Autologous Peripheral Blood Lymphocytes Research Articles

FANCA promotes lung adenocarcinoma progression and is a potential target for epitope vaccine immunotherapy

BackgroundFANCA mutations have been detected in a variety of cancers and found to be pro-carcinogenic. However, no functional studies have been identified regarding the involvement of FANCA in the occurrence and the immune response of LUAD.MethodsThe mRNA expression and overall survival rates of FANCA were evaluated by the TIMER, PrognoScan and TCGA database in LUAD tissues, and FANCA expression was further validated by clinical serum samples using ELISA. The correlation between FANCA and immune infiltration level was investigated via TISIDB database and CIBERSORT algorithm. The Kaplan–Meier plotter was used to further evaluate the prognostic value based on the expression levels of FANCA in related immune cells. Then, the influence of FANCA knockout on the proliferation, migration, and invasion of A549 and H1299 cells was validated using CCK8, cloning formation, and Transwell assays. Subsequently, HLA-A2-restricted FANCA antigenic peptides were predicted and synthesized by NetMHC4.0 and SYFPEITHI, and DCs were induced and cultured in vitro. Finally, DCs loaded with HLA-A2-restricted FANCA antigenic peptides were co-cultured with autologous peripheral blood lymphocyte to generate specific CTLs. The killing effects of different CTLs on LUAD cells were studied.ResultsThe results showed that high levels of FANCA in patients with LUAD were significantly correlated with worse OS survival, which was correlated with age, clinical stage, pathological T stage, M stage, and N stage in LUAD. Knockdown of FANCA in A549 and H1299 cells significantly inhibited proliferation, metastasis, and invasion in vitro. In addition, FANCA was significantly related to immune infiltrate, genomic alterations and TMB. FANCA expression infuenced the prognosis of LUAD patients by directly affecting immune cell infltration. Finally, HLA-A2-restricted FANCA antigenic peptides were synthesized. And FANCA 146–154 (SLLEFAQYL) antigenic peptide exhibit a stronger affinity for DCs, and induce CTLs to produce stronger targeted killing ability for LUAD cells at an effector-to-target ratio of 40:1.ConclusionThese results demonstrated that the elevation of FANCA promotes malignant phenotype of LUAD, and the potential peptide P2 (SLLEFAQYL) derived from FANCA may be used as an epitope vaccine for the treatment of LUAD.

Adoptive transfer of personalized neoantigen-reactive TCR-transduced T cells in metastatic colorectal cancer: phase 2 trial interim results.

Adoptive cell transfer (ACT) with neoantigen-reactive T lymphocytes can mediate cancer regression. Here we isolated unique, personalized, neoantigen-reactive T cell receptors (TCRs) from tumor-infiltrating lymphocytes of patients with metastatic gastrointestinal cancers and incorporated the TCR α and β chains into gamma retroviral vectors. We transduced autologous peripheral blood lymphocytes and adoptively transferred these cells into patients after lymphodepleting chemotherapy. In a phase 2 single-arm study, we treated seven patients with metastatic, mismatch repair-proficient colorectal cancers who had progressive disease following multiple previous therapies. The primary end point of the study was the objective response rate as measured using RECIST 1.1, and the secondary end points were safety and tolerability. There was no prespecified interim analysis defined in this study. Three patients had objective clinical responses by RECIST criteria including regressions of metastases to the liver, lungs and lymph nodes lasting 4 to 7 months. All patients received T cell populations containing ≥50% TCR-transduced cells, and all T cell populations were polyfunctional in that they secreted IFNγ, GM-CSF, IL-2 and granzyme B specifically in response to mutant peptides compared with wild-type counterparts. TCR-transduced cells were detected in the peripheral blood of five patients, including the three responders, at levels ≥10% of CD3+ cells 1 month post-ACT. In one patient who responded to therapy, ~20% of CD3+ peripheral blood lymphocytes expressed transduced TCRs more than 2 years after treatment. This study provides early results suggesting that ACT with T cells genetically modified to express personalized neoantigen-reactive TCRs can be tolerated and can mediate tumor regression in patients with metastatic colorectal cancers. ClinicalTrials.gov registration: NCT03412877 .

Tumor Organoids: The Era of Personalized Medicine.

The strategies of future medicine are aimed to modernize and integrate quality approaches including early molecular-genetic profiling, identification of new therapeutic targets and adapting design for clinical trials, personalized drug screening (PDS) to help predict and individualize patient treatment regimens. In the past decade, organoid models have emerged as an innovative in vitro platform with the potential to realize the concept of patient-centered medicine. Organoids are spatially restricted three-dimensional clusters of cells ex vivo that self-organize into complex functional structures through genetically programmed determination, which is crucial for reconstructing the architecture of the primary tissue and organs. Currently, there are several strategies to create three-dimensional (3D) tumor systems using (i) surgically resected patient tissue (PDTOs, patient-derived tumor organoids) or (ii) single tumor cells circulating in the patient's blood. Successful application of 3D tumor models obtained by co-culturing autologous tumor organoids (PDTOs) and peripheral blood lymphocytes have been demonstrated in a number of studies. Such models simulate a 3D tumor architecture in vivo and contain all cell types characteristic of this tissue, including immune system cells and stem cells. Components of the tumor microenvironment, such as fibroblasts and immune system cells, affect tumor growth and its drug resistance. In this review, we analyzed the evolution of tumor models from two-dimensional (2D) cell cultures and laboratory animals to 3D tissue-specific tumor organoids, their significance in identifying mechanisms of antitumor response and drug resistance, and use of these models in drug screening and development of precision methods in cancer treatment.

Abstract IA001: Patient-derived tumor organoids to probe the cancer immune interface

Abstract The development of rational (combination) immunotherapies is limited by an incomplete understanding of what determines a tumor’s sensitivity to immune attack. Despite great heterogeneity in resistance mechanisms between patients, functional model systems to study cancer/immune cell interactions at the level of individual patients have been lacking. Here we highlight the potential of organoid/immune cell co-cultures as individualized models of anti-tumor immunity. First, we established a co-culture platform of autologous tumor organoids and peripheral blood lymphocytes from patients with mismatch-repair deficient colorectal cancer (dMMR-CRC) and non-small-cell lung cancer (NSCLC). This allows enriching tumor-reactive T cells from peripheral blood of individual patients, and creating personalized test systems to evaluate their capacity to kill matched tumor organoids. Second, subclonal immune escape is a major barrier to achieving complete responses to cancer immunotherapies. While most tumours consist of multiple genetically distinct clones, directly investigating the potential for immune escape at the clonal level has been impossible so far, due to challenges in isolating and propagating individual tumour subclones from human cancers for functional studies. By leveraging the multi-region TRACERx study, we established multi-region organoid libraries of >20 parallel sublines from patients with NSCLC. Co-cultures of each subline with autologous tumour infiltrating lymphocytes (TIL) reveal substantial heterogeneity in the capacity of individual clonal sublines to elicit a T-cell response. We show that subclonal immune escape can be mediated by antigen-dependent and -independent mechanisms. These results show (i) that tumour evolution can give rise to distinct cancer clones with intrinsic differences in immune evasion capacity and (ii) provide an approach to prospectively identify and isolate immune evading subclones from cancer patients. Third, analysis of a clinical cohort of patients treated with anti-PD1 therapy revealed that gamma delta T cells are critical mediators of the response in dMMR-CRC patients with genetic inactivation of beta-2-microglobulin (B2M). Co-cultures of organoids and gamma delta T cells provide direct evidence that loss of B2M increases reactivity of patient-derived gamma delta T cells towards tumor organoids. This highlights the need for functional models that move beyond conventional T cells. Future developments are directed towards establishment of complex co-culture systems to study how communication between organoids and immune cells is mediated by cell surface factors and secreted molecules, and how cellular crosstalk is altered in cancer. Citation Format: Krijn K Dijkstra. Patient-derived tumor organoids to probe the cancer immune interface [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr IA001.

Abstract 1818: Developing an autologous humanized mouse model to assess responses to novel immunotherapeutics

Abstract Introduction: The development and FDA approval of drugs targeting the PD-1/PD-L1 axis has greatly shifted the paradigm of immunotherapy and has improved the clinical outcome for patients diagnosed with advanced melanoma. Despite the recent advancement of immune checkpoint inhibitors, more than 50% of patients still fail to show a sustained response to therapy. Therefore, there is a critical need to develop therapies specific to novel immune targets as well as combination drug regimens. Current immunocompetent preclinical models do not adequately model the responses to immunotherapy, reflecting the urgent need to develop models with greater translational utility. To address this need, we developed a humanized xenograft melanoma model engrafted with autologous peripheral blood lymphocytes (PBLs) to evaluate the utility of nemvaleukin alfa, an engineered IL-2 cytokine which preferentially activates and expands cytotoxic NK cells and antigen-experienced CD8 T cells. Methods: Melanoma-bearing mice were injected intravenously with matched melanoma patient PBLs and treated for four cycles (1 cycle/week) with anti-PD-1, nemvaleukin, or rhIL-2. Tumor growth was monitored, and correlative analyses were done on blood, spleen, and tumors by flow cytometry after 4 cycles of treatment. Results: This humanized melanoma mouse model exhibited good T cell engraftment and was characterized by expression of the proliferative marker Ki-67 in 50-60% of CD8+ and CD4+ T cell populations. Treatment with nemvaleukin resulted in significant tumor growth inhibition rates (TGIs) of an average of 48% compared to vehicle-treated mice. In addition, nemvaleukin promoted expansion of CD8+ and CD4+ T cells, without expansion of CD4+ regulatory T cells, which are known to suppress immunological responses. Compared to rhIL-2 and vehicle, nemvaleukin treatment delayed tumor growth and enhanced survival with a median survival time of 56 days compared to 21 and 50 days, respectively. Nemvaleukin-treated mice had higher CD45+ cell engraftment in spleens and tumors compared to anti-PD-1 treatment, indicating that nemvaleukin boosts T cell proliferation and expansion. Of note, we observed better antitumor efficacy in response to nemvaleukin or anti-PD-1 when mice were engrafted with tumors from patients who responded to checkpoint immunotherapy with TGIs of 36% and 51%, respectively, compared to those who did not with TGIs of 16% and 12%, respectively. These data indicate that treatment responses in our humanized model correlates with clinical outcomes. Conclusion: In this all autologous humanized melanoma mouse model, nemvaleukin treatment not only enhances T cell engraftment, it significantly augments anti-tumor efficacy. These data support the use of this personalized melanoma xenograft model to analyze the underlying immune responses in responders and non-responders to novel immunotherapies. Citation Format: Yee Peng Phoon, Jared E. Lopes, Lukas W. Pfannenstiel, C. Marcela Diaz-Montero, Ye F. Tian, Marc S. Ernstoff, Pauline Funchain, XiaoXia Li, Heather C. Losey, Raymond J. Winquist, Brian R. Gastman. Developing an autologous humanized mouse model to assess responses to novel immunotherapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1818.

Generation of murine tumour-reactive T cells by co-culturing murine pancreatic cancer organoids and peripheral blood lymphocytes

Pancreatic ductal adenocarcinoma (PDAC) is commonly diagnosed at a late stage and becomes resistant to several treatments. Significant clinical effects have been reported for cancer immunotherapies on a subset of patients diagnosed with epithelial cancers. Cancer organoid co-culture with autologous peripheral blood lymphocytes offers an innovative immunotherapeutic approach that is increasingly being tested, although there is a lack of cutting-edge platforms enabling the investigation of cancer-T cell interactions for individual patients. In this study, a pancreatic cancer organoid culture from a genetically engineered pancreatic cancer murine model was established and co-cultured with autologous peripheral blood lymphocytes to induce a tumour-specific T cell response to pancreatic cancer. Co-culturing autologous peripheral blood lymphocytes with cancer organoids can be an effective strategy to enrich tumour-reactive T cells from the peripheral blood of murine models; this approach could potentially be transferred to humans. Co-culture of peripheral blood lymphocytes and cancer organoids could provide an unbiased approach to evaluating the sensitivity of tumour cells to T cell-mediated priming on an individual patient level.

Catfish lymphocytes expressing CC41-reactive leukocyte immune-type receptors (LITRs) proliferate in response to Edwardsiella ictaluri infection in vitro

Monoclonal antibodies (mAbs) CC34 and CC41 recognize overlapping subsets of leukocyte immune-type receptors (LITRs). The mAb CC34 was raised against the clonal TS32.15 cytotoxic T cell line and the mAb CC41 was raised against the clonal NK cell line TS10.1. In this study, an in vitro model was developed to monitor CC34- and CC41-reactive cells in response to Edwardsiella ictaluri infection. Briefly, head kidney leukocytes and peripheral blood lymphocytes (PBL) were isolated from individual catfish and labeled with CellTrace Violet and CellTrace FarRed dye, respectively. Head kidney-derived macrophages were infected with E. ictaluri and then cocultured with autologous PBL. The combined cell cultures were then analyzed using flow cytometry. A significant increase in CC41 staining was observed in the PBL population at 2, 5 and 7 days after culture, which suggest that LITRs are involved in cell-mediated immunity to E. ictaluri.

Tumor organoid-T-cell coculture systems.

T cells are key players in cancer immunotherapy, but strategies to expand tumor-reactive cells and study their interactions with tumor cells at the level of an individual patient are limited. Here we describe the generation and functional assessment of tumor-reactive T cells based on cocultures of tumor organoids and autologous peripheral blood lymphocytes. The procedure consists of an initial coculture of 2 weeks, in which tumor-reactive T cells are first expanded in the presence of (IFNγ-stimulated) autologous tumor cells. Subsequently, T cells are evaluated for their capacity to carry out effector functions (IFNγ secretion and degranulation) after recognition of tumor cells, and their capacity to kill tumor organoids. This strategy is unique in its use of peripheral blood as a source of tumor-reactive T cells in an antigen-agnostic manner. In 2 weeks, tumor-reactive CD8+ T-cell populations can be obtained from ~33-50% of samples from patients with non-small-cell lung cancer (NSCLC) and microsatellite-instable colorectal cancer (CRC). This enables the establishment of ex vivo test systems for T-cell-based immunotherapy at the level of the individual patient.

Study of Autologous Peripheral Blood Lymphocytes in the Treatment of Patients With CLL or SLL

Study of Autologous Peripheral Blood Lymphocytes in the Treatment of Patients With CLL or SLL

Assessment of the presence and anti-tumor potential of tumor-infiltrating lymphocytes in patients with acute myeloid leukemia.

Purpose: Assessing the possibility of finding tumor-infiltrating lymphocytes (TIL) in bone marrow of acute myeloid leukemia (AML) patients and evaluating the anti-tumor activity of these TIL against autologous AML cells.Patients and methods: TIL were immunomagnetically isolated by using anti-CD3 from bone marrow samples of 20 patients at the presentation of AML or four weeks upon completion of chemotherapy. TIL were ex vivo expanded for two weeks and immunophenotyped. Functionality in terms of cytokine secretion and cytotoxicity was assessed by γ-interferon quantitation and Elispot assay, respectively.Results: TIL were detected in bone marrow samples of 50% (10/20) of the patient cohort. They were noted to highly express CD137 and PD-1 and display a significantly higher anti-tumor reactivity compared to that of autologous peripheral blood lymphocytes. TIL could be expanded in co-cultures with irradiated feeder cells supplemented with interleukin (IL)-7 and IL-15.Conclusion: Data suggested the presence of reactive γ-interferon-secreting TIL in AML patients. They are expandable and possess anti-tumor activity, which might have a great potential in the development of adoptive cellular therapy for AML.

Abstract P2-09-13: A phase I study of adoptive immunotherapy for ROR1+ advanced triple negative breast cancer (TNBC) with defined subsets of autologous T cells expressing a ROR1-specific chimeric antigen receptor (ROR1-CAR)

Abstract BACKGROUND: ROR1 is a type 1 transmembrane tyrosine kinase receptor that plays a critical role in embryonic and fetal development. ROR1 has been described as a possible oncogene and is expressed in numerous malignancies including TNBC and non-small cell lung cancer (NSCLC). We are conducting a first-in-human trial targeting ROR1 with CAR-T cells in patients with advanced TNBC and NSCLC. The cellular construct employed targets the Ig/Fz portion of the extracellular domain of ROR1 and contains 4-1BB/CD3ζ intracellular signaling domain. The manufacturing process utilizes autologous peripheral blood lymphocytes, separated into CD4 and CD8 subsets, which are independently cultured with anti-CD3/anti-CD28 beads and IL-2, then transduced with a lentiviral vector encoding the ROR1 CAR. The CAR-T cell product is formulated in a 1:1 ratio of CD4+ and CD8+ CAR-T cells. METHODS: This ongoing phase I trial (NCT02706392) is evaluating the safety of administering ROR1 CAR-T cells in escalating doses (3.3x105, 1x106, 3.3x106 and 1x107 cells/kg) following lymphodepletion with cyclophosphamide-containing regimens using a continual reassessment method (CRM) for dose escalation. TNBC patients with adequate organ function and performance status, measurable disease, and tumors expressing ROR1 (>20% by IHC) are eligible for enrollment. Persistence of CAR-T cells in blood, cytokine levels, measures of immunogenicity and multi-parametric flow cytometry are being evaluated at multiple time points. Imaging assessments by RECIST 1.1 are performed day 28 - 90, then at 6 and 12 months, and every 6 months as clinically indicated to estimate efficacy. RESULTS: To date, 4 TNBC patients (age range 38-67) have been enrolled, treated and are evaluable for response. Patients had received prior therapies for metastatic disease (range 3-11). 3 of 4 had visceral metastases. No dose-limiting toxicities, severe neurotoxicity or severe cytokine release syndrome (sCRS) were observed at dose levels 1 and 2. Two patients experienced grade 1 CRS. 2 of 4 patients had evidence of CAR-T cell expansion between days 14 and 20, with peak CD8+ CAR-T up to 232.1 cells/uL. Analysis of surface phenotype revealed upregulation of inhibitory receptors on CAR-T cells at the peak of expansion, confirmed by RNA seq. Post-treatment tumor biopsy in patient with partial response revealed an influx of CD3+ T cells and macrophages suggesting ROR1 CAR-T cell trafficking. Two patients received 2 CAR-T cell infusions. Two patients had confirmed stable disease at 15 weeks and 19 weeks, respectively. One patient has stable disease after 1st CAR-T cell infusion, then confirmed partial response after 2nd CAR-T cell infusion which has persisted for 14 weeks. Results will be updated. CONCLUSIONS: ROR1+ CAR-T cells can be safely transferred, expand in vivo in patients with TNBC. Current efforts are directed at understanding and overcoming the mechanisms that limit homing, persistence, and/or function at tumor sites. The trial is continuing with dose-escalation. Funding provided by 8RO1 CA114536-11 and Juno Therapeutics. Citation Format: Specht JM, Lee SM, Turtle C, Berger C, Balakrishnan A, Srivastava S, Viollet V, Veatch J, Gooley T, Mullane E, Chaney CN, Rader C, Pierce RH, Gottardo R, Maloney DG, Riddell SR. A phase I study of adoptive immunotherapy for ROR1+ advanced triple negative breast cancer (TNBC) with defined subsets of autologous T cells expressing a ROR1-specific chimeric antigen receptor (ROR1-CAR) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-09-13.

Generation of Tumor-Reactive T Cells by Co-culture of Peripheral Blood Lymphocytes and Tumor Organoids

Cancer immunotherapies have shown substantial clinical activity for a subset of patients with epithelial cancers. Still, technological platforms to study cancer T-cell interactions for individual patients and understand determinants of responsiveness are presently lacking. Here, we establish and validate a platform to induce and analyze tumor-specific Tcell responses to epithelial cancers in a personalized manner. We demonstrate that co-cultures of autologous tumor organoids and peripheral blood lymphocytes can be used to enrich tumor-reactive Tcells from peripheral blood of patients with mismatch repair-deficient colorectal cancer and non-small-cell lung cancer. Furthermore, we demonstrate that these Tcells can be used to assess the efficiency of killing of matched tumor organoids. This platform provides an unbiased strategy for the isolation of tumor-reactive Tcells and provides a means by which to assess the sensitivity of tumor cells to T cell-mediated attack at the level of the individual patient.

Abstract CT131: A phase I study of adoptive immunotherapy for advanced ROR1+ malignancies with defined subsets of autologous T cells expressing a ROR1-specific chimeric antigen receptor (ROR1-CAR)

Abstract INTRODUCTION: ROR1 is a type 1 transmembrane tyrosine kinase receptor that plays a critical role in embryonic and fetal development. ROR1 has been described as a possible oncogene and is expressed in numerous malignancies, including a subset of non-small cell lung cancer (NSCLC) and triple negative breast cancer (TNBC). We are conducting a first-in-human trial targeting ROR1 with CAR-T cells in patients with advanced NSCLC and TNBC. The cellular construct employed targets the Ig/Fz portion of the extracellular domain of ROR1 and contains 4-1BB/CD3ζ intracellular signaling domain. The manufacturing process utilizes autologous peripheral blood lymphocytes, separated into CD4 and CD8 subsets, which are independently cultured with anti-CD3/anti-CD28 beads and IL-2, then transduced with a lentiviral vector encoding the ROR1 CAR. The CAR-T cell product is formulated in a 1:1 ratio of CD4+ and CD8+ CAR-T cells. STUDY DESIGN: This ongoing phase 1 trial (NCT02706392) is evaluating the safety of administering ROR1 CAR-T cells in escalating doses (3.3x105, 1x106, 3.3x106 and 1x107 cells/kg) following lymphodepletion with cyclophosphamide-containing regimens using a continual reassessment method (CRM) for dose escalation. NSCLC and TNBC patients with adequate organ function and PS, measurable disease, and tumors expressing ROR1 (>20% by IHC) are eligible for enrollment. Persistence of CAR-T cells in blood, cytokine levels, measures of immunogenicity and multi-parametric flow cytometry are being evaluated at multiple time points. Imaging assessments by RECIST 1.1 are performed day 28 - 90, then at 6 and 12 months, and every 6 months as clinically indicated to estimate efficacy. RESULTS: To date, 6 patients (4-TNBC, 2-NSCLC) have been enrolled and treated. Five patients are evaluable for response. No dose-limiting toxicities, severe neurotoxicity or severe cytokine release syndrome (sCRS) were observed at dose levels 1 and 2. Three patients experienced grade 1 CRS. Three patients had evidence of CAR-T cell expansion between days 14 and 20, with peak CD8+ CAR-T up to 747/uL. Analysis of surface phenotype revealed upregulation of inhibitory receptors on CAR-T cells at the peak of expansion, confirmed by RNA seq. Post-treatment biopsies in 2 patients revealed a low level of CAR-T cell tumor infiltration. Four patients (2-NSCLC; 2-TNBC) demonstrated a mixed response with decreased disease burden at some metastatic sites at first disease assessment. One TNBC patient received 2 CAR-T cell infusions and remains with stable disease 56 days after second infusion with heavy burden of liver metastases. CONCLUSIONS: ROR1+ CAR-T cells can be safely transferred, expand in vivo in patients with NSCLC and TNBC. Current efforts are directed at understanding and overcoming the mechanisms that limit homing, persistence, and/or function at tumor sites. The trial is continuing enrollment with dose-escalation. Funding provided by 8R01 CA114536-11 and Juno Therapeutics. Citation Format: Jennifer M. Specht, Sylvia Lee, Cameron J. Turtle, Carolina Berger, Ashwini Baladrishnan, Shivani Srivastava, Valentin Voillet, Josh Veatch, Ted Gooley, Erin Mullane, Colette Chaney, Christoph Rader, Robert H. Pierce, Raphael Gottardo, David G. Maloney, Stanley R. Riddell. A phase I study of adoptive immunotherapy for advanced ROR1+ malignancies with defined subsets of autologous T cells expressing a ROR1-specific chimeric antigen receptor (ROR1-CAR) [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 CT131.

Recent Advances in Targeting CD8 T-Cell Immunity for More Effective Cancer Immunotherapy.

Recent advances in cancer treatment have emerged from new immunotherapies targeting T-cell inhibitory receptors, including cytotoxic T-lymphocyte associated antigen (CTLA)-4 and programmed cell death (PD)-1. In this context, anti-CTLA-4 and anti-PD-1 monoclonal antibodies have demonstrated survival benefits in numerous cancers, including melanoma and non-small-cell lung carcinoma. PD-1-expressing CD8+ T lymphocytes appear to play a major role in the response to these immune checkpoint inhibitors (ICI). Cytotoxic T lymphocytes (CTL) eliminate malignant cells through recognition by the T-cell receptor (TCR) of specific antigenic peptides presented on the surface of cancer cells by major histocompatibility complex class I/beta-2-microglobulin complexes, and through killing of target cells, mainly by releasing the content of secretory lysosomes containing perforin and granzyme B. T-cell adhesion molecules and, in particular, lymphocyte-function-associated antigen-1 and CD103 integrins, and their cognate ligands, respectively, intercellular adhesion molecule 1 and E-cadherin, on target cells, are involved in strengthening the interaction between CTL and tumor cells. Tumor-specific CTL have been isolated from tumor-infiltrating lymphocytes and peripheral blood lymphocytes (PBL) of patients with varied cancers. TCRβ-chain gene usage indicated that CTL identified in vitro selectively expanded in vivo at the tumor site compared to autologous PBL. Moreover, functional studies indicated that these CTL mediate human leukocyte antigen class I-restricted cytotoxic activity toward autologous tumor cells. Several of them recognize truly tumor-specific antigens encoded by mutated genes, also known as neoantigens, which likely play a key role in antitumor CD8 T-cell immunity. Accordingly, it has been shown that the presence of T lymphocytes directed toward tumor neoantigens is associated with patient response to immunotherapies, including ICI, adoptive cell transfer, and dendritic cell-based vaccines. These tumor-specific mutation-derived antigens open up new perspectives for development of effective second-generation therapeutic cancer vaccines.

Twenty-Five Years of Gene Therapy for ADA-SCID: FromBubble Babiesto an Approved Drug

Twenty-five years have passed since first attempts of gene therapy (GT) in children affected by severe combined immunodeficiency (SCID) due to adenosine deaminase (ADA) defect, also known by the general public as bubble babies. ADA-SCID is fatal early in life if untreated. Unconditioned hematopoietic stem cell (HSC) transplant from matched sibling donor represents a curative treatment but is available for few patients. Enzyme replacement therapy can be life-saving, but its chronic use has many drawbacks. This review summarizes the history of ADA-SCID GT over the last 25 years, starting from first pioneering studies in the early 1990s using gamma-retroviral vectors, based on multiple infusions of genetically corrected autologous peripheral blood lymphocytes. HSC represented the ideal target for gene correction to guarantee production of engineered multi-lineage progeny, but it required a decade to achieve therapeutic benefit with this approach. Introduction of low-intensity conditioning represented a crucial step in achieving stable gene-corrected HSC engraftment and therapeutic levels of ADA-expressing cells. Recent clinical trials demonstrated that gamma-retroviral GT for ADA-SCID has a favorable safety profile and is effective in restoring normal purine metabolism and immune functions in patients >13 years after treatment. No abnormal clonal proliferation or leukemia development have been observed in >40 patients treated experimentally in five different centers worldwide. In 2016, the medicinal product Strimvelis™ received marketing approval in Europe for patients affected by ADA-SCID without a suitable human leukocyte antigen-matched related donor. Positive safety and efficacy results have been obtained in GT clinical trials using lentiviral vectors encoding ADA. The results obtained in last 25 years in ADA-SCID GT development fundamentally contributed to improve patients' prognosis, together with earlier diagnosis thanks to newborn screening. These advances open the way to further clinical development of GT as treatment for broader applications, from inherited diseases to cancer.

Phase 1 study to evaluate the safety and efficacy of immunotherapy with tremelimumab and durvalumab in multiple myeloma patients receiving high dose chemotherapy and autologous stem cell transplant (HDT/ASCT) + peripheral blood lymphocyte (PBL) reinfusion.

TPS8051 Background: Multiple myeloma (MM) remains an incurable hematologic malignancy despite the advent of new classes of drugs, including immunomodulatory agents, proteasome inhibitors, and monoclonal antibodies. The success and synergistic activity of immunotherapy (IMT) in solid tumors and hematologic malignancies has fueled their investigation in MM. HDT/ASCT as consolidation or as treatment for relapse remains a cornerstone for improving overall survival. HDT/ASCT transiently eliminates immune-suppressive cell populations and provides a viable IMT platform. Reinfusion of PBLs harvested pre-HDT induces immune responses, supporting its inclusion in IMT combinations. This study evaluates the effect of IMT, using tremelimumab (T), an anti-CTLA-4 monoclonal antibody, and durvalumab (D), an anti-PD-L1 monoclonal antibody, together with autologous PBL reinfusion and starting T ± D at Day 100 and earlier (Day 30) post-ASCT. Methods: This ongoing Phase 1, open-label, multicenter study (NCT02716805) evaluates the safety and preliminary efficacy of T and D administered on 2 schedules in MM patients at high risk for relapse as outlined below. Cohort initiation requires dose-limiting toxicity in < 2/6 patients in the previous cohort. The primary endpoint is safety. Secondary endpoints are objective response rate per IMWG, minimal residual disease, progression free and overall survival, and 100-day ASCT-related mortality. Exploratory endpoints include immunological effects and immune response. Enrollment opened 18 Nov 2016. As of 31 Dec 2016, 1 patient is enrolled in Cohort 1; enrollment is ongoing. Clinical trial information: NCT02716805. [Table: see text]

BET Bromodomain Inhibition Selectively Targets the Epstein-Barr Virus Oncogene LMP1 While Promoting Virus-Specific, Adaptive Anti-Tumor Activity

Epstein-Barr virus (EBV) is a human herpesvirus that infects over 90% of the world's population and is associated with a wide-range of diseases. EBV has evolved to manipulate host cellular networks in the absence of proper immune function and is linked to malignant lymphoproliferative disorders (LPD) such as Hodgkin's lymphoma and diffuse large B-cell lymphomas. The lack of standard or effective therapeutic approaches for individuals with these aggressive and clinically complicated diseases represents an important unmet need.We have recently demonstrated EBV-induced B cell transformation to be dependent upon the dysregulation of protein arginine methyltransferase 5 (PRMT5), an epigenetic writer that functions as a global transcriptional repressor. We hypothesize that epigenetic readers, writers and erasers are similarly dysregulated following EBV infection of B cells and that these enzymes represent novel therapeutic candidates. The bromodomain and extra-terminal (BET) protein bromodomain 4 (BRD4) is an epigenetic reader that binds acetylated lysine residues and promotes transcription of genes that drive cell growth and survival. Here, we evaluate the relevance of BRD4 to EBV-driven B cell transformation and identify it as a potential therapeutic target for EBV-LPD. We used the highly selective and potent BRD4 inhibitor (BRD4i), JQ1, as a model to examine the relevance of BRD4 in EBV-driven lymphoproliferative disease (LPD).JQ1 treatment of, EBV-transformed lymphoblastoid cell lines (LCLs) led to reduced proliferation, but not direct cytotoxicity. However, similar concentrations of JQ1 in a more physiologically-relevant co-culture setting of EBV-LCLs cultured with autologous peripheral blood lymphocytes (PBMC), resulted in a robust depletion of LCLs, loss of suppressive myeloid populations (TAM/M2 phenotype) and expansion of anti-tumor adaptive, memory CD3/CD8 immune effector cells. Using a single dose of JQ1 (500nM) in a co-culture of LCLs and autologous PBMCs, there was a marked expansion of activated, effector memory (CD3+, CD8+, CD45RO+, CD62L-) cytotoxic T-cell population as compared to vehicle-treated co-cultures. Furthermore, a dramatic reduction in both CD19+ LCLs and suppressive myeloid populations (CD33+, CD11b, HLADR+, CD206+, PD-L1+) was observed after 10 days. Despite the depletion of myeloid cell subsets by CD33 depletion, we still saw the outgrowth of cytotoxic T cell population in the presence of JQ1.In order to delve into the causes of this heightened immune response, we looked at the changes in the expression of highly immunogenic EBV specific proteins. BRD4 inhibition led to upregulation of the EBV oncoprotein, latent membrane protein 1(LMP1), as well as MHC class I antigen presentation machinery in LCLs. The enhanced expression of LMP1 in LCLs with BRD4i, led to modulation of downstream signaling networks driven by this oncogene including PI3K/pAKT (decreased), NF-ĸB (canonical down/non canonical up) and pSTAT3 (decreased). Because LMP1 oncogenic activity is vital for EBV-driven B cell immortalization and transformation, we next conducted in-vitro assays where primary, naïve B cells were infected with EBV in the presence and absence of JQ1 (50, 500nM) treatment. Selective BRD4i was introduced in separate transformation cultures in seven-day intervals following EBV infection and absolute cell counts were monitored. At all timepoints BRD4i prevented EBV-driven transformation of purified naïve B-lymphocytes.Our findings highlight the utility of BRD4i as an experimental therapeutic strategy for EBV-driven lymphomas as it functions to target pathways initiating and sustaining transformed B cell outgrowth and survival while supporting anti-tumor host memory, immune networks. Collectively, these experiments identify BRD4 as a key driver of cell cycle progression, oncogene activation, and a potential immune checkpoint modulator in transformed B lymphoblasts. Furthermore, BRD4i enhances host immunity by eliminating suppressive myeloid cell populations permitting the activation and expansion of memory CD3+/CD8+ cytotoxic T cells. This work demonstrates BRD4i is an attractive therapeutic strategy as it sensitizes malignant cells while enhancing the responsiveness of the host immune system, making it an ideal candidate to be used in future trials with T cell immune therapies. DisclosuresBaiocchi:Essanex: Research Funding.

Phenotypic analysis of lamina propria lymphocytes. Predominance of helper-inducer and cytolytic T-cell phenotypes and deficiency of suppressor-inducer phenotypes in Crohn's disease and control patients.

To better define the nature of intestinal T cells, the phenotypes of isolated lamina propria lymphocytes (LPL) were determined in both Crohn's disease patients and control patients using combinations of monoclonal antibodies that have been found to correlate with particular immunoregulatory functions. Isolated LPL and autologous peripheral blood lymphocytes (PBL) were stained with multiple combinations of monoclonal antibodies and studied by dual immunofluorescence flow cytometry. In LPL, compared with PBL, there was a significant increase in the proportion of T cells having the Leu-3+, Leu-8- and Leu-3+, 2H4- phenotypes (associated with helper-inducer function) and a corresponding decrease in the proportion of T cells having the Leu-3+, Leu-8+ and Leu-3+, 2H4+ phenotypes (associated with suppressor-inducer function). It was also found that in LPL, compared with PBL, the percentage of cells with the Leu-2+, Leu-15+ phenotype (associated with suppressor-effector function) was significantly lower. However, the percentage of T cells with the Leu-2+, 9.3+ phenotype (associated with cytolytic function) was similar in PBL and LPL in control patients. There were no major differences comparing Crohn's disease patients with control patients, except that the proportion of Leu-2+, 9.3+ lymphocytes was higher in PBL in Crohn's disease patients. These results show that the lymphocyte subpopulations in the lamina propria differ from those in peripheral blood in having predominantly the phenotypes of helper-inducer and cytolytic T cells, whereas the phenotypes of suppressor-inducer cells and activated suppressor cells are less frequently observed.

Recombinant MAGE-A3 protein immunotherapy and peripheral blood lymphocyte (PBL) reconstitution induce strong antigen-specific humoral and cellular immune responses in patients undergoing autologous stem cell transplantation (ASCT) for consolidation of multiple myeloma (MM)

BACKGROUND: MAGE-A3 is an immunogenic tumor-associated antigen detected in 1/3 of newly diagnosed MM patients, and confers a poor prognosis, making it a rational target for immunotherapy. We previously reported (Cohen et al, ASH 2013, #154) that pre- and post-ASCT administration of recMAGE-A3 + AS15 adjuvant (containing MPL, QS21, and CpG7909) and infusion of vaccine-primed autologous peripheral blood lymphocytes (PBL) in the early post-ASCT period had an acceptable safety profile and induced robust antibody responses against MAGE-A3. We now report our initial cellular immune response data, and update the humoral response and clinical outcome data. METHODS: The composition of recMAGE-A3 +AS15 and the immunization schedule (Fig. 1) for this pilot study have been described (ASH 2013, #154). Antibody responses were assessed by ELISA. CD4 and CD8 T cell responses were assessed by ELISpot and intracellular cytokine release assays after in vitro re-stimulation with MAGE-A3 overlapping peptide pools or controls and autologous antigen-presenting cells. Clinical responses were determined by IMWG criteria. M3H67 mAb (specific for MAGE-A3 and homologous MAGE-A family members) was used for assessing expression in MM cells by immunohistochemistry (IHC). RESULTS: Thirteen patients enrolled (med. age 56; 45% high-risk cytogenetics; 42% ISS II/III). All had MAGE-A+ myeloma cells and had achieved at least VGPR following induction. Twelve of 12 (100%) subjects tested to date developed high-titer (1:104-106) antibodies against MAGE-A3 that persisted to at least 1 year post-SCT. These titers were 10-100-fold higher than those seen in a prior study in lung cancer patients with recMAGE-A3 + AS02b, an older adjuvant lacking CpG7909 (PNAS 2008; 105:1650). Epitope mapping identified at least 7 distinct MAGE-A3 epitopes clustering in the hydrophobic regions from aa. 1-100 and 220-300. Isotyping and IgG subclass analysis demonstrated IgG class switching in all patients, with IgG1 and IgG3 subclasses most prevalent. Peripheral blood T cell responses have been evaluated in 3 subjects to date. All had MAGE-A3-specific CD4 responses by IFNγ ELISpot starting as early as d+31 after ASCT, with significant expansion after booster vaccinations and persistence through 1 year post-ASCT. Intracellular cytokine staining confirmed a polyfunctional, Th1-biased CD4 T cell response (IFNγ+, TNFα+, IL5-) in all 3 patients. No CD8 responses against MAGE-A3 have been detected to date. Clinical response assessments were as follows: there were 12 VGPR and 1 CR at enrollment, 7 VGPR and 6 CR (3 stringent CR) at 3 months (mos.) post-ASCT, and 3 VGPR and 5 CR (4 sCR) at 1 year post-ASCT, with 4 patients relapsing at or before 1 year, and 1 not yet evaluable. With a median follow-up of 19 mos. (range 6-32), 6 patients have relapsed (estimated median PFS is 24 mos.) and 1 died of progressive MM. There was no difference between progressors and non-progressors with regard to cytogenetics, baseline MAGE-A expression, antibody titers, hematologic response, or use of lenalidomide maintenance (n=4). MAGE-A expression was assessed by IHC in 3 relapse bone marrow biopsies, and all were negative. CONCLUSIONS: RecMAGE-A3 immunotherapy and PBL reconstitution is well-tolerated, feasible, and induces antibody and Th1-biased CD4 T cell responses, but not CD8 responses, in the setting of ASCT for MM. Cellular immune assessments are ongoing. The magnitudes of antibody and CD4 responses appear greater than those seen historically with older formulations of recMAGE-A3 in other cancers, despite significant immune compromise after ASCT, suggesting a benefit from the new AS15 adjuvant formulation, or from immunization and autologous PBL transfer in the peri-ASCT setting, or both. The loss of MAGE-A3 expression in relapsing patients implies antigen-specific immune selective pressure even in the absence of CD8 T cell responses, and also suggests that combination strategies aimed at limiting immune escape (eg multi-antigen vaccines) should be investigated. Clinical outcomes are promising for this high-risk patient population. These results support advanced phase clinical trials to investigate clinical efficacy of recMAGE-A3 vaccine immunotherapy in MM. ![Figure 1][1] Figure 1 Disclosures Cohen: Onyx Pharmaceuticals: Advisory Board, Advisory Board Other; Bristol-Myers Squibb: Advisory Board, Advisory Board Other, Research Funding; Janssen: Advisory Board, Advisory Board Other; Celgene: Member, Independent Response Adjudication Committee Other. Bertolini: Ludwig Institute for Cancer Research: Employment. Pan: Ludwig Institute for Cancer Research: Employment. Venhaus: Ludwig Institute for Cancer Research: Employment. Fellague-Chebra: GlaxoSmithKline: Employment. Gruselle: GlaxoSmithKline: Employment. [1]: pending:yes

Abstract LB-116: Strong MAGE-A3-specific humoral and cellular immune responses in multiple myeloma patients receiving MAGE-A3 protein immunotherapy and peripheral blood lymphocyte reconstitution

Abstract MAGE-A3 is an immunogenic tumor-associated antigen expressed in multiple myeloma (MM) patients and conferring poor prognosis, making it a rational target for immunotherapy. Recombinant MAGE-A3 protein was administered in AS15 immunostimulant (containing MPL, QS-21, and CpG 7909) to 13 MM patients pre- and post-autologous stem cell transplant (ASCT) coupled with infusion of vaccine-primed autologous peripheral blood lymphocytes (PBL) early post-ASCT (NCT01380145). All patients had MAGE-A+ myeloma cells at baseline and had an acceptable safety profile during immunotherapy. Robust antibody responses against MAGE-A3 (assessed by ELISA) were induced in all 13 subjects, with high antibody titers (1:10^4-10^6) that persisted to at least 1-year post-ASCT. Subclass analysis demonstrated a prevalence of IgG1 and IgG3. Epitope mapping identified 7 distinct epitopes clustering in hydrophilic regions of MAGE-A3. Peripheral blood T cell responses were evaluated in 8 subjects by IFNγ-ELISpot after in vitro re-stimulation with MAGE-A3 overlapping peptide pools. All patients quickly developed strong MAGE-A3-specific CD4 responses post-vaccination and ASCT, persisting 1-year post-ASCT. Intracellular cytokine staining confirmed polyfunctional, Th1-biased CD4 T cell responses. One patient developed CD8 responses against MAGE-A3 that recognized naturally processed antigen. To date, 6 patients relapsed and 1 died of progressive MM, with no notable difference in cytogenetics or antibody titers compared to non-progressors. MAGE-A expression was assessed by immunohistochemistry in relapse bone marrow biopsies, and interestingly, 4/6 were negative. MAGE-A3 protein-based immunotherapy and PBL reconstitution is generally well tolerated, feasible, and induces antibody and Th1-biased CD4 T cell responses, but only rare CD8 responses, in the setting of ASCT for MM. Cellular immune assessments are ongoing. The magnitudes of antibody and CD4 responses appear greater than those seen historically with older immunostimulant formulations of MAGE-A3 in other cancers, despite significant immune compromise after ASCT, suggesting a benefit from the new AS15 immunostimulant formulation, or from immunization and autologous PBL transfer in the peri-ASCT setting, or both. The frequent loss of MAGE-A3 expression in relapsing patients implies antigen-specific immune selective pressure, and suggests that combination strategies aimed at limiting immune escape should be investigated. Funding Sources: GlaxoSmithKline Biologicals SA, Ludwig Institute for Cancer Research, Cancer Research Institute. Citation Format: Sacha Gnjatic, Sarah Nataraj, Naoko Imai, Achim A. Jungbluth, Linda Pan, Ralph Venhaus, Rafik Fellague-Chebra, Olivier Gruselle, Adam Cohen, Nikoletta Lendvai, Hearn J. Cho. Strong MAGE-A3-specific humoral and cellular immune responses in multiple myeloma patients receiving MAGE-A3 protein immunotherapy and peripheral blood lymphocyte reconstitution. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-116. doi:10.1158/1538-7445.AM2015-LB-116