mediated anti-tumor responses counteracting the tumorigenesis process. The T-lymphocyte pool, especially its capacity for antigen-directed cytotoxicity, has become a central focus for engaging the immune system in defeating cancer. The adoptive cell transfer of autologous tumor-infiltrating lymphocytes has been used in humans for over 30 years to treat metastatic melanoma. In this review, we provide a brief history of ACT-TIL and discuss the current state of ACT-TIL clinical development in solid tumors. We also discuss how key advances in understanding genetic intratumor heterogeneity, to accurately identify neoantigens, and new strategies designed to overcome T-cell exhaustion and tumor immunosuppression have improved the efficacy of the TIL-therapy infusion. Characteristics of the TIL products will be discussed, as well as new strategies, including the selective expansion of specific fractions from the cell product or the genetic manipulation of T cells for improving the in-vivo survival and functionality. In summary, this review outlines the potential of ACT-TIL as a personalized approach for epithelial tumors and continued discoveries are making it increasingly more effective against other types of cancers.

Immunotherapy Resistance in Glioblastoma

Glioblastoma is the most common malignant primary brain tumor in adults. Despite treatment consisting of surgical resection followed by radiotherapy and adjuvant chemotherapy, survival remains poor at a rate of 26.5% at 2 years. Recent successes in using immunotherapies to treat a number of solid and hematologic cancers have led to a growing interest in harnessing the immune system to target glioblastoma. Several studies have examined the efficacy of various immunotherapies, including checkpoint inhibitors, vaccines, adoptive transfer of lymphocytes, and oncolytic virotherapy in both pre-clinical and clinical settings. However, these therapies have yielded mixed results at best when applied to glioblastoma. While the initial failures of immunotherapy were thought to reflect the immunoprivileged environment of the brain, more recent studies have revealed immune escape mechanisms created by the tumor itself and adaptive resistance acquired in response to therapy. Several of these resistance mechanisms hijack key signaling pathways within the immune system to create a protumoral microenvironment. In this review, we discuss immunotherapies that have been trialed in glioblastoma, mechanisms of tumor resistance, and strategies to sensitize these tumors to immunotherapies. Insights gained from the studies summarized here may help pave the way for novel therapies to overcome barriers that have thus far limited the success of immunotherapy in glioblastoma.

B cells do not play a role in vaccine-mediated immunity against Marek’s disease

BackgroundMarek’s disease virus (MDV), a highly oncogenic α-herpesvirus, is the etiological agent of Marek’s disease (MD) in chickens. The antiviral activity of vaccine-induced immunity against MD reduces the level of early cytolytic infection, production of cell-free virions in the feather follicle epithelial cells (FFE), and lymphoma formation. Despite the success of several vaccines that have greatly reduced the economic losses from MD, the mechanism of vaccine-induced immunity is poorly understood. MethodsTo provide insight into possible role of B cells in vaccine-mediated protection, we bursectomized birds on day of hatch and vaccinated them eight days later. The birds were challenged 10 days post vaccination with or without receiving adoptive lymphocytes from age-matched control birds prior to inoculation. The study also included vaccinated/challenged and non-vaccinated challenged intact birds. Flowcytometric analysis of PBMN cells were conducted twice post bursectomy to confirm B cell depletion and assess the effect of surgery on T cell population. Immunohistochemical analysis and viral genome copy number assessment in the skin samples at termination was performed to measure the replication rate of MDV in the FFE of the skin tissues of the challenged birds. ResultsThe non-vaccinated/challenged birds developed typical clinical signs of MD while the vaccinated/challenged and bursectomized, vaccinated/challenged groups with or without adoptive lymphocyte transfer, were fully protected with no sign of transient paralysis, weight loss, or T cell lymphomas. Immunohistochemical analysis and viral genome copy number evaluation in the skin samples revealed that unlike the vaccinated/challenged birds a significant number of virus particles were produced in the FFE of the non-vaccinated/challenged birds at termination. In the bursectomized, vaccinated/challenged groups, only a few replicating virions were detected in the skin of birds that received adoptive lymphocytes prior to challenge. ConclusionsThe study shows that B cells do not play a critical role in MD vaccine-mediated immunity.

Alloantigen-activated (AAA) CD4+ T cells reinvigorate host endogenous T cell immunity to eliminate pre-established tumors in mice

BackgroundCancer vaccines that induce endogenous antitumor immunity represent an ideal strategy to overcome intractable cancers. However, doing this against a pre-established cancer using autologous immune cells has proven to be challenging. “Allogeneic effects” refers to the induction of an endogenous immune response upon adoptive transfer of allogeneic lymphocytes without utilizing hematopoietic stem cell transplantation. While allogeneic lymphocytes have a potent ability to activate host immunity as a cell adjuvant, novel strategies that can activate endogenous antitumor activity in cancer patients remain an unmet need. In this study, we established a new method to destroy pre-developed tumors and confer potent antitumor immunity in mice using alloantigen-activated CD4+ (named AAA-CD4+) T cells.MethodsAAA-CD4+ T cells were generated from CD4+ T cells isolated from BALB/c mice in cultures with dendritic cells (DCs) induced from C57BL/6 (B6) mice. In this culture, allogeneic CD4+ T cells that recognize and react to B6 mouse-derived alloantigens are preferentially activated. These AAA-CD4+ T cells were directly injected into the pre-established melanoma in B6 mice to assess their ability to elicit antitumor immunity in vivo.ResultsUpon intratumoral injection, these AAA-CD4+ T cells underwent a dramatic expansion in the tumor and secreted high levels of IFN-γ and IL-2. This was accompanied by markedly increased infiltration of host-derived CD8+ T cells, CD4+ T cells, natural killer (NK) cells, DCs, and type-1 like macrophages. Selective depletion of host CD8+ T cells, rather than NK cells, abrogated this therapeutic effect. Thus, intratumoral administration of AAA-CD4+ T cells results in a robust endogenous CD8+ T cell response that destroys pre-established melanoma. This locally induced antitumor immunity elicited systemic protection to eliminate tumors at distal sites, persisted over 6 months in vivo, and protected the animals from tumor re-challenge. Notably, the injected AAA-CD4+ T cells disappeared within 7 days and caused no adverse reactions.ConclusionsOur findings indicate that AAA-CD4+ T cells reinvigorate endogenous cytotoxic T cells to eradicate pre-established melanoma and induce long-term protective antitumor immunity. This approach can be immediately applied to patients with advanced melanoma and may have broad implications in the treatment of other types of solid tumors.

Adoptive Transfer of Tumor-Infiltrating Lymphocytes in Metastatic Melanoma

Adoptive Transfer of Tumor-Infiltrating Lymphocytes in Metastatic Melanoma

Impact of Prior Treatment on the Efficacy of Adoptive Transfer of Tumor-Infiltrating Lymphocytes in Patients with Metastatic Melanoma.

Adoptive cell transfer (ACT) of autologous tumor-infiltrating lymphocytes (TIL) can mediate durable responses in patients with metastatic melanoma. This retrospective analysis provides long-term follow-up and describes the effect of prior therapy on outcomes after ACT-TIL. Patients with metastatic melanoma underwent surgical resection of a tumor for generation of TILs and were treated with a lymphodepleting preparative regimen followed by adoptive transfer of TILs and intravenous IL2. Clinical characteristics of enrolled patients and treatment characteristics of TIL infusion products over two decades of ACT were analyzed to identify predictors of objective response. Adoptive transfer of TILs mediated an objective response rate of 56% (108/192) and median melanoma-specific survival of 28.5 months in patients naïve to anti-programmed cell death-1 (PD-1) therapy compared with 24% (8/34) and 11.6 months in patients refractory to anti-PD-1 (aPD-1). Among patients with BRAF V600E/K-mutated disease, prior treatment with targeted molecular therapy was also associated with a decreased response rate (21% vs. 60%) and decreased survival (9.3 vs. 50.7 months) when compared with those patients naïve to targeted therapy. With a median potential follow-up of 89 months, 46 of 48 complete responders in the aPD-1-naïve cohort have ongoing responses after a single treatment and 10-year melanoma-specific survival of 96%. Patients previously treated with PD-1 or MAPK inhibition are significantly less likely to develop durable objective responses to ACT-TIL. While ACT-TIL is currently being investigated for treatment-refractory patients, it should also be considered as an initial treatment option for eligible patients with metastatic melanoma. See related commentary by Sznol, p. 5156.