Publications by authors named "Begonya Comin-Anduix"

35 Publications

Key Parameters of Tumor Epitope Immunogenicity Revealed Through a Consortium Approach Improve Neoantigen Prediction.

Cell 2020 Oct 9;183(3):818-834.e13. Epub 2020 Oct 9.

Parker Institute for Cancer Immunotherapy, San Francisco, CA, USA. Electronic address:

Many approaches to identify therapeutically relevant neoantigens couple tumor sequencing with bioinformatic algorithms and inferred rules of tumor epitope immunogenicity. However, there are no reference data to compare these approaches, and the parameters governing tumor epitope immunogenicity remain unclear. Here, we assembled a global consortium wherein each participant predicted immunogenic epitopes from shared tumor sequencing data. 608 epitopes were subsequently assessed for T cell binding in patient-matched samples. By integrating peptide features associated with presentation and recognition, we developed a model of tumor epitope immunogenicity that filtered out 98% of non-immunogenic peptides with a precision above 0.70. Pipelines prioritizing model features had superior performance, and pipeline alterations leveraging them improved prediction performance. These findings were validated in an independent cohort of 310 epitopes prioritized from tumor sequencing data and assessed for T cell binding. This data resource enables identification of parameters underlying effective anti-tumor immunity and is available to the research community.
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http://dx.doi.org/10.1016/j.cell.2020.09.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7652061PMC
October 2020

Antiestrogens in combination with immune checkpoint inhibitors in breast cancer immunotherapy.

J Steroid Biochem Mol Biol 2019 10 19;193:105415. Epub 2019 Jun 19.

UCLA David Geffen School of Medicine, Department of Medicine, Division of Hematology-Oncology, Los Angeles CA 90095, USA; UCLA Jonsson Comprehensive Cancer Center, Los Angeles CA 90095, USA. Electronic address:

Breast cancers (BCs) with expression of estrogen receptor-alpha (ERα) occur in more than 70% of newly-diagnosed patients in the U.S. Endocrine therapy with antiestrogens or aromatase inhibitors is an important intervention for BCs that express ERα, and it remains one of the most effective targeted treatment strategies. However, a substantial proportion of patients with localized disease, and essentially all patients with metastatic BC, become resistant to current endocrine therapies. ERα is present in most resistant BCs, and in many of these its activity continues to regulate BC growth. Fulvestrant represents one class of ERα antagonists termed selective ER downregulators (SERDs). Treatment with fulvestrant causes ERα down-regulation, an event that helps overcome several resistance mechanisms. Unfortunately, full antitumor efficacy of fulvestrant is limited by its poor bioavailability in clinic. We have designed and tested a new generation of steroid-like SERDs. Using ERα-positive BC cells in vitro, we find that these compounds suppress ERα protein levels with efficacy similar to fulvestrant. Moreover, these new SERDs markedly inhibit ERα-positive BC cell transcription and proliferation in vitro even in the presence of estradiol-17β. In vivo, the SERD termed JD128 significantly inhibited tumor growth in MCF-7 xenograft models in a dose-dependent manner (P < 0.001). Further, our findings indicate that these SERDs also interact with ER-positive immune cells in the tumor microenvironment such as myeloid-derived suppressor cells (MDSC), tumor infiltrating lymphocytes and other selected immune cell subpopulations. SERD-induced inhibition of MDSCs and concurrent actions on CD8+ and CD4 + T-cells promotes interaction of immune checkpoint inhibitors with BC cells in preclinical models, thereby leading to enhanced tumor killing even among highly aggressive BCs such as triple-negative BC that lack ERα expression. Since monotherapy with immune checkpoint inhibitors has not been effective for most BCs, combination therapies with SERDs that enhance immune recognition may increase immunotherapy responses in BC and improve patient survival. Hence, ERα antagonists that also promote ER downregulation may potentially benefit patients who are unresponsive to current endocrine therapies.
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http://dx.doi.org/10.1016/j.jsbmb.2019.105415DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6903431PMC
October 2019

Antiestrogens in combination with immune checkpoint inhibitors in breast cancer immunotherapy.

J Steroid Biochem Mol Biol 2019 10 19;193:105415. Epub 2019 Jun 19.

UCLA David Geffen School of Medicine, Department of Medicine, Division of Hematology-Oncology, Los Angeles CA 90095, USA; UCLA Jonsson Comprehensive Cancer Center, Los Angeles CA 90095, USA. Electronic address:

Breast cancers (BCs) with expression of estrogen receptor-alpha (ERα) occur in more than 70% of newly-diagnosed patients in the U.S. Endocrine therapy with antiestrogens or aromatase inhibitors is an important intervention for BCs that express ERα, and it remains one of the most effective targeted treatment strategies. However, a substantial proportion of patients with localized disease, and essentially all patients with metastatic BC, become resistant to current endocrine therapies. ERα is present in most resistant BCs, and in many of these its activity continues to regulate BC growth. Fulvestrant represents one class of ERα antagonists termed selective ER downregulators (SERDs). Treatment with fulvestrant causes ERα down-regulation, an event that helps overcome several resistance mechanisms. Unfortunately, full antitumor efficacy of fulvestrant is limited by its poor bioavailability in clinic. We have designed and tested a new generation of steroid-like SERDs. Using ERα-positive BC cells in vitro, we find that these compounds suppress ERα protein levels with efficacy similar to fulvestrant. Moreover, these new SERDs markedly inhibit ERα-positive BC cell transcription and proliferation in vitro even in the presence of estradiol-17β. In vivo, the SERD termed JD128 significantly inhibited tumor growth in MCF-7 xenograft models in a dose-dependent manner (P < 0.001). Further, our findings indicate that these SERDs also interact with ER-positive immune cells in the tumor microenvironment such as myeloid-derived suppressor cells (MDSC), tumor infiltrating lymphocytes and other selected immune cell subpopulations. SERD-induced inhibition of MDSCs and concurrent actions on CD8+ and CD4 + T-cells promotes interaction of immune checkpoint inhibitors with BC cells in preclinical models, thereby leading to enhanced tumor killing even among highly aggressive BCs such as triple-negative BC that lack ERα expression. Since monotherapy with immune checkpoint inhibitors has not been effective for most BCs, combination therapies with SERDs that enhance immune recognition may increase immunotherapy responses in BC and improve patient survival. Hence, ERα antagonists that also promote ER downregulation may potentially benefit patients who are unresponsive to current endocrine therapies.
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http://dx.doi.org/10.1016/j.jsbmb.2019.105415DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6903431PMC
October 2019

A kinetic investigation of interacting, stimulated T cells identifies conditions for rapid functional enhancement, minimal phenotype differentiation, and improved adoptive cell transfer tumor eradication.

PLoS One 2018 23;13(1):e0191634. Epub 2018 Jan 23.

NanoSystems Biology Cancer Center, California Institute of Technology, Pasadena, California, United States of America.

For adoptive cell transfer (ACT) immunotherapy of tumor-reactive T cells, an effective therapeutic outcome depends upon cell dose, cell expansion in vivo through a minimally differentiated phenotype, long term persistence, and strong cytolytic effector function. An incomplete understanding of the biological coupling between T cell expansion, differentiation, and response to stimulation hinders the co-optimization of these factors. We report on a biophysical investigation of how the short-term kinetics of T cell functional activation, through molecular stimulation and cell-cell interactions, competes with phenotype differentiation. T cells receive molecular stimulation for a few minutes to a few hours in bulk culture. Following this priming period, the cells are then analyzed at the transcriptional level, or isolated as single cells, with continuing molecular stimulation, within microchambers for analysis via 11-plex secreted protein assays. We resolve a rapid feedback mechanism, promoted by T cell-T cell contact interactions, which strongly amplifies T cell functional performance while yielding only minimal phenotype differentiation. When tested in mouse models of ACT, optimally primed T cells lead to complete tumor eradication. A similar kinetic process is identified in CD8+ and CD4+ T cells collected from a patient with metastatic melanoma.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0191634PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5779691PMC
February 2018

Single-cell analysis resolves the cell state transition and signaling dynamics associated with melanoma drug-induced resistance.

Proc Natl Acad Sci U S A 2017 12 11;114(52):13679-13684. Epub 2017 Dec 11.

NanoSystems Biology Cancer Center, California Institute of Technology, Pasadena, CA 91125;

Continuous BRAF inhibition of mutant melanomas triggers a series of cell state changes that lead to therapy resistance and escape from immune control before establishing acquired resistance genetically. We used genome-wide transcriptomics and single-cell phenotyping to explore the response kinetics to BRAF inhibition for a panel of patient-derived -mutant melanoma cell lines. A subset of plastic cell lines, which followed a trajectory covering multiple known cell state transitions, provided models for more detailed biophysical investigations. Markov modeling revealed that the cell state transitions were reversible and mediated by both Lamarckian induction and nongenetic Darwinian selection of drug-tolerant states. Single-cell functional proteomics revealed activation of certain signaling networks shortly after BRAF inhibition, and before the appearance of drug-resistant phenotypes. Drug targeting those networks, in combination with BRAF inhibition, halted the adaptive transition and led to prolonged growth inhibition in multiple patient-derived cell lines.
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http://dx.doi.org/10.1073/pnas.1712064115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5748184PMC
December 2017

Interferon Receptor Signaling Pathways Regulating PD-L1 and PD-L2 Expression.

Cell Rep 2017 05;19(6):1189-1201

Division of Hematology-Oncology, Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles (UCLA), Los Angeles, CA 90095, USA; Department of Molecular and Medical Pharmacology, UCLA, Los Angeles, CA 90095, USA; Jonsson Comprehensive Cancer Center, UCLA, Los Angeles, CA 90095, USA; Division of Surgical Oncology, Department of Surgery, UCLA, Los Angeles, CA 90095, USA. Electronic address:

PD-L1 and PD-L2 are ligands for the PD-1 immune inhibiting checkpoint that can be induced in tumors by interferon exposure, leading to immune evasion. This process is important for immunotherapy based on PD-1 blockade. We examined the specific molecules involved in interferon-induced signaling that regulates PD-L1 and PD-L2 expression in melanoma cells. These studies revealed that the interferon-gamma-JAK1/JAK2-STAT1/STAT2/STAT3-IRF1 axis primarily regulates PD-L1 expression, with IRF1 binding to its promoter. PD-L2 responded equally to interferon beta and gamma and is regulated through both IRF1 and STAT3, which bind to the PD-L2 promoter. Analysis of biopsy specimens from patients with melanoma confirmed interferon signature enrichment and upregulation of gene targets for STAT1/STAT2/STAT3 and IRF1 in anti-PD-1-responding tumors. Therefore, these studies map the signaling pathway of interferon-gamma-inducible PD-1 ligand expression.
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http://dx.doi.org/10.1016/j.celrep.2017.04.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6420824PMC
May 2017

Combined treatment with dabrafenib and trametinib with immune-stimulating antibodies for BRAF mutant melanoma.

Oncoimmunology 2016 Jul 17;5(7):e1052212. Epub 2015 Aug 17.

Department of Medicine; Division of Hematology/Oncology; University of California, Los Angeles (UCLA); Los Angeles, CA USA; Department of Molecular and Medical Pharmacology; University of California, Los Angeles (UCLA); Los Angeles, CA USA; The Jonsson Comprehensive Cancer Center (JCCC); University of California, Los Angeles (UCLA); Los Angeles, CA USA; Surgery; Division of Surgical Oncology; University of California, Los Angeles (UCLA); Los Angeles, CA USA.

The combination of targeted therapy with BRAF and MEK inhibitors has become the standard of care in patients with BRAF (V600E) mutant melanoma, but responses are not durable. In addition, the impressive clinical benefits with anti-PD-1 and anti-PD-L1 antibodies (Ab) in patients with heavily pretreated metastatic melanoma and the synergistic effect of dabrafenib, trametinib and anti-PD-1 compared with single therapy alone groups support the idea that combining dabrafenib, trametinib and immunotherapy based on PD-1 blockade could be an interesting approach in the treatment of metastatic melanoma. With our mouse model of syngeneic BRAF (V600E) driven melanoma (SM1), we tested whether the addition of an immunostimulatory Ab targeting CD137 (4-1BB) and/or CD134 (OX40) would enhance the antitumor effect of dabrafenib, trametinib and anti-PD-1 or anti-PD-L1 therapy. In vitro studies showed that the combination group of dabrafenib, trametinib and anti-PD-1 increases CD8(+) tumor infiltrating lymphocytes (TILs), as well as CD4(+) T cells and tumor-associated macrophages (TAMs). An upregulation of PD-L1 was observed in the combination of dabrafenib, trametinib and anti-PD-1 therapy. Combination of dabrafenib, trametinib and anti-PD-1, with either anti-CD137 or anti-CD134, showed a superior antitumor effect, but the five-agent combination was not superior to the four-agent combinations. In conclusion, the combination of dabrafenib, trametinib, anti-PD1 or anti-PD-L1 therapy results in robust antitumor activity, which is further improved by adding the immune-stimulating Ab anti-CD137 or anti-CD134. Our findings support the testing of these combinations in patients with BRAF (V600E) mutant metastatic melanoma.
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http://dx.doi.org/10.1080/2162402X.2015.1052212DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5006894PMC
July 2016

Combination of pan-RAF and MEK inhibitors in NRAS mutant melanoma.

Mol Cancer 2015 Feb 3;14:27. Epub 2015 Feb 3.

Department of Medicine, Division of Hematology-Oncology, University of California Los Angeles (UCLA), Los Angeles, CA, USA.

Background: Approximately 20% of melanomas contain a mutation in NRAS. However no direct inhibitor of NRAS is available. One of the main signaling pathways downstream of NRAS is the MAPK pathway. In this study we investigated the possibility of blocking oncogenic signaling of NRAS by inhibiting two signaling points in the MAPK pathway.

Methods: Fourteen NRAS mutated human melanoma cell lines were treated with a pan-RAF inhibitor (PRi, Amgen Compd A), a MEK inhibitor (MEKi, trametinib) or their combination and the effects on proliferation, cell cycle progression, apoptosis, transcription profile and signaling of the cells were investigated.

Results: The majority of the cell lines showed a significant growth inhibition, with high levels of synergism of the PRi and MEKi combination. Sensitive cell lines showed induction of apoptosis by the combination treatment and there was a correlation between p-MEK levels and synergistic effect of the combination treatment. Proliferation of sensitive cell lines was blocked by the inhibition of the MAPK pathway, which also blocked expression of cyclin D1. However, in resistant cell lines, proliferation was blocked by combined inhibition of the MAPK pathway and cyclin D3, which is not regulated by the MAPK pathway. Resistant cell lines also showed higher levels of p-GSK3β and less perturbation of the apoptotic profile upon the treatment in comparison with the sensitive cell lines.

Conclusions: The combination of PRi + MEKi can be an effective regimen for blocking proliferation of NRAS mutant melanomas when there is higher activity of the MAPK pathway and dependence of proliferation and survival on this pathway.
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http://dx.doi.org/10.1186/s12943-015-0293-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4320814PMC
February 2015

Antitumor activity of the ERK inhibitor SCH772984 [corrected] against BRAF mutant, NRAS mutant and wild-type melanoma.

Mol Cancer 2014 Aug 20;13:194. Epub 2014 Aug 20.

Department of Medicine, Division of Hematology-Oncology, University of California Los Angeles (UCLA), 11-934 Factor Building, Los Angeles, CA, USA.

Background: In melanoma, dysregulation of the MAPK pathway, usually via BRAF(V600) or NRAS(Q61) somatic mutations, leads to constitutive ERK signaling. While BRAF inhibitors are initially effective for BRAF-mutant melanoma, no FDA-approved targeted therapies exist for BRAF-inhibitor-resistant BRAF(V600), NRAS mutant, or wild-type melanoma.

Methods: The 50% inhibitory concentration (IC50) of SCH772984, a novel inhibitor of ERK1/2, was determined in a panel of 50 melanoma cell lines. Effects on MAPK and AKT signaling by western blotting and cell cycle by flow cytometry were determined.

Results: Sensitivity fell into three groups: sensitive, 50% inhibitory concentration (IC50) < 1 μM; intermediately sensitive, IC50 1-2 μM; and resistant, >2 μM. Fifteen of 21 (71%) BRAF mutants, including 4 with innate vemurafenib resistance, were sensitive to SCH772984. All three (100%) BRAF/NRAS double mutants, 11 of 14 (78%) NRAS mutants and 5 of 7 (71%) wild-type melanomas were sensitive. Among BRAF(V600) mutants with in vitro acquired resistance to vemurafenib, those with MAPK pathway reactivation as the mechanism of resistance were sensitive to SCH772984. SCH772984 caused G1 arrest and induced apoptosis.

Conclusions: Combining vemurafenib and SCH722984 in BRAF mutant melanoma was synergistic in a majority of cell lines and significantly delayed the onset of acquired resistance in long term in vitro assays. Therefore, SCH772984 may be clinically applicable as a treatment for non-BRAF mutant melanoma or in BRAF-mutant melanoma with innate or acquired resistance, alone or in combination with BRAF inhibitors.
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http://dx.doi.org/10.1186/1476-4598-13-194DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4155088PMC
August 2014

Distinct immunological mechanisms of CTLA-4 and PD-1 blockade revealed by analyzing TCR usage in blood lymphocytes.

Oncoimmunology 2014;3:e29244. Epub 2014 Jun 25.

Department of Medicine (Division of Hematology-Oncology); University of California Los Angeles (UCLA); Los Angeles, CA USA ; Department of Surgery (Division of Surgical-Oncology); University of California Los Angeles (UCLA); Los Angeles, CA USA ; Jonsson Comprehensive Cancer Center (JCCC); University of California Los Angeles (UCLA); Los Angeles, CA USA.

Targeting immune inhibitory receptors has brought excitement, innovation and hope to cancer patients. Our recent work revealed the immunological effects of blocking the CTLA4 and PD-1 immune checkpoints on T cell receptor usage among peripheral blood cells, and further uncovers how the expansion of the T cell repertoire matches the immunotoxicity profile of the therapy.
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http://dx.doi.org/10.4161/onci.29244DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4108466PMC
February 2021

Effects of MAPK and PI3K pathways on PD-L1 expression in melanoma.

Clin Cancer Res 2014 Jul 8;20(13):3446-57. Epub 2014 May 8.

Authors' Affiliations: Division of Hematology-Oncology, Department of Medicine; Division of Surgical-Oncology, Department of Surgery; Department of Molecular and Medical Pharmacology; and Jonsson Comprehensive Cancer Center, University of California Los Angeles, Los Angeles, California

Purpose: PD-L1 is the main ligand for the immune inhibitory receptor PD-1. This ligand is frequently expressed by melanoma cells. In this study, we investigated whether PD-L1 expression is controlled by melanoma driver mutations and modified by oncogenic signaling inhibition.

Experimental Design: Expression of PD-L1 was investigated in a panel of 51 melanoma cell lines containing different oncogenic mutations, including cell lines with innate and acquired resistance to BRAF inhibitors (BRAFi). The effects of targeted therapy drugs on expression of PD-L1 by melanoma cells were investigated.

Results: No association was found between the level of PD-L1 expression and mutations in BRAF, NRAS, PTEN, or amplification of AKT. Resistance to vemurafenib due to the activation of alternative signaling pathways was accompanied with the induction of PD-L1 expression, whereas the resistance due to the reactivation of the MAPK pathway had no effect on PD-L1 expression. In melanoma cell lines, the effects of BRAF, MEK, and PI3K inhibitors on expression of PD-L1 were variable from reduction to induction, particularly in the presence of INFγ. In PD-L1-exposed lymphocytes, vemurafenib paradoxically restored activity of the MAPK pathway and increased the secretion of cytokines.

Conclusions: In melanoma cell lines, including BRAFi-resistant cells, PD-L1 expression is variably regulated by oncogenic signaling pathways. PD-L1-exposed lymphocytes decrease MAPK signaling, which is corrected by exposure to vemurafenib, providing potential benefits of combining this drug with immunotherapies.
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http://dx.doi.org/10.1158/1078-0432.CCR-13-2797DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4079734PMC
July 2014

Effects of AKT inhibitor therapy in response and resistance to BRAF inhibition in melanoma.

Mol Cancer 2014 Apr 16;13:83. Epub 2014 Apr 16.

Department of Medicine, Division of Hematology-Oncology, David Geffen School of Medicine, University of California Los Angeles (UCLA), Los Angeles, CA, USA.

Background: The clinical use of BRAF inhibitors for treatment of metastatic melanoma is limited by the development of drug resistance. In this study we investigated whether co-targeting the MAPK and the PI3K-AKT pathway can prevent emergence of resistance or provide additional growth inhibitory effects in vitro.

Methods: Anti-tumor effects of the combination of the BRAF inhibitor (BRAFi) dabrafenib and GSK2141795B (AKTi) in a panel of 23 BRAF mutated melanoma cell lines were evaluated on growth inhibition by an ATP-based luminescent assay, on cell cycle and apoptosis by flow cytometry and on cell signaling by western blot. Moreover, we investigated the possibilities of delaying or reversing resistance or achieving further growth inhibition by combining AKTi with dabrafenib and/or the MEK inhibitor (MEKi) trametinib by using long term cultures.

Results: More than 40% of the cell lines, including PTEN-/- and AKT mutants showed sensitivity to AKTi (IC50 < 1.5 μM). The combination of dabrafenib and AKTi synergistically potentiated growth inhibition in the majority of cell lines with IC50 > 5 nM dabrafenib. Combinatorial treatment induced apoptosis only in cell lines sensitive to AKTi. In long term cultures of a PTEN-/- cell line, combinatorial treatment with the MAPK inhibitors, dabrafenib and trametinib, and AKTi markedly delayed the emergence of drug resistance. Moreover, combining AKTi with the MAPK inhibitors from the beginning provided superior growth inhibitory effects compared to addition of AKTi upon development of resistance to MAPK inhibitors in this particular cell line.

Conclusions: AKTi combined with BRAFi-based therapy may benefit patients with tumors harboring BRAF mutations and particularly PTEN deletions or AKT mutations.
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http://dx.doi.org/10.1186/1476-4598-13-83DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4021505PMC
April 2014

COX-2 inhibition prevents the appearance of cutaneous squamous cell carcinomas accelerated by BRAF inhibitors.

Mol Oncol 2014 Mar 1;8(2):250-60. Epub 2013 Dec 1.

Department of Medicine (Division of Hematology-Oncology), David Geffen School of Medicine, University of California Los Angeles (UCLA), Los Angeles, CA, USA; Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California Los Angeles (UCLA), Los Angeles, CA, USA; Department of Surgery (Division of Surgical-Oncology), David Geffen School of Medicine, University of California Los Angeles (UCLA), Los Angeles, CA, USA; Jonsson Comprehensive Cancer Center, Los Angeles, CA, USA. Electronic address:

Keratoacanthomas (KAs) and cutaneous squamous cell carcinomas (cuSCCs) develop in 15-30% of patients with BRAF(V600E) metastatic melanoma treated with BRAF inhibitors (BRAFi). These lesions resemble mouse skin tumors induced by the two-stage DMBA/TPA skin carcinogenesis protocol; in this protocol BRAFi accelerates tumor induction. Since prior studies demonstrated cyclooxygenase 2 (COX-2) is necessary for DMBA/TPA tumor induction, we hypothesized that COX-2 inhibition might prevent BRAFi-accelerated skin tumors. Celecoxib, a COX-2 inhibitor, significantly delayed tumor acceleration by the BRAFi inhibitor PLX7420 and decreased tumor number by 90%. Tumor gene expression profiling demonstrated that celecoxib partially reversed the PLX4720-induced gene signature. In PDV cuSCC cells, vemurafenib (a clinically approved BRAFi) increased ERK phosphorylation and soft agar colony formation; both responses were greatly decreased by celecoxib. In clinical trials trametinib, a MEK inhibitor (MEKi) increases BRAFi therapy efficacy in BRAF(V600E) melanomas and reduces BRAFi-induced KA and cuSCC frequency. Trametinib also reduced vemurafenib-induced PDV soft agar colonies, but less efficiently than celecoxib. The trametinb/celecoxib combination was more effective than either inhibitor alone. In conclusion, celecoxib suppressed both BRAFi-accelerated skin tumors and soft-agar colonies, warranting its testing as a chemopreventive agent for non-melanoma skin lesions in patients treated with BRAFi alone or in combination with MEKi.
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http://dx.doi.org/10.1016/j.molonc.2013.11.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3943738PMC
March 2014

Multifunctional T-cell analyses to study response and progression in adoptive cell transfer immunotherapy.

Cancer Discov 2013 Apr 21;3(4):418-29. Epub 2013 Mar 21.

NanoSystems Biology Cancer Center, Division of Physics, California Institute of Technology, Pasadena, CA 91125, USA.

Unlabelled: Adoptive cell transfer (ACT) of genetically engineered T cells expressing cancer-specific T-cell receptors (TCR) is a promising cancer treatment. Here, we investigate the in vivo functional activity and dynamics of the transferred cells by analyzing samples from 3 representative patients with melanoma enrolled in a clinical trial of ACT with TCR transgenic T cells targeted against the melanosomal antigen MART-1. The analyses included evaluating 19 secreted proteins from individual cells from phenotypically defined T-cell subpopulations, as well as the enumeration of T cells with TCR antigen specificity for 36 melanoma antigens. These analyses revealed the coordinated functional dynamics of the adoptively transferred, as well as endogenous, T cells, and the importance of highly functional T cells in dominating the antitumor immune response. This study highlights the need to develop approaches to maintaining antitumor T-cell functionality with the aim of increasing the long-term efficacy of TCR-engineered ACT immunotherapy.

Significance: A longitudinal functional study of adoptively transferred TCR–engineered lymphocytes yielded revealing snapshots for understanding the changes of antitumor responses over time in ACT immunotherapy of patients with advanced melanoma.
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http://dx.doi.org/10.1158/2159-8290.CD-12-0383DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3716460PMC
April 2013

BRAF inhibitor vemurafenib improves the antitumor activity of adoptive cell immunotherapy.

Cancer Res 2012 Aug 12;72(16):3928-37. Epub 2012 Jun 12.

Department of Surgery, Division of Surgical Oncology, Crump Institute for Molecular Imaging, UCLA Biomedical Physics Interdepartmental Graduate Program, Los Angeles, University of California Los Angeles, Los Angeles, California 90095-1782, USA.

Combining immunotherapy with targeted therapy blocking oncogenic BRAFV600 may result in improved treatments for advanced melanoma. In this study, we developed a BRAFV600E-driven murine model of melanoma, SM1, which is syngeneic to fully immunocompetent mice. SM1 cells exposed to the BRAF inhibitor vemurafenib (PLX4032) showed partial in vitro and in vivo sensitivity resulting from the inhibition of MAPK pathway signaling. Combined treatment of vemurafenib plus adoptive cell transfer therapy with lymphocytes genetically modified with a T-cell receptor (TCR) recognizing chicken ovalbumin (OVA) expressed by SM1-OVA tumors or pmel-1 TCR transgenic lymphocytes recognizing gp100 endogenously expressed by SM1 resulted in superior antitumor responses compared with either therapy alone. T-cell analysis showed that vemurafenib did not significantly alter the expansion, distribution, or tumor accumulation of the adoptively transferred cells. However, vemurafenib paradoxically increased mitogen-activated protein kinase (MAPK) signaling, in vivo cytotoxic activity, and intratumoral cytokine secretion by adoptively transferred cells. Taken together, our findings, derived from 2 independent models combining BRAF-targeted therapy with immunotherapy, support the testing of this therapeutic combination in patients with BRAFV600 mutant metastatic melanoma.
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http://dx.doi.org/10.1158/0008-5472.CAN-11-2837DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422880PMC
August 2012

Reversing melanoma cross-resistance to BRAF and MEK inhibitors by co-targeting the AKT/mTOR pathway.

PLoS One 2011 14;6(12):e28973. Epub 2011 Dec 14.

Department of Medicine, Division of Hematology/Oncology, University of California Los Angeles, Los Angeles, California, United States of America.

Background: The sustained clinical activity of the BRAF inhibitor vemurafenib (PLX4032/RG7204) in patients with BRAF(V600) mutant melanoma is limited primarily by the development of acquired resistance leading to tumor progression. Clinical trials are in progress using MEK inhibitors following disease progression in patients receiving BRAF inhibitors. However, the PI3K/AKT pathway can also induce resistance to the inhibitors of MAPK pathway.

Methodology/principal Findings: The sensitivity to vemurafenib or the MEK inhibitor AZD6244 was tested in sensitive and resistant human melanoma cell lines exploring differences in activation-associated phosphorylation levels of major signaling molecules, leading to the testing of co-inhibition of the AKT/mTOR pathway genetically and pharmacologically. There was a high degree of cross-resistance to vemurafenib and AZD6244, except in two vemurafenib-resistant cell lines that acquired a secondary mutation in NRAS. In other cell lines, acquired resistance to both drugs was associated with persistence or increase in activity of AKT pathway. siRNA-mediated gene silencing and combination therapy with an AKT inhibitor or rapamycin partially or completely reversed the resistance.

Conclusions/significance: Primary and acquired resistance to vemurafenib in these in vitro models results in frequent cross resistance to MEK inhibitors, except when the resistance is the result of a secondary NRAS mutation. Resistance to BRAF or MEK inhibitors is associated with the induction or persistence of activity within the AKT pathway in the presence of these drugs. This resistance can be potentially reversed by the combination of a RAF or MEK inhibitor with an AKT or mTOR inhibitor. These combinations should be available for clinical testing in patients progressing on BRAF inhibitors.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0028973PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3237573PMC
August 2012

Immunomodulation by imiquimod in patients with high-risk primary melanoma.

J Invest Dermatol 2012 Jan 18;132(1):163-9. Epub 2011 Aug 18.

Division of Dermatology, Los Angeles Biomedical Research Institute, Harbor-UCLA Medical Center, Torrance, California 90502, USA.

Imiquimod is a synthetic Toll-like receptor 7 (TLR7) agonist approved for the topical treatment of actinic keratoses, superficial basal cell carcinoma, and genital warts. Imiquimod leads to an 80-100% cure rate of lentigo maligna; however, studies of invasive melanoma are lacking. We conducted a pilot study to characterize the local, regional, and systemic immune responses induced by imiquimod in patients with high-risk melanoma. After treatment of the primary melanoma biopsy site with placebo or imiquimod cream, we measured immune responses in the treated skin, sentinel lymph nodes (SLNs), and peripheral blood. Treatment of primary melanomas with 5% imiquimod cream was associated with an increase in both CD4+ and CD8+ T cells in the skin, and CD4+ T cells in the SLN. Most of the CD8+ T cells in the skin were CD25 negative. We could not detect any increases in CD8+ T cells specifically recognizing HLA-A(*)0201-restricted melanoma epitopes in the peripheral blood. The findings from this small pilot study demonstrate that topical imiquimod treatment results in enhanced local and regional T-cell numbers in both the skin and SLN. Further research into TLR7 immunomodulating pathways as a basis for effective immunotherapy against melanoma in conjunction with surgery is warranted.
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http://dx.doi.org/10.1038/jid.2011.247DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3229834PMC
January 2012

Combination therapy with vemurafenib (PLX4032/RG7204) and metformin in melanoma cell lines with distinct driver mutations.

J Transl Med 2011 May 24;9:76. Epub 2011 May 24.

Department of Medicine, Division of Hematology/Oncology, University of California Los Angeles (UCLA), USA.

Background: A molecular linkage between the MAPK and the LKB1-AMPK energy sensor pathways suggests that combined MAPK oncogene inhibition and metabolic modulation of AMPK would be more effective than either manipulation alone in melanoma cell lines.

Materials And Methods: The combination of the BRAF inhibitor vemurafenib (formerly PLX4032) and metformin were tested against a panel of human melanoma cell lines with defined BRAF and NRAS mutations for effects on viability, cell cycle and apoptosis. Signaling molecules in the MAPK, PI3K-AKT and LKB1-AMPK pathways were studied by Western blot.

Results: Single agent metformin inhibited proliferation in 12 out of 19 cell lines irrespective of the BRAF mutation status, but in one NRASQ61K mutant cell line it powerfully stimulated cell growth. Synergistic anti-proliferative effects of the combination of metformin with vemurafenib were observed in 6 out of 11 BRAFV600E mutants, including highly synergistic effects in two BRAFV600E mutant melanoma cell lines. Antagonistic effects were noted in some cell lines, in particular in BRAFV600E mutant cell lines resistant to single agent vemurafenib. Seven out of 8 BRAF wild type cell lines showed marginally synergistic anti-proliferative effects with the combination, and one cell line had highly antagonistic effects with the combination. The differential effects were not dependent on the sensitivity to each drug alone, effects on cell cycle or signaling pathways.

Conclusions: The combination of vemurafenib and metformin tended to have stronger anti-proliferative effects on BRAFV600E mutant cell lines. However, determinants of vemurafenib and metformin synergism or antagonism need to be understood with greater detail before any potential clinical utility of this combination.
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http://dx.doi.org/10.1186/1479-5876-9-76DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3152784PMC
May 2011

A clinical microchip for evaluation of single immune cells reveals high functional heterogeneity in phenotypically similar T cells.

Nat Med 2011 Jun 22;17(6):738-43. Epub 2011 May 22.

NanoSystems Biology Cancer Center, California Institute of Technology, Pasadena, California, USA.

Cellular immunity has an inherent high level of functional heterogeneity. Capturing the full spectrum of these functions requires analysis of large numbers of effector molecules from single cells. We report a microfluidic platform designed for highly multiplexed (more than ten proteins), reliable, sample-efficient (∼1 × 10(4) cells) and quantitative measurements of secreted proteins from single cells. We validated the platform by assessment of multiple inflammatory cytokines from lipopolysaccharide (LPS)-stimulated human macrophages and comparison to standard immunotechnologies. We applied the platform toward the ex vivo quantification of T cell polyfunctional diversity via the simultaneous measurement of a dozen effector molecules secreted from tumor antigen-specific cytotoxic T lymphocytes (CTLs) that were actively responding to tumor and compared against a cohort of healthy donor controls. We observed profound, yet focused, functional heterogeneity in active tumor antigen-specific CTLs, with the major functional phenotypes quantitatively identified. The platform represents a new and informative tool for immune monitoring and clinical assessment.
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http://dx.doi.org/10.1038/nm.2375DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3681612PMC
June 2011

A beta-camera integrated with a microfluidic chip for radioassays based on real-time imaging of glycolysis in small cell populations.

J Nucl Med 2011 May;52(5):815-21

Crump Institute for Molecular Imaging, University of California, Los Angeles, California 90095-1770, USA.

Unlabelled: An integrated β-camera and microfluidic chip was developed that is capable of quantitative imaging of glycolysis radioassays using (18)F-FDG in small cell populations down to a single cell. This paper demonstrates that the integrated system enables digital control and quantitative measurements of glycolysis in B-Raf(V600E)-mutated melanoma cell lines in response to specific B-Raf inhibition.

Methods: The β-camera uses a position-sensitive avalanche photodiode to detect charged particle-emitting probes within a microfluidic chip. The integrated β-camera and microfluidic chip system was calibrated, and the linearity was measured using 4 different melanoma cell lines (M257, M202, M233, and M229). Microfluidic radioassays were performed with cell populations ranging from hundreds of cells down to a single cell. The M229 cell line has a homozygous B-Raf(V600E) mutation and is highly sensitive to a B-Raf inhibitor, PLX4032. A microfluidic radioassay was performed over the course of 3 days to assess the cytotoxicity of PLX4032 on cellular (18)F-FDG uptake.

Results: The β-camera is capable of imaging radioactive uptake of (18)F-FDG in microfluidic chips. (18)F-FDG uptake for a single cell was measured using a radioactivity concentration of 37 MBq/mL during the radiotracer incubation period. For in vitro cytotoxicity monitoring, the β-camera showed that exposure to 1 μM PLX4032 for 3 days decreased the (18)F-FDG uptake per cell in highly sensitive M229 cells, compared with vehicle controls.

Conclusion: The integrated β-camera and microfluidic chip can provide digital control of live cell cultures and allow in vitro quantitative radioassays for multiple samples simultaneously.
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http://dx.doi.org/10.2967/jnumed.110.078725DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3270819PMC
May 2011

Intra-lymph node prime-boost vaccination against Melan A and tyrosinase for the treatment of metastatic melanoma: results of a phase 1 clinical trial.

Clin Cancer Res 2011 May 8;17(9):2987-96. Epub 2011 Mar 8.

Jonsson Comprehensive Cancer Center, University of California Los Angeles, Los Angeles, CA 90095, USA.

Purpose: The goal of this study was to test the safety and activity of a therapeutic vaccine, MKC1106-MT, in patients with metastatic melanoma.

Experimental Design: MKC1106-MT comprises a plasmid (pMEL-TYR) and two peptides (E-MEL and E-TYR), corresponding to Melan A and tyrosinase, administered by intra-lymph node injection in a prime-boost sequence. All 18 patients were HLA-A*0201 positive and received a fixed priming dose of plasmid and a low or a high peptide dose. Enumeration of antigen-specific T cells was done prior to and throughout the treatment. Patients who did not exhibit disease progression remained on study and could receive up to eight cycles of treatment.

Results: The MKC1106-MT regimen was well tolerated and resulted in an overall immune response rate of 50%. The treatment showed disease control, defined as stable disease that lasted for 8 weeks or more in 6 of 18 (33%) of the patients: 14% and 46% in the low and high peptide dose, respectively. Interestingly, four patients, all with tumor burden largely confined to lymph nodes and Melan A-specific T cells at baseline, showed durable disease control associated with radiologic evidence of tumor regression. There was no noticeable correlation between the expansion of antigen-specific T cells in blood and the clinical outcome; yet, there was evidence of active tumor-infiltrating lymphocytes (TIL) in two regressing lesions.

Conclusions: MKC1106-MT showed immunogenicity and evidence of disease control in a defined patient population. These findings support further development of this investigational agent and the concept of therapeutic vaccination in metastatic melanoma.
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http://dx.doi.org/10.1158/1078-0432.CCR-10-3272DOI Listing
May 2011

MHC-I-restricted melanoma antigen specific TCR-engineered human CD4+ T cells exhibit multifunctional effector and helper responses, in vitro.

Clin Immunol 2010 Sep 23;136(3):338-47. Epub 2010 May 23.

Department of Medicine, University of Connecticut Health Center, Farmington, CT 06030, USA.

MHC class I-restricted human melanoma epitope MART-1(27-35) specific TCR-engineered CD4+CD25- T cells synthesize Th1 type cytokines and exhibit cytolytic effector function upon cognate stimulation. A detailed characterization of such TCR-engineered CD4+CD25- T cells now reveals that they are multifunctional. For example, they undergo multiple rounds of division, synthesize cytokines (IFN-gamma, TNF-alpha, IL-2, and MIP1ss), lyse target cells, and "help" the expansion of the MART-1(27-35) specific CD8+ T cells when stimulated by the MART-1(27-35) peptide pulsed DC. Multiparametric analyses reveal that a single TCR-engineered CD4+ T cell can perform as many as five different functions. Nearly 100% MART-1(27-35) specific TCR expressing CD4+ T cells can be generated through retroviral vector-based transduction and one round of in vitro stimulation by the peptide pulsed DC. MHC class I-restricted tumor epitope specific TCR transduced CD4+ T cells, therefore, could be useful in immunotherapeutic strategies for melanoma or other human malignancies.
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http://dx.doi.org/10.1016/j.clim.2010.04.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2917536PMC
September 2010

Modular nucleic acid assembled p/MHC microarrays for multiplexed sorting of antigen-specific T cells.

J Am Chem Soc 2009 Jul;131(28):9695-703

NanoSystems Biology Cancer Center, California Institute of Technology, Pasadena, California 91125, USA.

The human immune system consists of a large number of T cells capable of recognizing and responding to antigens derived from various sources. The development of peptide-major histocompatibility (p/MHC) tetrameric complexes has enabled the direct detection of these antigen-specific T cells. With the goal of increasing throughput and multiplexing of T cell detection, protein microarrays spotted with defined p/MHC complexes have been reported, but studies have been limited due to the inherent instability and reproducibility of arrays produced via conventional spotted methods. Herein, we report on a platform for the detection of antigen-specific T cells on glass substrates that offers significant advantages over existing surface-bound schemes. In this approach, called "Nucleic Acid Cell Sorting (NACS)", single-stranded DNA oligomers conjugated site-specifically to p/MHC tetramers are employed to immobilize p/MHC tetramers via hybridization to a complementary-printed substrate. Fully assembled p/MHC arrays are used to detect and enumerate T cells captured from cellular suspensions, including primary human T cells collected from cancer patients. NACS arrays outperform conventional spotted arrays assessed in key criteria such as repeatability and homogeneity. The versatility of employing DNA sequences for cell sorting is exploited to enable the programmed, selective release of target populations of immobilized T cells with restriction endonucleases for downstream analysis. Because of the performance, facile and modular assembly of p/MHC tetramer arrays, NACS holds promise as a versatile platform for multiplexed T cell detection.
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http://dx.doi.org/10.1021/ja9006707DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2720314PMC
July 2009

CTLA4 blockade increases Th17 cells in patients with metastatic melanoma.

J Transl Med 2009 May 20;7:35. Epub 2009 May 20.

Department of Surgery, Division of Surgical Oncology, University of California, Los Angeles, Los Angeles, California, USA.

Background: Th17 cells are CD4+ cells that produce interleukin 17 (IL-17) and are potent inducers of tissue inflammation and autoimmunity. We studied the levels of this T cell subset in peripheral blood of patients treated with the anti-CTLA4 antibody tremelimumab since its major dose limiting toxicities are inflammatory and autoimmune in nature.

Methods: Peripheral blood mononuclear cells (PBMC) were collected before and after receiving tremelimumab within two clinical trials, one with tremelimumab alone (21 patients) and another together with autologous dendritic cells (DC) pulsed with the melanoma epitope MART-126-35 (6 patients). Cytokines were quantified directly in plasma from patients and after in vitro stimulation of PBMC. We also quantified IL-17 cytokine-producing cells by intracellular cytokine staining (ICS).

Results: There were no significant changes in 13 assayed cytokines, including IL-17, when analyzing plasma samples obtained from patients before and after administration of tremelimumab. However, when PBMC were activated in vitro, IL-17 cytokine in cell culture supernatant and Th17 cells, detected as IL-17-producing CD4 cells by ICS, significantly increased in post-dosing samples. There were no differences in the levels of Th17 cells between patients with or without an objective tumor response, but samples from patients with inflammatory and autoimmune toxicities during the first cycle of therapy had a significant increase in Th17 cells.

Conclusion: The anti-CTLA4 blocking antibody tremelimumab increases Th17 cells in peripheral blood of patients with metastatic melanoma. The relation between increases in Th17 cells and severe autoimmune toxicity after CTLA4 blockade may provide insights into the pathogenesis of anti-CTLA4-induced toxicities.
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http://dx.doi.org/10.1186/1479-5876-7-35DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2697137PMC
May 2009

HMGB1 mediates endogenous TLR2 activation and brain tumor regression.

PLoS Med 2009 Jan;6(1):e10

Department of Medicine, Board of Governors' Gene Therapeutics Research Institute, Cedars Sinai Medical Center, Los Angeles, California, United States of America

Background: Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor that carries a 5-y survival rate of 5%. Attempts at eliciting a clinically relevant anti-GBM immune response in brain tumor patients have met with limited success, which is due to brain immune privilege, tumor immune evasion, and a paucity of dendritic cells (DCs) within the central nervous system. Herein we uncovered a novel pathway for the activation of an effective anti-GBM immune response mediated by high-mobility-group box 1 (HMGB1), an alarmin protein released from dying tumor cells, which acts as an endogenous ligand for Toll-like receptor 2 (TLR2) signaling on bone marrow-derived GBM-infiltrating DCs.

Methods And Findings: Using a combined immunotherapy/conditional cytotoxic approach that utilizes adenoviral vectors (Ad) expressing Fms-like tyrosine kinase 3 ligand (Flt3L) and thymidine kinase (TK) delivered into the tumor mass, we demonstrated that CD4(+) and CD8(+) T cells were required for tumor regression and immunological memory. Increased numbers of bone marrow-derived, tumor-infiltrating myeloid DCs (mDCs) were observed in response to the therapy. Infiltration of mDCs into the GBM, clonal expansion of antitumor T cells, and induction of an effective anti-GBM immune response were TLR2 dependent. We then proceeded to identify the endogenous ligand responsible for TLR2 signaling on tumor-infiltrating mDCs. We demonstrated that HMGB1 was released from dying tumor cells, in response to Ad-TK (+ gancyclovir [GCV]) treatment. Increased levels of HMGB1 were also detected in the serum of tumor-bearing Ad-Flt3L/Ad-TK (+GCV)-treated mice. Specific activation of TLR2 signaling was induced by supernatants from Ad-TK (+GCV)-treated GBM cells; this activation was blocked by glycyrrhizin (a specific HMGB1 inhibitor) or with antibodies to HMGB1. HMGB1 was also released from melanoma, small cell lung carcinoma, and glioma cells treated with radiation or temozolomide. Administration of either glycyrrhizin or anti-HMGB1 immunoglobulins to tumor-bearing Ad-Flt3L and Ad-TK treated mice, abolished therapeutic efficacy, highlighting the critical role played by HMGB1-mediated TLR2 signaling to elicit tumor regression. Therapeutic efficacy of Ad-Flt3L and Ad-TK (+GCV) treatment was demonstrated in a second glioma model and in an intracranial melanoma model with concomitant increases in the levels of circulating HMGB1.

Conclusions: Our data provide evidence for the molecular and cellular mechanisms that support the rationale for the clinical implementation of antibrain cancer immunotherapies in combination with tumor killing approaches in order to elicit effective antitumor immune responses, and thus, will impact clinical neuro-oncology practice.
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http://dx.doi.org/10.1371/journal.pmed.1000010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2621261PMC
January 2009

Immunosensitization with a Bcl-2 small molecule inhibitor.

Cancer Immunol Immunother 2009 May 20;58(5):699-708. Epub 2008 Sep 20.

Department of Surgery, University of California at Los Angeles, UCLA Medical Center, 10833 Le Conte Avenue, Los Angeles, CA 90095-1782, USA.

Several tumor immunotherapy approaches result in a low percentage of durable responses in selected cancers. We hypothesized that the insensitivity of cancer cells to immunotherapy may be related to an anti-apoptotic cancer cell milieu, which could be pharmacologically reverted through the inhibition of antiapoptotic Bcl-2 family proteins in cancer cells. ABT-737, a small molecule inhibitor of the antiapoptotic proteins Bcl-2, Bcl-w and Bcl-x(L), was tested for the ability to increase antitumor immune responses in two tumor immunotherapy animal models. The addition of systemic therapy with ABT-737 to the immunization of BALB/c mice with tumor antigen peptide-pulsed dendritic cells (DC) resulted in a significant delay in CT26 murine colon carcinoma tumor growth and improvement in survival. However, the addition of ABT-737 to either a vaccine strategy involving priming with TRP-2 melanoma antigen peptide-pulsed DC and boosting with recombinant Listeria monocytogenes expressing the same melanoma antigen, or the adoptive transfer of TCR transgenic cells, did not result in superior antitumor activity against B16 murine melanoma. In vitro studies failed to demonstrate increased cytotoxic lytic activity when testing the combination of ABT-737 with lymphokine activated killer (LAK) cells, or the death receptor agonists Fas, TRAIL-ligand or TNF-alpha against the CT26 and B16 cell lines. In conclusion, the Bcl-2 inhibitor ABT-737 sensitized cancer cells to the antitumor effect of antigen-specific immunotherapy in a vaccine model for the CT26 colon carcinoma in vivo but not in two immunotherapy strategies against B16 melanoma.
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http://dx.doi.org/10.1007/s00262-008-0592-4DOI Listing
May 2009

T-cell responses to survivin in cancer patients undergoing radiation therapy.

Clin Cancer Res 2008 Aug;14(15):4883-90

Department of Radiation Oncology, University of California at Los Angeles, Los Angeles, California 90095-1714, USA.

Purpose: The goal of this study was to determine if radiation therapy (RT) of human cancer enhances or diminishes tumor-specific T-cell reactivity. This is important if immunotherapy is to be harnessed to improve the outcome of cancer radiotherapy.

Experimental Design: Lymphocytes were isolated from colorectal cancer (CRC) patients before, during, and after presurgical chemoradiotherapy. Similar samples were taken from prostate cancer patients receiving standard RT. The level of CD8(+) T cells capable of binding tetramers for the tumor-associated antigen survivin, which is overexpressed in both cancer types, was enumerated in HLA-A*0201 patient samples. CD4(+), CD25(high), Foxp3(+) cells were also enumerated to evaluate therapy-induced changes in T(regulatory) cells. For CRC patients, most of whom were enrolled in a clinical trial, pathologic response data were available, as well as biopsy and resection specimens, which were stained for cytoplasmic and intranuclear survivin.

Results: Survivin-specific CD8(+) T lymphocytes were detected in the peripheral blood of CRC and prostate cancer patients and increased after therapy in some, but not all, patients. Increases were more common in CRC patients whose tumor was downstaged after chemoradiotherapy. Biopsy specimens from this cohort generally had higher nuclear to cytoplasmic survivin expression. T(regulatory) cells generally increased in the circulation following therapy but only in CRC patients.

Conclusion: This study indicates that RT may increase the likelihood of some cancer patients responding to immunotherapy and lays a basis for future investigations aimed at combining radiation and immunotherapy.
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http://dx.doi.org/10.1158/1078-0432.CCR-07-4462DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2748652PMC
August 2008

Adenovirus MART-1-engineered autologous dendritic cell vaccine for metastatic melanoma.

J Immunother 2008 Apr;31(3):294-309

Department of Medicine, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213, USA.

We performed a phase 1/2 trial testing the safety, toxicity, and immune response of a vaccine consisting of autologous dendritic cells (DCs) transduced with a replication-defective adenovirus (AdV) encoding the full-length melanoma antigen MART-1/Melan-A (MART-1). This vaccine was designed to activate MART-1-specific CD+8 and CD4+ T cells. Metastatic melanoma patients received 3 injections of 10(6) or 10(7) DCs, delivered intradermally. Cell surface phenotype and cytokine production of the DCs used for the vaccines were tested, and indicated intermediate maturity. CD8+ T-cell responses to MART-1 27-35 were assessed by both major histocompatibility complex class I tetramer and interferon (IFN)-gamma enzyme-linked immunosorbent spot (ELISPOT) before, during, and after each vaccine and CD4+ T-cell responses to MART-1 51-73 were followed by IFN-gamma ELISPOT. We also measured antigen response breadth. Determinant spreading from the immunizing antigen MART-1 to other melanoma antigens [gp100, tyrosinase, human melanoma antigen-A3 (MAGE-A3)] was assessed by IFN-gamma ELISPOT. Twenty-three patients were enrolled and 14 patients received all 3 scheduled DC vaccines. Significant CD8+ and/or CD4+ MART-1-specific T-cell responses were observed in 6/11 and 2/4 patients evaluated, respectively, indicating that the E1-deleted adenovirus encoding the cDNA for MART-1/Melan-A (AdVMART1)/DC vaccine activated both helper and killer T cells in vivo. Responses in CD8+ and CD4+ T cells to additional antigens were noted in 2 patients. The AdVMART1-transduced DC vaccine was safe and immunogenic in patients with metastatic melanoma.
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http://dx.doi.org/10.1097/CJI.0b013e31816a8910DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3651040PMC
April 2008