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1
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Greiner D, Xue Q, Waddell TQ, Kurudza E, Chaudhary P, Belote RL, Dotti G, Judson-Torres RL, Reeves MQ, Cheshier SH, Roh-Johnson M. Human CSPG4-targeting CAR-macrophages inhibit melanoma growth. Oncogene 2025:10.1038/s41388-025-03332-0. [PMID: 40082557 DOI: 10.1038/s41388-025-03332-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/28/2024] [Revised: 01/12/2025] [Accepted: 02/24/2025] [Indexed: 03/16/2025]
Abstract
Approximately half of melanoma patients relapse or fail to respond to current standards of care, highlighting the need for new treatment options. Engineering T-cells with chimeric antigen receptors (CARs) has revolutionized the treatment of hematological malignancies but has been clinically less effective in solid tumors. We therefore sought to engineer alternative immune cell types to inhibit melanoma progression. Engineering macrophages with CARs has emerged as a promising approach to overcome some of the challenges faced by CAR-T cells; however, whether these engineered macrophages can effectively inhibit melanoma growth is unknown. To determine whether CAR-macrophages (CAR-Ms) specifically target and kill melanoma cells, we engineered CAR-Ms targeting chondroitin sulfate proteoglycan 4 (CSPG4), an antigen expressed in melanoma. CSPG4-targeting CAR-Ms exhibited specific phagocytosis of CSPG4-expressing melanoma cells. We developed 3D approaches to show that CSPG4-targeting CAR-Ms efficiently infiltrated melanoma spheroids. Furthermore, combining CSPG4-targeting CAR-Ms with strategies inhibiting CD47/SIRPα "don't eat me" signaling synergistically enhanced CAR-M-mediated phagocytosis and robustly inhibited melanoma spheroid growth in 3D. Importantly, CSPG4-targeting CAR-Ms inhibited melanoma tumor growth in mouse models. These results suggest engineering macrophages against melanoma antigens is a promising solid tumor immunotherapy approach for treating melanoma.
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Affiliation(s)
- Daniel Greiner
- Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, UT, 84112, USA
| | - Qian Xue
- Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, UT, 84112, USA
| | - Trinity Qa Waddell
- Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, UT, 84112, USA
| | - Elena Kurudza
- Department of Neurosurgery, Clinical Neurosciences Center, University of Utah, Salt Lake City, UT, 84112, USA
| | - Piyush Chaudhary
- Huntsman Cancer Institute, University of Utah School of Medicine, Salt Lake City, UT, 84112, USA
- Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT, 84112, USA
| | - Rachel L Belote
- Huntsman Cancer Institute, University of Utah School of Medicine, Salt Lake City, UT, 84112, USA
- Department of Molecular Genetics, The Ohio State University College of Arts and Sciences, Columbus, OH, 43210, USA
| | - Gianpietro Dotti
- Department of Microbiology and Immunology, University of North Carolina, Chapel Hill, NC, 27599, USA
| | - Robert L Judson-Torres
- Huntsman Cancer Institute, University of Utah School of Medicine, Salt Lake City, UT, 84112, USA
- Department of Dermatology, University of Utah School of Medicine, Salt Lake City, UT, 84112, USA
- Department of Oncological Sciences, University of Utah School of Medicine, Salt Lake City, UT, 84112, USA
| | - Melissa Q Reeves
- Huntsman Cancer Institute, University of Utah School of Medicine, Salt Lake City, UT, 84112, USA
- Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT, 84112, USA
| | - Samuel H Cheshier
- Department of Neurosurgery, Clinical Neurosciences Center, University of Utah, Salt Lake City, UT, 84112, USA
- Huntsman Cancer Institute, University of Utah School of Medicine, Salt Lake City, UT, 84112, USA
- Division of Pediatric Neurosurgery, Intermountain Primary Children's Hospital, Salt Lake City, UT, 84112, USA
| | - Minna Roh-Johnson
- Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, UT, 84112, USA.
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Safarzadeh Kozani P, Safarzadeh Kozani P, O'Connor RS. In Like a Lamb; Out Like a Lion: Marching CAR T Cells Toward Enhanced Efficacy in B-ALL. Mol Cancer Ther 2021; 20:1223-1233. [PMID: 33903140 PMCID: PMC8285067 DOI: 10.1158/1535-7163.mct-20-1089] [Citation(s) in RCA: 26] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2020] [Revised: 02/26/2021] [Accepted: 04/19/2021] [Indexed: 11/16/2022]
Abstract
Combining synthetic biology with adoptive T-cell transfer has led to promising advances in the treatment of relapsed/refractory B-cell acute lymphoblastic leukemia (R/R B-ALL), diffuse large B-cell lymphoma (DLBCL), and mantle cell lymphoma (MCL). Chimeric antigen receptors (CARs) are synthetic receptors that redirect T-cell specificity against cancer. CARs include "built-in" signaling domains that reprogram T-cell metabolism, enhance effector function, and support long-term persistence. Despite their success in blood-based malignancies, relapse can occur in CD19-redirected CAR T-cell therapies for several reasons, including poor engraftment, impaired in vivo proliferation, and T-cell senescence. Herein, we explain how subtle alterations in CAR design may overcome barriers to effective adoptive immunotherapy. We also discuss how the physiochemical properties of the single-chain variable fragment (scFv) affect differentiation and persistence. Moreover, we describe innovative advances in CAR engineering and provide insight into the development of humanized scFvs whose proposed benefits include increased persistence and improved clinical outcomes. Tumor cells can evade CAR T-cell-mediated detection and elimination due to the emergence or presence of CD19-negative leukemic cell subpopulations. We also discuss the opportunities and challenges in targeting other B-ALL-associated antigens. Identifying alternate targets is fundamentally necessary to restore the success of CAR T-cell therapies in CD19-negative patients with B-ALL.
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MESH Headings
- Animals
- Antigens, CD19/immunology
- Antigens, Neoplasm/immunology
- Disease Management
- Genetic Engineering
- Humans
- Immunity
- Immunotherapy, Adoptive/adverse effects
- Immunotherapy, Adoptive/methods
- Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/diagnosis
- Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/etiology
- Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/therapy
- Receptors, Antigen, T-Cell/genetics
- Receptors, Antigen, T-Cell/immunology
- Receptors, Chimeric Antigen/genetics
- Receptors, Chimeric Antigen/immunology
- Research Design
- T-Cell Antigen Receptor Specificity/immunology
- T-Lymphocytes/immunology
- T-Lymphocytes/metabolism
- Treatment Outcome
- Tumor Escape/immunology
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Affiliation(s)
- Pouya Safarzadeh Kozani
- Department of Medical Biotechnology, Faculty of Paramedicine, Guilan University of Medical Sciences, Rasht, Iran
- Student Research Committee, Medical Biotechnology Research Center, School of Nursing, Midwifery, and Paramedicine, Guilan University of Medical Sciences, Rasht, Iran
| | - Pooria Safarzadeh Kozani
- Department of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
| | - Roddy S O'Connor
- Center for Cellular Immunotherapies, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania.
- Department of Pathology and Laboratory Medicine, Perelman School of Medicine of the University of Pennsylvania, Philadelphia, Pennsylvania
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3
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Antibody Identification for Antigen Detection in Formalin-Fixed Paraffin-Embedded Tissue Using Phage Display and Naïve Libraries. Antibodies (Basel) 2021; 10:antib10010004. [PMID: 33466676 PMCID: PMC7839037 DOI: 10.3390/antib10010004] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/28/2020] [Revised: 12/15/2020] [Accepted: 01/07/2021] [Indexed: 02/07/2023] Open
Abstract
Immunohistochemistry is a widely used technique for research and diagnostic purposes that relies on the recognition by antibodies of antigens expressed in tissues. However, tissue processing and particularly formalin fixation affect the conformation of these antigens through the formation of methylene bridges. Although antigen retrieval techniques can partially restore antigen immunoreactivity, it is difficult to identify antibodies that can recognize their target especially in formalin-fixed paraffin-embedded tissues. Most of the antibodies currently used in immunohistochemistry have been obtained by animal immunization; however, in vitro display techniques represent alternative strategies that have not been fully explored yet. This review provides an overview of phage display-based antibody selections using naïve antibody libraries on various supports (fixed cells, dissociated tissues, tissue fragments, and tissue sections) that have led to the identification of antibodies suitable for immunohistochemistry.
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Wiesinger M, März J, Kummer M, Schuler G, Dörrie J, Schuler-Thurner B, Schaft N. Clinical-Scale Production of CAR-T Cells for the Treatment of Melanoma Patients by mRNA Transfection of a CSPG4-Specific CAR under Full GMP Compliance. Cancers (Basel) 2019; 11:cancers11081198. [PMID: 31426437 PMCID: PMC6721485 DOI: 10.3390/cancers11081198] [Citation(s) in RCA: 48] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2019] [Revised: 07/14/2019] [Accepted: 08/14/2019] [Indexed: 02/06/2023] Open
Abstract
Chimeric antigen receptor (CAR)-T cells already showed impressive clinical regressions in leukemia and lymphoma. However, the development of CAR-T cells against solid tumors lags behind. Here we present the clinical-scale production of CAR-T cells for the treatment of melanoma under full GMP compliance. In this approach a CAR, specific for chondroitin sulfate proteoglycan 4 (CSPG4) is intentionally transiently expressed by mRNA electroporation for safety reasons. The clinical-scale protocol was optimized for: (i) expansion of T cells, (ii) electroporation efficiency, (iii) viability, (iv) cryopreservation, and (v) potency. Four consistency runs resulted in CAR-T cells in clinically sufficient numbers, i.e., 2.4 × 109 CAR-expressing T cells, starting from 1.77x108 PBMCs, with an average expansion of 13.6x, an electroporation efficiency of 88.0% CAR-positive cells, a survival of 74.1% after electroporation, and a viability of 84% after cryopreservation. Purity was 98.7% CD3+ cells, with 78.1% CD3+/CD8+ T cells and with minor contaminations of 1.2% NK cells and 0.6% B cells. The resulting CAR-T cells were tested for cytolytic activity after cryopreservation and showed antigen-specific and very efficient lysis of tumor cells. Although our work is descriptive rather than investigative in nature, we expect that providing this clinically applicable protocol to generate sufficient numbers of mRNA-transfected CAR-T cells will help in moving the field of adoptive cell therapy of cancer forward.
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Affiliation(s)
- Manuel Wiesinger
- Department of Dermatology, Universtitätsklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Hartmannstraße 14, 91052 Erlangen, Germany
| | - Johannes März
- Department of Dermatology, Universtitätsklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Hartmannstraße 14, 91052 Erlangen, Germany
| | - Mirko Kummer
- Department of Dermatology, Universtitätsklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Hartmannstraße 14, 91052 Erlangen, Germany
| | - Gerold Schuler
- Department of Dermatology, Universtitätsklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Hartmannstraße 14, 91052 Erlangen, Germany
| | - Jan Dörrie
- Department of Dermatology, Universtitätsklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Hartmannstraße 14, 91052 Erlangen, Germany
| | - Beatrice Schuler-Thurner
- Department of Dermatology, Universtitätsklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Hartmannstraße 14, 91052 Erlangen, Germany
| | - Niels Schaft
- Department of Dermatology, Universtitätsklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Hartmannstraße 14, 91052 Erlangen, Germany.
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CSPG4-Specific CAR T Cells for High-Risk Childhood B Cell Precursor Leukemia. Int J Mol Sci 2019; 20:ijms20112764. [PMID: 31195686 PMCID: PMC6600602 DOI: 10.3390/ijms20112764] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/16/2019] [Revised: 05/24/2019] [Accepted: 05/27/2019] [Indexed: 02/06/2023] Open
Abstract
The advent of CD19-specific chimeric antigen receptor (CAR) T cells has proven to be a powerful asset in the arsenal of cancer immunotherapy of acute lymphoblastic leukemia and certain B cell lymphomas. However, a sizable portion of patients treated with CD19-CAR T cells relapse with CD19-negative cancer cells, necessitating the quest for back-up antigens. Chondroitin sulfate proteoglycan 4 (CSPG4) expression has been reported on leukemic blasts bearing the ill-fated MLL 11q23 rearrangement. We aimed at exploring the use of CSPG4-specific CAR T cells against mixed-lineage leukemia (MLL)-rearranged leukemic blasts, using the precursor B cell leukemia cell line KOPN8 (MLL–MLLT1 translocation) as a model. First, we confirmed CSPG4 expression on KOPN8 cells. Bulk T cells electroporated with mRNA encoding a CSPG4-specific CAR upregulated activation markers and secreted the Th1 cytokines TNF and IFNγ in an antigen-specific manner upon co-culture with KOPN8 cells. More importantly, CSPG4-specific CAR T cells evinced specific degranulation towards KOPN8 cells and specifically lysed KOPN8 target cells in chromium lysis experiments. CSPG4 is a well-established CAR target in cutaneous melanoma. Here, we provide proof-of-principle data for the use of CSPG4-specific CAR T cells against MLL-translocated leukemias.
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6
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Kumar R, Parray HA, Shrivastava T, Sinha S, Luthra K. Phage display antibody libraries: A robust approach for generation of recombinant human monoclonal antibodies. Int J Biol Macromol 2019; 135:907-918. [PMID: 31170490 DOI: 10.1016/j.ijbiomac.2019.06.006] [Citation(s) in RCA: 65] [Impact Index Per Article: 10.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2019] [Revised: 06/02/2019] [Accepted: 06/02/2019] [Indexed: 12/29/2022]
Abstract
Monoclonal antibodies (mAbs) and their derivatives have achieved remarkable success as medicine, targeting both diagnostic and therapeutic applications associated with communicable and non-communicable diseases. In the last 3 to 4 decades, tremendous success has been manifested in the field of cancer therapy, autoimmune diseases, cardiovascular and infectious diseases. MAbs are the fastest growing class of biopharmaceuticals, with more than 25 derivatives are in clinical use and 7 of these have been isolated through phage display technology. Phage display technology has gained impetus in the field of medical and health sciences, as a large repertoire of diverse recombinant antibodies, targeting various antigens have been generated in a short span of time. A prominent number of phage display derived antibodies are already approved for therapy and significant numbers are currently in clinical trials. In this review we have discussed the various strategies employed for generation of monoclonal antibodies; their advantages, limitations and potential therapeutic applications. We also discuss the potential of phage display antibody libraries in isolation of monoclonal antibodies.
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Affiliation(s)
- Rajesh Kumar
- Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, India; Translational Health Science & Technology Institute, NCR Biotech Science Cluster, Faridabad, Haryana 121001, India.
| | - Hilal Ahmed Parray
- Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, India
| | - Tripti Shrivastava
- Translational Health Science & Technology Institute, NCR Biotech Science Cluster, Faridabad, Haryana 121001, India
| | - Subrata Sinha
- Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, India
| | - Kalpana Luthra
- Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, India.
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7
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Karamanos NK, Piperigkou Z, Theocharis AD, Watanabe H, Franchi M, Baud S, Brézillon S, Götte M, Passi A, Vigetti D, Ricard-Blum S, Sanderson RD, Neill T, Iozzo RV. Proteoglycan Chemical Diversity Drives Multifunctional Cell Regulation and Therapeutics. Chem Rev 2018; 118:9152-9232. [PMID: 30204432 DOI: 10.1021/acs.chemrev.8b00354] [Citation(s) in RCA: 253] [Impact Index Per Article: 36.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Affiliation(s)
- Nikos K. Karamanos
- Biochemistry, Biochemical Analysis & Matrix Pathobiology Research Group, Laboratory of Biochemistry, Department of Chemistry, University of Patras, Patras 26110, Greece
- Foundation for Research and Technology-Hellas (FORTH)/Institute of Chemical Engineering Sciences (ICE-HT), Patras 26110, Greece
| | - Zoi Piperigkou
- Biochemistry, Biochemical Analysis & Matrix Pathobiology Research Group, Laboratory of Biochemistry, Department of Chemistry, University of Patras, Patras 26110, Greece
- Foundation for Research and Technology-Hellas (FORTH)/Institute of Chemical Engineering Sciences (ICE-HT), Patras 26110, Greece
| | - Achilleas D. Theocharis
- Biochemistry, Biochemical Analysis & Matrix Pathobiology Research Group, Laboratory of Biochemistry, Department of Chemistry, University of Patras, Patras 26110, Greece
| | - Hideto Watanabe
- Institute for Molecular Science of Medicine, Aichi Medical University, Aichi 480-1195, Japan
| | - Marco Franchi
- Department for Life Quality Studies, University of Bologna, Rimini 47100, Italy
| | - Stéphanie Baud
- Université de Reims Champagne-Ardenne, Laboratoire SiRMa, CNRS UMR MEDyC 7369, Faculté de Médecine, 51 rue Cognacq Jay, Reims 51100, France
| | - Stéphane Brézillon
- Université de Reims Champagne-Ardenne, Laboratoire de Biochimie Médicale et Biologie Moléculaire, CNRS UMR MEDyC 7369, Faculté de Médecine, 51 rue Cognacq Jay, Reims 51100, France
| | - Martin Götte
- Department of Gynecology and Obstetrics, Münster University Hospital, Münster 48149, Germany
| | - Alberto Passi
- Department of Medicine and Surgery, University of Insubria, Varese 21100, Italy
| | - Davide Vigetti
- Department of Medicine and Surgery, University of Insubria, Varese 21100, Italy
| | - Sylvie Ricard-Blum
- University Claude Bernard Lyon 1, CNRS, UMR 5246, Institute of Molecular and Supramolecular Chemistry and Biochemistry, Villeurbanne 69622, France
| | - Ralph D. Sanderson
- Department of Pathology, Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, Alabama 35294, United States
| | - Thomas Neill
- Department of Pathology, Anatomy and Cell Biology, Sidney Kimmel Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 10107, United States
| | - Renato V. Iozzo
- Department of Pathology, Anatomy and Cell Biology, Sidney Kimmel Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 10107, United States
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8
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Harrer DC, Simon B, Fujii SI, Shimizu K, Uslu U, Schuler G, Gerer KF, Hoyer S, Dörrie J, Schaft N. RNA-transfection of γ/δ T cells with a chimeric antigen receptor or an α/β T-cell receptor: a safer alternative to genetically engineered α/β T cells for the immunotherapy of melanoma. BMC Cancer 2017; 17:551. [PMID: 28818060 PMCID: PMC5561563 DOI: 10.1186/s12885-017-3539-3] [Citation(s) in RCA: 91] [Impact Index Per Article: 11.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2017] [Accepted: 08/10/2017] [Indexed: 12/16/2022] Open
Abstract
Background Adoptive T-cell therapy relying on conventional T cells transduced with T-cell receptors (TCRs) or chimeric antigen receptors (CARs) has caused substantial tumor regression in several clinical trials. However, genetically engineered T cells have been associated with serious side-effects due to off-target toxicities and massive cytokine release. To obviate these concerns, we established a protocol adaptable to GMP to expand and transiently transfect γ/δ T cells with mRNA. Methods PBMC from healthy donors were stimulated using zoledronic-acid or OKT3 to expand γ/δ T cells and bulk T cells, respectively. Additionally, CD8+ T cells and γ/δ T cells were MACS-isolated from PBMC and expanded with OKT3. Next, these four populations were electroporated with RNA encoding a gp100/HLA-A2-specific TCR or a CAR specific for MCSP. Thereafter, receptor expression, antigen-specific cytokine secretion, specific cytotoxicity, and killing of the endogenous γ/δ T cell-target Daudi were analyzed. Results Using zoledronic-acid in average 6 million of γ/δ T cells with a purity of 85% were generated from one million PBMC. MACS-isolation and OKT3-mediated expansion of γ/δ T cells yielded approximately ten times less cells. OKT3-expanded and CD8+ MACS-isolated conventional T cells behaved correspondingly similar. All employed T cells were efficiently transfected with the TCR or the CAR. Upon respective stimulation, γ/δ T cells produced IFNγ and TNF, but little IL-2 and the zoledronic-acid expanded T cells exceeded MACS-γ/δ T cells in antigen-specific cytokine secretion. While the cytokine production of γ/δ T cells was in general lower than that of conventional T cells, specific cytotoxicity against melanoma cell lines was similar. In contrast to OKT3-expanded and MACS-CD8+ T cells, mock-electroporated γ/δ T cells also lysed tumor cells reflecting the γ/δ T cell-intrinsic anti-tumor activity. After transfection, γ/δ T cells were still able to kill MHC-deficient Daudi cells. Conclusion We present a protocol adaptable to GMP for the expansion of γ/δ T cells and their subsequent RNA-transfection with tumor-specific TCRs or CARs. Given the transient receptor expression, the reduced cytokine release, and the equivalent cytotoxicity, these γ/δ T cells may represent a safer complementation to genetically engineered conventional T cells in the immunotherapy of melanoma (Exper Dermatol 26: 157, 2017, J Investig Dermatol 136: A173, 2016). Electronic supplementary material The online version of this article (doi:10.1186/s12885-017-3539-3) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Dennis C Harrer
- Department of Dermatology, Universitätsklinikum Erlangen, Hartmannstraße 14, D-91052, Erlangen, Germany.,Department of Dermatology, Faculty of Medicine, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Erlangen, Germany
| | - Bianca Simon
- Department of Dermatology, Universitätsklinikum Erlangen, Hartmannstraße 14, D-91052, Erlangen, Germany.,Department of Genetics, Friedrich-Alexander-Universität Erlangen-Nürnberg Erlangen-Nürnberg, Erlangen, Germany
| | - Shin-Ichiro Fujii
- Laboratory for Immunotherapy, RIKEN Center for Integrative Medical Sciences (IMS), 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan
| | - Kanako Shimizu
- Laboratory for Immunotherapy, RIKEN Center for Integrative Medical Sciences (IMS), 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan
| | - Ugur Uslu
- Department of Dermatology, Universitätsklinikum Erlangen, Hartmannstraße 14, D-91052, Erlangen, Germany
| | - Gerold Schuler
- Department of Dermatology, Universitätsklinikum Erlangen, Hartmannstraße 14, D-91052, Erlangen, Germany.,Department of Dermatology, Faculty of Medicine, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Erlangen, Germany
| | - Kerstin F Gerer
- Department of Dermatology, Universitätsklinikum Erlangen, Hartmannstraße 14, D-91052, Erlangen, Germany.,Department of Genetics, Friedrich-Alexander-Universität Erlangen-Nürnberg Erlangen-Nürnberg, Erlangen, Germany
| | - Stefanie Hoyer
- Department of Dermatology, Universitätsklinikum Erlangen, Hartmannstraße 14, D-91052, Erlangen, Germany
| | - Jan Dörrie
- Department of Dermatology, Universitätsklinikum Erlangen, Hartmannstraße 14, D-91052, Erlangen, Germany.,Department of Dermatology, Faculty of Medicine, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Erlangen, Germany
| | - Niels Schaft
- Department of Dermatology, Universitätsklinikum Erlangen, Hartmannstraße 14, D-91052, Erlangen, Germany. .,Department of Dermatology, Faculty of Medicine, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Erlangen, Germany.
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9
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Rolih V, Barutello G, Iussich S, De Maria R, Quaglino E, Buracco P, Cavallo F, Riccardo F. CSPG4: a prototype oncoantigen for translational immunotherapy studies. J Transl Med 2017; 15:151. [PMID: 28668095 PMCID: PMC5494135 DOI: 10.1186/s12967-017-1250-4] [Citation(s) in RCA: 52] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/13/2017] [Accepted: 06/21/2017] [Indexed: 12/21/2022] Open
Abstract
Thanks to striking progress in both the understanding of anti-tumor immune response and the characterization of several tumor associated antigens (TAA), a more rational design and more sophisticated strategies for anti-tumor vaccination have been possible. However, the effectiveness of cancer vaccines in clinical trial is still partial, indicating that additional studies are needed to optimize their design and their pre-clinical testing. Indeed, anti-tumor vaccination success relies on the choice of the best TAA to be targeted and on the translational power of the pre-clinical model used to assess its efficacy. The chondroitin sulfate proteoglycan-4 (CSPG4) is a cell surface proteoglycan overexpressed in a huge range of human and canine neoplastic lesions by tumor cells, tumor microenvironment and cancer initiating cells. CSPG4 plays a central role in the oncogenic pathways required for malignant progression and metastatization. Thanks to these features and to its poor expression in adult healthy tissues, CSPG4 represents an ideal oncoantigen and thus an attractive target for anti-tumor immunotherapy. In this review we explore the potential of CSPG4 immune-targeting. Moreover, since it has been clearly demonstrated that spontaneous canine tumors mimic the progression of human malignancies better than any other pre-clinical model available so far, we reported also our results indicating that CSPG4 DNA vaccination is safe and effective in significantly increasing the survival of canine melanoma patients. Therefore, anti-CSPG4 vaccination strategy could have a substantial impact for the treatment of the wider population of spontaneous CSPG4-positive tumor affected dogs with a priceless translational value and a revolutionary implication for human oncological patients.
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Affiliation(s)
- Valeria Rolih
- Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center, University of Torino, 10126 Turin, Italy
| | - Giuseppina Barutello
- Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center, University of Torino, 10126 Turin, Italy
| | - Selina Iussich
- Department of Veterinary Sciences, University of Torino, 10095 Grugliasco, Italy
| | - Raffaella De Maria
- Department of Veterinary Sciences, University of Torino, 10095 Grugliasco, Italy
| | - Elena Quaglino
- Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center, University of Torino, 10126 Turin, Italy
| | - Paolo Buracco
- Department of Veterinary Sciences, University of Torino, 10095 Grugliasco, Italy
| | - Federica Cavallo
- Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center, University of Torino, 10126 Turin, Italy
| | - Federica Riccardo
- Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center, University of Torino, 10126 Turin, Italy
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10
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Pero SC, Sun YJ, Shukla GS, Carman CL, Krag CC, Teuscher C, Krementsov DN, Krag DN. Vaccine draining lymph nodes are a source of antigen-specific B cells. Vaccine 2017; 35:1259-1265. [PMID: 28161423 DOI: 10.1016/j.vaccine.2017.01.036] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/11/2016] [Revised: 12/21/2016] [Accepted: 01/18/2017] [Indexed: 12/31/2022]
Abstract
PURPOSE Our research is focused on using vaccine draining lymph nodes as a source of immune cells to better understand the immune response and to attempt to generate new anti-cancer reagents. Following a vaccine, harvesting the lymph node can only be done once. We endeavored to determine the range of times that B cells secreting anti-KLH antibodies were present in the node of KLH-vaccinated mice. RESULTS Following vaccination the total number of mononuclear cells (MNCs) increased in the vaccine-draining lymph node (VDN). The percentage of MNCs that were B cells nearly doubled. B cells recovered from the node that secreted anti-KLH antibodies were evident by day 7. The number continued to increase and then slowly decreased over the observed time range to 28days after vaccination. The VDN, compared to the spleen, the bone marrow and the nonVDN, contained a higher percentage of B cells that secreted anti-KLH antibodies. CONCLUSIONS After a vaccine, there is a multi-week window of time when an increasing number of B cells are present in a VDN that secrete anti-KLH antibodies. These results support using the VDN as a source for B cells that secrete anti-vaccine antibodies.
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Affiliation(s)
- Stephanie C Pero
- Department of Surgery, Vermont Cancer Center, University of Vermont College of Medicine, 89 Beaumont Avenue, Given Building E310, Burlington, VT 05405, USA
| | - Yu-Jing Sun
- Department of Surgery, Vermont Cancer Center, University of Vermont College of Medicine, 89 Beaumont Avenue, Given Building E310, Burlington, VT 05405, USA
| | - Girja S Shukla
- Department of Surgery, Vermont Cancer Center, University of Vermont College of Medicine, 89 Beaumont Avenue, Given Building E310, Burlington, VT 05405, USA
| | - Chelsea L Carman
- Department of Surgery, Vermont Cancer Center, University of Vermont College of Medicine, 89 Beaumont Avenue, Given Building E310, Burlington, VT 05405, USA
| | - Christopher C Krag
- Department of Surgery, Vermont Cancer Center, University of Vermont College of Medicine, 89 Beaumont Avenue, Given Building E310, Burlington, VT 05405, USA
| | - Cory Teuscher
- Department of Medicine, University of Vermont College of Medicine, 89 Beaumont Avenue, Given Building C329, Burlington, VT 05405, USA
| | - Dimitry N Krementsov
- Department of Medicine, University of Vermont College of Medicine, 89 Beaumont Avenue, Given Building C329, Burlington, VT 05405, USA
| | - David N Krag
- Department of Surgery, Vermont Cancer Center, University of Vermont College of Medicine, 89 Beaumont Avenue, Given Building E310, Burlington, VT 05405, USA.
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11
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Torres BA, Kominsky S, Perrin GQ, Hobeika AC, Johnson HM. Superantigens: The Good, the Bad, and the Ugly. Exp Biol Med (Maywood) 2016; 226:164-76. [PMID: 11361034 DOI: 10.1177/153537020122600303] [Citation(s) in RCA: 49] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022] Open
Abstract
Increasing evidence suggests that superantigens play a role in Immune-mediated diseases. Superantigens are potent activators of CD4* T cells, causing rapid and massive proliferation of cells and cytokine production. This characteristic of superantigens can be exploited in diseases where strong immunologic responses are required, such as in the B16F10 animal model of melanoma. Superantigen administration is able to significantly enhance Ineffective anti-tumor Immune responses, resulting in potent and long-lived protective anti-tumor immunity. However, superantigens are more well-known for the role they play in diseases. Studies using an animal model for neurologic demy-elinatlng diseases such as multiple sclerosis show that superantigens can induce severe relapses and activate auto-reactive T cells not involved in the Initial bout of disease. This may also involve epitope spreading of disease. Superantigens have also been implicated in acute diseases such as food poisoning and TSS, and in chronic diseases such as psoriasis and rheumatoid arthritis. Viral superantigens are also involved in the disease process, including superantigens derived from human Immunodeficiency virus and mouse mammary tumor virus. Finally, immunotherapies that ameliorate the role played by superantigens in disease are discussed.
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Affiliation(s)
- B A Torres
- Department of Microbiology and Cell Science, University of Florida, Gainesville 32611, USA
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12
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Krug C, Birkholz K, Paulus A, Schwenkert M, Schmidt P, Hoffmann N, Hombach A, Fey G, Abken H, Schuler G, Schuler-Thurner B, Dörrie J, Schaft N. Stability and activity of MCSP-specific chimeric antigen receptors (CARs) depend on the scFv antigen-binding domain and the protein backbone. Cancer Immunol Immunother 2015; 64:1623-35. [PMID: 26515978 PMCID: PMC11028909 DOI: 10.1007/s00262-015-1767-4] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2014] [Accepted: 10/16/2015] [Indexed: 12/26/2022]
Abstract
Chimeric antigen receptor (CAR)-modified T cells emerged as effective tools in the immunotherapy of cancer but can produce severe on-target off-tissue toxicities. This risk can conceivably be overcome, at least partially, by transient transfection. The design of CARs, however, has so far not been optimized for use in non-permanent T cell modification. Here we compared the performance of T cells modified with three different first- and second-generation CARs, each specific for MCSP (HMW-MAA) which is commonly expressed by melanoma cells. Upon RNA transfer, the expression of all receptors was limited in time. The second-generation CARs, which combined CD28-CD3ζ signaling, were expressed at higher levels and more prolonged than first-generation CARs with CD3ζ only. The CD28 domain increased the cytokine production, but had only an indirect effect on the lytic capacity, by prolonging the CAR expression. Especially for the second-generation CARs, the scFv clearly impacted the level and duration of CAR expression and the T cell performance. Thus, we identified a CAR high in both expression and anti-tumor cell reactivity. T cells transfected with this CAR increased the mean survival time of mice after challenge with melanoma cells. To facilitate clinical application, this CAR was used to redirect T cells from late-stage melanoma patients by RNA transfection. These T cells mediated effective antigen-specific tumor cell lysis and release of pro-inflammatory cytokines, even after cryoconservation of the transfected T cells. Taken together, the analysis identified a CAR with superior anti-melanoma performance after RNA transfer which is a promising candidate for clinical exploration.
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MESH Headings
- Animals
- Carrier Proteins/immunology
- Cell Line, Tumor
- Disease Models, Animal
- Gene Expression Regulation, Neoplastic/immunology
- Humans
- Melanoma/immunology
- Melanoma/physiopathology
- Mice
- Mitochondrial Proteins/genetics
- Mitochondrial Proteins/immunology
- Protein Stability
- Protein Structure, Tertiary
- Receptors, Antigen, T-Cell/genetics
- Receptors, Antigen, T-Cell/immunology
- Receptors, Antigen, T-Cell/metabolism
- Single-Chain Antibodies/metabolism
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Affiliation(s)
- Christian Krug
- Department of Dermatology, Universitätsklinikum Erlangen, Hartmannstraße 14, 91052, Erlangen, Germany
- Department of Biology, Friedrich-Alexander-University of Erlangen-Nuremberg, Erlangen, Germany
| | - Katrin Birkholz
- Department of Dermatology, Universitätsklinikum Erlangen, Hartmannstraße 14, 91052, Erlangen, Germany
| | - Alexander Paulus
- Department of Dermatology, Universitätsklinikum Erlangen, Hartmannstraße 14, 91052, Erlangen, Germany
| | - Michael Schwenkert
- Department of Biology, Friedrich-Alexander-University of Erlangen-Nuremberg, Erlangen, Germany
| | - Patrick Schmidt
- Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany
- Department I Internal Medicine, University Hospital Cologne, Cologne, Germany
| | - Nicole Hoffmann
- Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany
- Department I Internal Medicine, University Hospital Cologne, Cologne, Germany
| | - Andreas Hombach
- Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany
- Department I Internal Medicine, University Hospital Cologne, Cologne, Germany
| | - Georg Fey
- Department of Biology, Friedrich-Alexander-University of Erlangen-Nuremberg, Erlangen, Germany
| | - Hinrich Abken
- Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany
- Department I Internal Medicine, University Hospital Cologne, Cologne, Germany
| | - Gerold Schuler
- Department of Dermatology, Universitätsklinikum Erlangen, Hartmannstraße 14, 91052, Erlangen, Germany
| | - Beatrice Schuler-Thurner
- Department of Dermatology, Universitätsklinikum Erlangen, Hartmannstraße 14, 91052, Erlangen, Germany
| | - Jan Dörrie
- Department of Dermatology, Universitätsklinikum Erlangen, Hartmannstraße 14, 91052, Erlangen, Germany
| | - Niels Schaft
- Department of Dermatology, Universitätsklinikum Erlangen, Hartmannstraße 14, 91052, Erlangen, Germany.
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13
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Nicolosi PA, Dallatomasina A, Perris R. Theranostic impact of NG2/CSPG4 proteoglycan in cancer. Theranostics 2015; 5:530-44. [PMID: 25767619 PMCID: PMC4350014 DOI: 10.7150/thno.10824] [Citation(s) in RCA: 57] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2014] [Accepted: 12/03/2014] [Indexed: 12/27/2022] Open
Abstract
NG2/CSPG4 is an unusual cell-membrane integral proteoglycan widely recognized to be a prognostic factor, a valuable tool for ex vivo and non-invasive molecular diagnostics and, by virtue of its tight association with malignancy, a tantalizing therapeutic target in several tumour types. Although the biology behind its involvement in cancer progression needs to be better understood, implementation of NG2/CSPG4 in the routine clinical practice is attainable and has the potential to contribute to an improved individualized management of cancer patients. In this context, its polymorphic nature seems to be particularly valuable in the effort to standardize informative diagnostic procedures and consolidate forcible immunotherapeutic treatment strategies. We discuss here the underpinnings for this potential and highlight the benefits of taking advantage of the intra-tumour and inter-patient variability in the regulation of NG2/CSPG4 expression. We envision that NG2/CSPG4 may effectively be exploited in therapeutic interventions aimed at averting resistance to target therapy agents and at interfering with secondary lesion formation and/or tumour recurrence.
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14
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Zhao A, Tohidkia MR, Siegel DL, Coukos G, Omidi Y. Phage antibody display libraries: a powerful antibody discovery platform for immunotherapy. Crit Rev Biotechnol 2014; 36:276-89. [PMID: 25394539 DOI: 10.3109/07388551.2014.958978] [Citation(s) in RCA: 70] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023]
Abstract
Phage display technology (PDT), a combinatorial screening approach, provides a molecular diversity tool for creating libraries of peptides/proteins and discovery of new recombinant therapeutics. Expression of proteins such as monoclonal antibodies (mAbs) on the surface of filamentous phage can permit the selection of high affinity and specificity therapeutic mAbs against virtually any target antigen. Using a number of diverse selection platforms (e.g. solid phase, solution phase, whole cell and in vivo biopannings), phage antibody libraries (PALs) from the start point provides great potential for the isolation of functional mAb fragments with diagnostic and/or therapeutic purposes. Given the pivotal role of PDT in the discovery of novel therapeutic/diagnostic mAbs, in the current review, we provide an overview on PALs and discuss their impact in the advancement of engineered mAbs.
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Affiliation(s)
- Aizhi Zhao
- a Ovarian Cancer Research Center, Perelman School of Medicine, University of Pennsylvania , Philadelphia , PA , USA
| | - Mohammad R Tohidkia
- b Research Center for Pharmaceutical Nanotechnology, Faculty of Pharmacy, Tabriz University of Medical Sciences , Tabriz , Iran
| | - Donald L Siegel
- c Division of Transfusion Medicine, Department of Pathology & Laboratory Medicine , University of Pennsylvania School of Medicine , Philadelphia , PA , USA , and
| | - George Coukos
- a Ovarian Cancer Research Center, Perelman School of Medicine, University of Pennsylvania , Philadelphia , PA , USA .,d Ludwig Center for Cancer Research, University of Lausanne , Lausanne , Switzerland
| | - Yadollah Omidi
- a Ovarian Cancer Research Center, Perelman School of Medicine, University of Pennsylvania , Philadelphia , PA , USA .,b Research Center for Pharmaceutical Nanotechnology, Faculty of Pharmacy, Tabriz University of Medical Sciences , Tabriz , Iran
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15
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Garusi E, Rossi S, Perris R. Antithetic roles of proteoglycans in cancer. Cell Mol Life Sci 2012; 69:553-79. [PMID: 21964924 PMCID: PMC11114698 DOI: 10.1007/s00018-011-0816-1] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2011] [Revised: 09/01/2011] [Accepted: 09/05/2011] [Indexed: 12/15/2022]
Abstract
Proteoglycans (PGs), a family of complex post-translationally sculptured macromolecules, are fundamental regulators of most normal and aberrant cellular functions. The unparalleled structural-functional diversity of PGs endows them with the ability to serve as critical mediators of the tumor cells' interaction with the host microenvironment, while directly contributing to the organization and dynamic remodeling of this milieu. Despite their indisputable importance during embryonic development and in the adult organism, and their frequent dysregulation in tumor lesions, their precise involvement in tumorigenesis awaits a more decisive demonstration. Particularly challenging is to ascertain to what extent selected PGs may catalyze tumor progression and to what extent they may inhibit it, implying antithetic functions of individual PGs. Integrated efforts are needed to consolidate the routine use of PGs in the clinical monitoring of cancer patients and to broaden the exploitation of these macromolecules as therapeutic targets. Several PGs have the required attributes to be contemplated as effective antigens for immunotherapeutic approaches, while the tangible results obtained in recent clinical trials targeting the NG2/CSPG4 transmembrane PG urge further development of PG-based cancer treatment modalities.
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Affiliation(s)
- Elena Garusi
- COMT, Centre for Molecular and Translational Oncology, University of Parma, Via G.P. Usberti 11/A, 43100 Parma, Italy
| | - Silvia Rossi
- COMT, Centre for Molecular and Translational Oncology, University of Parma, Via G.P. Usberti 11/A, 43100 Parma, Italy
- Department of Genetic, Biology of Microorganism, Anthropology and Evolution, University of Parma, Via G.P. Usberti 11/A, 43100 Parma, Italy
| | - Roberto Perris
- COMT, Centre for Molecular and Translational Oncology, University of Parma, Via G.P. Usberti 11/A, 43100 Parma, Italy
- Department of Genetic, Biology of Microorganism, Anthropology and Evolution, University of Parma, Via G.P. Usberti 11/A, 43100 Parma, Italy
- S.O.C. of Experimental Oncology 2, The National Cancer Institute Aviano, CRO-IRCCS, Via Franco Gallini, 2, 33081 Aviano, PN Italy
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Abstract
The lack of effective conventional therapies for the treatment of advanced stage melanoma has stimulated interest in the development of novel strategies for the management of patients with malignant melanoma. Among them, immunotherapy has attracted much attention because of the potential role played by immunological events in the clinical course of melanoma. For many years, T cell-based immunotherapy has been emphasized in part because of the disappointing results of the monoclonal antibody (mAb)-based clinical trials conducted in the early 1980s and in part because of the postulated major role played by T cells in tumor growth control. More recently, mAb-based therapies have gained in popularity given their clinical and commercial success for a variety of malignant diseases. As a result, there has been increased interest in identifying and characterizing antibody-defined melanoma antigens. Among them, the chondroitin sulfate proteoglycan 4 (CSPG4), also known as high molecular weight-melanoma associated antigen (HMW-MAA) or melanoma chondroitin sulfate proteoglycan (MCSP), has attracted much attention in recent years because of the growing experimental evidence that it fulfills two requirements for immunotherapy to be therapeutically effective: (1) targeting of cancer stem cells (CSC) and (2) development of combinatorial therapies to counteract the escape mechanisms driven by the genetic instability of tumor cells. With this in mind, in this chapter, we have reviewed recent information related to the distribution of CSPG4 on various types of tumors, including CSC, its expression on pericytes in the tumor microenvironment, its recognition by T cells, its role in cell biology as well as the potential mechanisms underlying the ability of CSPG4-specific immunity to control malignant cell growth.
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17
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Superantigens increase the survival of mice bearing T cell lymphomas by inducing apoptosis of neoplastic cells. PLoS One 2010; 5:e15694. [PMID: 21203530 PMCID: PMC3008744 DOI: 10.1371/journal.pone.0015694] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2010] [Accepted: 11/23/2010] [Indexed: 11/29/2022] Open
Abstract
Superantigens bind to major histocompatibility complex class II molecules and interact with T cells expressing a particular T cell receptor Vβ inducing a strong proliferation/deletion response of the superantigen-reactive T cells. However, there have been no attempts to investigate the ability of Sags to induce apoptosis in neoplastic T cells by signaling through the Vβ region of their TCR. In the present study we show that bacterial and MMTV-encoded superantigens induce the apoptosis of AKR/J cognate lymphoma T cells both in vitro and in vivo. The Fas-Fas-L pathway was shown to be involved in the apoptosis of lymphoma T cells induced by bacterial superantigens. In vivo exposure to bacterial superantigens was able to improve the survival of lymphoma bearing mice. Moreover, the permanent expression of a retroviral encoded superantigen induced the complete remission of an aggressive lymphoma in a high percentage of mice. The possibility of a therapeutic use of superantigens in lymphoma/leukemia T cell malignancies is discussed.
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18
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Xu Q, Zhang X, Yue J, Liu C, Cao C, Zhong H, Ma Q. Human TGFalpha-derived peptide TGFalphaL3 fused with superantigen for immunotherapy of EGFR-expressing tumours. BMC Biotechnol 2010; 10:91. [PMID: 21176167 PMCID: PMC3018390 DOI: 10.1186/1472-6750-10-91] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2010] [Accepted: 12/22/2010] [Indexed: 11/10/2022] Open
Abstract
Background Monoclonal antibodies have been employed as targeting molecules of superantigen for the preclinical treatment of a variety of tumours. However, other targeting molecules, such as tumour-related ligands or peptides, are less exploited. Here, we tested other targeting molecules by genetically fusing the third loop of transforming growth factor alpha (TGFalphaL3) to mutant staphylococcal enterotoxin A (SEAD227A). Results The resultant fusion proteins were expressed in E. coli and purified to homogeneity through a Ni-NTA affinity column. Fusion protein TGFalphaL3SEAD227A can promote splenocyte proliferation to a level comparable to recombinant SEA (rSEA) and bind to EGFR-expressing tumour cells in an EGFR-dependent way. Consistent with these observations, TGFalphaL3SEAD227A exerted an inhibitory effect on the growth of EGFR-expressing tumour cells both in vitro and in vivo. Notably, significant infiltrations of CD8+ and CD4+ T cells were detected in the tumour tissues of these C57BL/6 mice treated with TGFalphaL3SEAD227A, suggesting the involvement of T cells in this tumour-inhibitory process. Conclusions The data here showed that TGFαL3 is capable of targeting superantigen to tumours and exerting an inhibitory effect on tumour growth, which enables TGFαL3SEAD227A to be an attractive candidate for the immunotherapy of EGFR-expressing tumours.
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Affiliation(s)
- Quanbin Xu
- Beijing Institute of Biotechnology, Taiping Road 27, Beijing, PR China.
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19
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Eriksson F, Culp WD, Massey R, Egevad L, Garland D, Persson MAA, Pisa P. Tumor specific phage particles promote tumor regression in a mouse melanoma model. Cancer Immunol Immunother 2007; 56:677-87. [PMID: 16967280 PMCID: PMC11031031 DOI: 10.1007/s00262-006-0227-6] [Citation(s) in RCA: 47] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2006] [Accepted: 08/09/2006] [Indexed: 11/25/2022]
Abstract
Within cancer research, phage display libraries have been widely used for the identification of tumor targeting peptides and antibodies. Additionally, phages are known to be highly immunogenic; therefore we evaluated the immunotherapeutic potential of tumor specific phages to treat established solid tumors in a mouse model of melanoma. We developed two tumor specific phages, one derived from a peptide phage display library and one Fab expressing phage with known specificity, for the treatment of mice bearing palpable B16-F10 or B16/A2K(b) tumors. Therapy in B16-F10 tumor bearing mice with tumor specific phages was superior to treatment with non-tumor specific phages and lead to delayed tumor growth and increased survival. In B16/A2K(b )tumor bearing mice, therapy with tumor specific phages resulted in complete tumor regression and long-term survival in 50% of the mice. Histological analysis of tumors undergoing treatment with tumor specific phages revealed that phage administration induced a massive infiltration of polymorphonuclear neutrophils. Furthermore, phages induced secretion of IL-12 (p70) and IFN-gamma as measured in mouse splenocyte culture supernatants. These results demonstrate a novel, immunotherapeutic cancer treatment showing that tumor specific phages can promote regression of established tumors by recruitment of inflammatory cells and induction of Th1 cytokines.
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Affiliation(s)
- Fredrik Eriksson
- Department of Oncology and Pathology, Immune and Gene Therapy Laboratory, Cancer Centre Karolinska, Karolinska Institute, Stockholm, Sweden.
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20
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Wei J, Liu Y, Yang S, Xu J, Kong H, Han B, Bao Y, Wu Y, Yin W, Li W, Yan G, Luo G, Xu HP, Li Y, Yang B. Screening of single-chain variable fragments against TSP50 from a phage display antibody library and their expression as soluble proteins. ACTA ACUST UNITED AC 2006; 11:546-52. [PMID: 16928985 DOI: 10.1177/1087057106287901] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
Abstract
A novel gene, testes-specific protease 50 (TSP50), is abnormally activated and differentially expressed in most patients with breast cancer, suggesting it as a novel biomarker for this disease. The possibility that TSP50 may be an oncogene is presently under investigation. In this study, the single-chain variable fragments (scFvs) against TSP50 were panned from a phage display antibody library using TSP50-specific peptide, pep-50, as a target antigen. After 4 rounds of panning, 3 clones (A1, A11, and C8) from the library were verified to show strong binding affinities for TSP50 by enzyme-linked immunosorbent assay (ELISA) and to contain the variable region genes of the light and heavy chains of scFv antibodies but different complementary determining regions by sequencing. The genes of scFv-A1 and scFv-A11 were cloned into expression vector pPELB and successfully expressed as a soluble protein inEscherichia coli Rosetta. The yields of expressions were about 4.0 to 5.0 mg of protein from 1 L of culture. The expressed proteins were purified by a 2-step procedure consisting of ion-exchange chromatography, followed by immobilized metal affinity chromatography. The purified proteins were shown a single band at the position of 31 KDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sandwich ELISA demonstrated that the expressed scFv proteins were able to specifically react with pep-50, laying a foundation for the investigation of the function of TSP50 in the development and treatment of breast cancer.
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Affiliation(s)
- Jingyan Wei
- College of Pharmaceutical Science, Jilin University, 1266 Fujin Road, Changchun, 130021, China.
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21
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Angenendt P, Wilde J, Kijanka G, Baars S, Cahill DJ, Kreutzberger J, Lehrach H, Konthur Z, Glökler J. Seeing Better through a MIST: Evaluation of Monoclonal Recombinant Antibody Fragments on Microarrays. Anal Chem 2004; 76:2916-21. [PMID: 15144205 DOI: 10.1021/ac035357a] [Citation(s) in RCA: 37] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
Automation is the key approach for genomewide and proteomewide screening of function and interaction. Especially for proteomics, antibody microarrays are a useful tool for massive parallel profiling of complex samples. To meet the requirements of antibody microarrays and to obtain a great variety of antibodies, new technologies such as phage display have partly replaced the classical hybridoma method. While the selection process for phage-displayed antibody fragments itself has been automated, the bottleneck was shifted further downstream to the identification of monoclonal binders obtained from the selections. Here, we present a new approach to reduce time, material, and waste to extend automation beyond the selection process by application of conventional microarray machinery. We were able to express recombinant antibody fragments in a single inoculation and expression step and subjected them without purification directly to an automated high-throughput screening procedure based on the multiple spotting technique (MIST). While obtaining comparable sensitivities to enzyme-linked immunosorbent assays, we minimized manual interaction steps and streamlined the technique to be accessible within the automated selection procedure.
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Affiliation(s)
- Philipp Angenendt
- Max Planck Institute for Molecular Genetics, Ihnestrasse 73, 14195 Berlin, Germany. angenend@ molgen.mpg.de
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22
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Huang C, Yu H, Wang Q, Ma W, Xia D, Yi P, Zhang L, Cao X. Potent antitumor effect elicited by superantigen-linked tumor cells transduced with heat shock protein 70 gene. Cancer Sci 2004; 95:160-167. [PMID: 14965367 PMCID: PMC11159597 DOI: 10.1111/j.1349-7006.2004.tb03198.x] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2003] [Revised: 11/27/2003] [Accepted: 11/28/2003] [Indexed: 11/26/2022] Open
Abstract
Heat shock proteins (HSP) induce antitumor-specific immunity via a unique mechanism, but HSP alone fails to produce a satisfactory antitumor efficacy. We considered that the potent immune-activation of superantigen (SAg) might assist HSP to elicit a strong tumor-antigen-specific immunity. We initially prepared B16 melanoma cells linked to SAg SEA via a fusion protein with a transmembrane sequence (TM), and demonstrated that SEA thus anchored on the tumor cell surface could elicit strong antitumor immunity. We then prepared cells transduced with an inducible heat shock protein 70 (HSP70) gene, and bearing SEA-TM fusion protein on the cell surface, and used these cells as a dual-modified vaccine. In this study, either in a therapeutic setting or in a pre-immune model, the SEA-anchored vaccine or the HSP70 gene-modified vaccine induced marked tumor suppression, prolonged survival, augmented lymphocyte proliferation and higher NK and CTL activity in C57BL/6 mice compared with their controls (P < 0.01), though they were less effective than the dual-modified vaccine. Among these vaccines, the dual-modified vaccine showed the best therapeutic efficacy in B16 melanoma-bearing mice and gave the greatest protection against wild-type B16 melanoma challenge. The results indicated that the dual-modified vaccine could induce a potent tumor-antigen-specific immune response in addition to an increase of non-specific immunity. This study offers a novel approach to bridging specific and non-specific immunity for cancer therapy.
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Affiliation(s)
- Changxin Huang
- Institute of Immunology, Zhejiang University School of Medicine, Hangzhou, P. R. China
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23
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Hjortland GO, Garman-Vik SS, Juell S, Olsen OE, Hirschberg H, Fodstad O, Engebraaten O. Immunotoxin treatment targeted to the high-molecular-weight melanoma-associated antigen prolonging the survival of immunodeficient rats with invasive intracranial human glioblastoma multiforme. J Neurosurg 2004; 100:320-7. [PMID: 15086240 DOI: 10.3171/jns.2004.100.2.0320] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
OBJECT The aim of this study was to target immunotoxin treatment to the high-molecular-weight melanoma-associated antigen (HMW-MAA) and thereby examine any changes in the survival of immunodeficient rats with human glioblastoma multiforme (GBM). METHODS To target treatment specifically to human glioma cells, Pseudomonas exotoxin A (PE) was conjugated to the 9.2.27 antibody, which recognizes the HMW-MAA. Treatment of the antigen-positive glioma cell line U87MG with the resulting 9.2.27-PE caused cytotoxicity with a median inhibitory concentration of 1 ng/ml. Intratumoral 9.2.27-PE treatment of intracranial U87MG tumors in nude rats prolonged the survival of these animals by 43% compared with controls. In additional studies on the use of this targeted treatment, the authors precultured freshly dissected glioblastoma multiforme (GBM) biopsy tissue for 1 to 2 weeks. Inoculation of this tissue into the rat brain resulted in diffuse infiltrative gliomas. The markers glial fibrillary acidic protein and S100 protein were found to be expressed in the original biopsy specimens, as well as in the glioma xenografts in nude rat brains. Intratumoral immunotoxin treatment of such established tumors with 9.2.27-PE was effective and prolonged survival time from 30% to as high as 90% in animals with tumors originating from four different GBM specimens. CONCLUSIONS Targeted treatment of highly invasive GBMs proved effective, and these results emphasize the clinical relevance of this antigen as a target molecule for immunotoxin treatment of human GBMs.
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Affiliation(s)
- Geir Olav Hjortland
- Department of Tumor Biology, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, Oslo, Norway.
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Berry JD, Popkov M, Gubbins M, Mandeville R. Recent Innovations and Analytical Applications of Phage Display Libraries. ANAL LETT 2003. [DOI: 10.1081/al-120026568] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/03/2022]
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Nettelbeck DM, Rivera AA, Kupsch J, Dieckmann D, Douglas JT, Kontermann RE, Alemany R, Curiel DT. Retargeting of adenoviral infection to melanoma: combining genetic ablation of native tropism with a recombinant bispecific single-chain diabody (scDb) adapter that binds to fiber knob and HMWMAA. Int J Cancer 2003; 108:136-45. [PMID: 14618628 DOI: 10.1002/ijc.11563] [Citation(s) in RCA: 63] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
Gene therapy is an emerging and promising modality for the treatment of malignant melanoma and other neoplasms for which conventional therapies are inadequate. Various therapeutic genes have shown promise for tumor cell killing. However, successful gene therapy depends on the development of efficient and targeted gene transfer vectors. Here we describe a novel strategy for targeting of adenovirus-mediated gene transfer to melanoma cells. This strategy combines genetic ablation of native adenoviral tropism with redirected viral binding to melanoma cells via a bispecific adapter molecule, a bacterially expressed single-chain diabody, scDb MelAd, that binds to both the adenoviral fiber protein and to the high molecular weight melanoma-associated antigen (HMWMAA). This antigen is widely and specifically expressed on the surface of melanoma cells and its expression is associated with tumor development and progression. Our results showed specific and strong binding of the anti-HMWMAA scFv RAFT3 and the bispecific adapter scDb MelAd to melanoma cells. In adenoviral infection experiments, we demonstrated i) substantially (>50-fold) reduced infectivity of capsid mutant adenoviruses, ii) restored (up to 367-fold increase), CAR-independent and HMWMAA-mediated infectivity of these mutant viruses by scDb MelAd specifically in melanoma cells, and iii) higher levels of transgene expression in melanoma cells by fiber mutant virus complexed with scDbMelAd, relative to a vector with wild-type fibers. We confirmed the utility of this targeting strategy with human primary melanoma cells that represent clinically relevant substrates. These experiments established that the retargeting strategy mediates up to 54-fold increased adenoviral gene transfer to CAR-negative melanoma cells compared to the vector with native tropism. Hence, the HMWMAA-targeted adenoviral vector lacking native tropism exhibits both enhanced specificity and augmented infectivity of gene transfer to melanoma cells, suggesting that it is feasible to use this vector to improve gene therapy for malignant melanoma.
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Affiliation(s)
- Dirk M Nettelbeck
- Division of Human Gene Therapy, Department of Medicine, and the Gene Therapy Center, University of Alabama at Birmingham, Birmingham, AL, USA.
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Erlandsson E, Andersson K, Cavallin A, Nilsson A, Larsson-Lorek U, Niss U, Sjöberg A, Wallén-Ohman M, Antonsson P, Walse B, Forsberg G. Identification of the antigenic epitopes in staphylococcal enterotoxins A and E and design of a superantigen for human cancer therapy. J Mol Biol 2003; 333:893-905. [PMID: 14583188 DOI: 10.1016/j.jmb.2003.09.009] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Monoclonal antibodies have a potential for cancer therapy that may be further improved by linking them to effector molecules such as superantigens. Tumor targeting of a superantigen leads to a powerful T cell attack against the tumour tissue. Encouraging results have been observed preclinically and in patients using the superantigen staphylococcal enterotoxin A, SEA. To further improve the concept, we have reduced the reactivity to antibodies against superantigens, which is found in all individuals. Using epitope mapping, antibody binding sites in SEA and SEE were found around their MHC class II binding sites. These epitopes were removed genetically and a large number of synthetic superantigens were produced in an iterative engineering procedure. Properties such as decreased binding to anti-SEA as well as higher selectivity to induce killing of tumour cells compared to MHC class II expressing cells, were sequentially improved. The lysine residues 79, 81, 83 and 84 are all part of major antigenic epitopes, Gln204, Lys74, Asp75 and Asn78 are important for optimal killing of tumour cells while Asp45 affects binding to MHC class II. The production properties were optimised by further engineering and a novel synthetic superantigen, SEA/E-120, was designed. It is recognised by approximately 15% of human anti-SEA antibodies and have more potent tumour cell killing properties than SEA. SEA/E-120 is likely to have a low toxicity due to its reduced capacity to mediate killing of MHC class II expressing cells. It is produced as a Fab fusion protein at approximately 35 mg/l in Escherichia coli.
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Affiliation(s)
- Eva Erlandsson
- Active Biotech Research AB, Box 724, 220 07 Lund, Sweden
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Thamm DH, Kurzman ID, Macewen EG, Feinmehl R, Towell TL, Longhofer SL, Johnson CM, Geoly FJ, Stinchcomb DT. Intralesional lipid-complexed cytokine/superantigen immunogene therapy for spontaneous canine tumors. Cancer Immunol Immunother 2003; 52:473-80. [PMID: 12768328 PMCID: PMC11032875 DOI: 10.1007/s00262-003-0387-6] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2002] [Accepted: 02/05/2003] [Indexed: 11/29/2022]
Abstract
These studies sought to determine the gene expression and short-term effects of intralesional lipid-complexed immunogene therapy with constructs encoding Staphylococcus aureus enterotoxin A and canine interleukin-2 (L-SEA/cIL-2) in dogs with tumors of various histotypes, and then to assess the safety and efficacy of repeated L-SEA/cIL-2 injections in dogs with spontaneous soft tissue sarcomas (STS). In the first study, pet dogs with a variety of tumors received a single intralesional injection of L-SEA/cIL-2, and surgical excision was performed 48 h later. In the second study, dogs with histologically confirmed STS were treated weekly for a maximum of 12 weeks with escalating doses of L-SEA/cIL-2. Tumors were then surgically excised and assessed histologically and immunohistochemically. Overall, treatments were well tolerated, with no dose-limiting toxicities encountered. At 48 h, in the single injection study, plasmid DNA was detected in 14 of 16 tumor samples, and plasmid-specific mRNA was detected in 3 of 14. In the multiple injection study, the overall response rate in dogs with STS was 25%, consisting of 3 complete responses (CR) and 1 partial response (PR). Diffuse lymphoplasmacytic inflammation was observed in all tumors from patients experiencing CR or PR, whereas these changes were not evident in tumors from nonresponders. The infiltrate was composed primarily of CD3(+) cells at 48 h from the single-injection study, and was composed of both CD3(+) and CD79a(+) cells at 12 weeks in responding dogs from the multiple-injection study. In conclusion, these studies suggests that intralesional L-SEA/cIL-2 immunotherapy is well tolerated, results in detectable transgene expression in canine tumors, and has antitumor activity in dogs with spontaneous STS.
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Affiliation(s)
- Douglas H Thamm
- Department of Medical Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, 2015 Linden Drive West, WI 53706, Madison, USA.
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Abstract
In the past two decades, the worldwide steadily rising incidence of melanoma, its dismal prognosis when locally advanced or metastatic, and the absence of clinically effective therapeutic options have prompted studies that generated extensive preclinical knowledge on the biology of melanoma cells and their interaction with the host's immune system. As a consequence, among solid tumors, melanoma represents a "model malignancy" to design and apply in the clinic new bioimmunotherapeutic approaches, that are eventually translated to solid tumors of different histotypes. Despite its waxing and waning appeal as a therapeutic strategy, antibody treatment still represents a promising clinical approach to melanoma. Europe is no exception in the clinical interest for antibodies as therapeutic tools in melanoma patients; European researchers have also focused on preclinical issues that may improve current antibody-based therapeutic approaches on the one hand, while providing novel clinical strategies on the other hand.
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Affiliation(s)
- Maresa Altomonte
- Cancer Bioimmunotherapy Unit, Department of Medical Oncology, Centro di Riferimento Oncologico, Istituto di Ricovero e Cura a Carattere Scientifico, Aviano, Italy
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Abstract
Phage display is a molecular diversity technology that allows the presentation of large peptide and protein libraries on the surface of filamentous phage. Phage display libraries permit the selection of peptides and proteins, including antibodies, with high affinity and specificity for almost any target. A crucial advantage of this technology is the direct link that exists between the experimental phenotype and its encapsulated genotype, which allows the evolution of the selected binders into optimized molecules. Phage display facilitates engineering of antibodies with regard to their size, valency, affinity, and effector functions. The selection of antibodies and peptides from libraries displayed on the surface of filamentous phage has proven significant for routine isolation of peptides and antibodies for diagnostic and therapeutic applications. This review serves as an introduction to phage display, antibody engineering, the development of phage-displayed peptides and antibody fragments into viable diagnostic reagents, and recent trends in display technology.
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Affiliation(s)
- Hassan M E Azzazy
- Department of Pathology, University of Maryland School of Medicine, Baltimore, 21201, USA.
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Nie YZ, He FT, Li ZK, Wu KC, Cao YX, Chen BJ, Fan DM. Identification of tumor associated single-chain Fv by panning and screening antibody phage library using tumor cells. World J Gastroenterol 2002; 8:619-23. [PMID: 12174367 PMCID: PMC4656309 DOI: 10.3748/wjg.v8.i4.619] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To study the feasibility of panning and screening phage-displaying recombinant single-chain variable fragment (ScFv) of anti-tumor monoclonal antibodies for fixed whole cells as the carriers of mAb-binding antigens.
METHODS: The recombinant phage displaying libraries for anti-colorectal tumor mAb MC3Ab, MC5Ab and anti-gastric tumor mAb MGD1 was constructed. Panning and screening were carried out by means of modified fixation of colorectal and gastric tumor cells expressed the mAb-binding antigens. Concordance of binding specificity to tumor cells between phage clones and parent antibodies was analyzed. The phage of positive clones was identified with competitive ELISA, and infected by E. coli HB2151 to express soluble ScFv.
RESULTS: The ratio of positive clones to MC3-ScF-MC5-ScFv and MGD1-ScFv were 60%, 24% and 30%. MC3-ScFv had Mr 32000 confirmed by Western blot. The specificity to antigen had no difference between 4 positive recombinant phage antibodies and MC3Ab.
CONCLUSION: The modified process of fixing whole tumor cells is efficient, convenient and feasible to pan and screen the phage-displaying ScFv of anti-tumor monoclonal antibodies.
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Affiliation(s)
- Yong-Zhan Nie
- Institute of Digestive Diseases,Xijing Hospital, Fourth Military Medical University, Changle West Road, Xi'an 710032,Shannxi Province China
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Ilbäck NG, Persson R, Gunnarsson K, Stålhandske T. A new screening model for safety evaluation of superantigen-antibody recombinant fusion proteins (mAb Fab-SEA/E) using telemetric monitoring in conscious rabbits. J Pharmacol Toxicol Methods 2002; 48:31-9. [PMID: 12750039 DOI: 10.1016/s1056-8719(03)00006-6] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022]
Abstract
INTRODUCTION Carcinoma recognising monoclonal antibodies (mAb) and mutated forms of the T-cell-activating bacterial staphylococcal enterotoxin A/E (SEA/E) have been combined in single hybrid constructs (mAb Fab-SEA/E). By introducing substitutions in an MHC class II binding site, these harmful toxins can be converted into tolerable immunotoxins. Rabbits and humans are sensitive to SE toxins, and cardiovascular effects in rabbits are similar to those seen in septic shock in man. A new screening model using telemetry in conscious rabbits was applied in the safety evaluation of different mAb Fab-SEA/E constructs administered intravenously. METHODS Telemetry transmitters were implanted in the peritoneal cavity of animals with a pressure catheter in the aorta and electrodes for ECG recording subcutaneously following administration of mAb Fab-SEA/E constructs intravenously. RESULTS The responses in body temperature, heart rate, and blood pressure varied depending on the treatment regimen and the mutations of the drug given. For example, 25 micro g/kg of C215 Fab-SEAmut9 were given as a first treatment cycle on days 1, 5, and 7 and as a second treatment cycle on days 13-15. The first dose induced high fever, whereas the second and third doses induced fever responses more rapidly and were of lower and shorter duration. The second treatment cycle, starting on day 13, did not induce any responses probably due to anti-SEA antibodies formed because of the treatment. Another construct, 5T4 Fab-SEA/E-11 at 50 micro g/kg, induced a similar response as C215 Fab-SEAmut9 on days 1, 5, and 7. In this case, the pharmacologic response was still present on days 13-15, though no clinical signs developed or no formation of anti-SEA antibodies occurred. When 50 micro g/kg of 5T4 Fab-SEA/E-11 was administered once daily for 4 days, body temperature after the first dose increased slowly during the first 24 h, whereas the second to fourth doses induced more rapid and higher responses. The fourth dose of another compound, K305 Fab-SEA/E-11 (50 micro g/kg), induced an even more pronounced response both in magnitude and in duration as well as in adverse clinical signs. DISCUSSION By using continuous telemetric registration in the rabbit as a tool in superantigen-antibody (mAb Fab-SEA/E) drug selection, it has been possible to evaluate the dynamics of drug-induced immune effects (fever) and concomitant engagement of the cardiovascular system, conditions that are essential before clinical trials can be initiated.
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Shinohara N, Fukuda H. Isolation of monoclonal antibodies recognizing rare and dominant epitopes in plant vascular cell walls by phage display subtraction. J Immunol Methods 2002; 264:187-94. [PMID: 12191521 DOI: 10.1016/s0022-1759(02)00088-1] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022]
Abstract
A combination of phage display antibody technology and a subtraction method provides a powerful tool for the isolation of novel biomarkers. However, the dilemma that high stringency screening often reduces the diversity in the subtracted phage antibody repertoires and that it is difficult to isolate phage antibody against rare epitopes remain. Therefore, we carefully monitored the occupancy of differentiation-specific clones in a phage antibody library through an enrichment process, and succeeded in isolating monoclonal antibodies against rare and dominant epitopes in plant vascular cell walls. We also report that clones with stop and frameshift mutations significantly survived the enrichment process, owing to noncanonical translation mechanisms.
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Affiliation(s)
- Naoki Shinohara
- Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo, Tokyo, Japan.
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Isolation of endotoxin-specific antibodies by selection of an single chain phage antibody library. Chin J Cancer Res 2002. [DOI: 10.1007/s11670-002-0026-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022] Open
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He FT, Nie YZ, Chen BJ, Qiao TD, Fan DM, Li RF, Kang YS, Zhang Y. Expression and identification of recombinant soluble single-chain variable fragment of monoclonal antibody MC3. World J Gastroenterol 2002; 8:258-62. [PMID: 11925603 PMCID: PMC4658362 DOI: 10.3748/wjg.v8.i2.258] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To generate soluble single chain variable fragments (ScFv) of monoclonal antibody MC3 recognizing colorectal and gastric carcinomas.
METHODS: mRNA was isolated from the hybridoma cell line producing MC3 and the DNAs encoding variable domains of heavy and light chains (VH and VL) of the antibody were amplified separately by RT-PCR and assembled into ScFv DNA with a linker DNA.The ScFv DNA was ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into E. coli TG1.The transformed cells were infected with M13KO7 helper phage to yield recombinant phages. After two rounds of panning with gastric carcinoma cell line AGS highly expressing MC3-binding antigen, the phage clones displaying ScFv fragments of the antibody were selected by ELISA. 4 phage clones showing strong signal in ELISA were used to infect E. coli HB2151 to express soluble ScFvs. The soluble ScFvs were identified by Dot blot and Western blot, and their antigen-binding activity was assayed by ELISA. The VH and VL DNAs of the ScFv DNA derived from phage clone 19 were sequenced.
RESULTS: The VH, VL and ScFv DNAs were about 340 bp, 320 bp and 750 bp respectively. After two rounds of panning to the recombinant phages, 18 antigen-positive phage clones were selected from 30 preselected phage clones by ELISA. All the soluble ScFvs derived from the 4 out of the 18 antigen-positive phage clones were about Mr32000 and concentrated in periplasmatic space under the given culture condition. The soluble ScFvs could bind the antigen, and they shared the same binding site with MC3. The sequences of the VH and VL DNAs of the MC3 ScFv showed that the variable antibody genes belonged to the IgG1 subgroup, κ-type.
CONCLUSION: The soluble ScFv of MC3 is successfully produced, which not only provides a possible novel targeting vehicle for in vivo and in vitro study on associated cancers, but also offers the anuibody a stable genetic source.
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Affiliation(s)
- Feng-Tian He
- Department of Biochemistry & Molecular Biology, Third Military Medical University, Chongqing 400038, China.
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O'Brien PM, Maxwell G, Campo MS. Bacterial expression and purification of recombinant bovine Fab fragments. Protein Expr Purif 2002; 24:43-50. [PMID: 11812221 DOI: 10.1006/prep.2001.1534] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
We have previously described a recombinant phagemid expression vector, pComBov, designed for the production of native sequence bovine monoclonal antibodies (mAb) generated by antibody phage display. Bovine mAb Fab fragments isolated from libraries constructed using pComBov in Escherichia coli strain XL1-Blue, which is routinely used for antibodies expressed on the surface of phage, were expressed at very low yields. Therefore, a study was undertaken to determine optimal growth conditions for maximal expression of bovine Fab fragments in E. coli. By varying the E. coli strain, and the temperature and length of the culture growth, we were able to substantially increase the yield of soluble Fab fragments. A high yield of Fab fragments was found in the culture growth medium, which enabled us to devise a rapid and simple single-step method for the purification of native (nondenatured) Fabs based on immobilized metal affinity chromatography against a six-histidine amino acid carboxyl-terminal extension of the heavy-chain constant region. Using these methods we were able to express and purify antigen-specific bovine Fab fragments from E. coli.
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Affiliation(s)
- Philippa M O'Brien
- Department of Veterinary Pathology, University of Glasgow, Bearsden, Glasgow, Scotland, United Kingdom.
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Stausbøl-Grøn B, Jensen KB, Jensen KH, Jensen MØ, Clark BF. De novo
identification of cell-type specific antibody-antigen pairs by phage display subtraction. ACTA ACUST UNITED AC 2001; 268:3099-107. [PMID: 11358530 DOI: 10.1046/j.1432-1327.2001.02210.x] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
The aim of this study was to identify novel antibodies directed against cytosolic keratinocyte-specific antigens from a phage display antibody repertoire by using phage display subtraction. Phage display is a method of displaying foreign molecules on the surface of filamentous bacteriophage particles. It allows the interaction between two cognate molecules to be analysed through affinity selections. Recently, large repertoires of phage displayed human antibody fragments have been constructed. From such repertoires, antibodies can be obtained in vitro without the need for immunization or the hybridoma technology. A novel subtractive strategy for selecting antibodies from phage libraries was applied. Phage antibodies were selected against immobilized crude lysates of cultured human keratinocytes, the target antigens being unknown beforehand. A competing cell lysate was used to reduce retrieval of phage antibodies with specificities to commonly non-differentially expressed antigens. A monoclonal single chain fragment variable (scFv) with specificity for crude lysates of cultured human keratinocytes was identified as demonstrated by ELISA assays and immunoblotting analysis. The cognate keratinocyte antigen was shown to be keratin 14 (K14) by using immunoblotting based on 2D PAGE and a corresponding 2D PAGE protein database. In accordance with the expected tissue localization of K14, the identified scFv stained the basal layer of human epidermis by indirect immunofluorescence analysis. Starting with crude cell lysates, phage display subtraction in combination with 2D PAGE and 2D PAGE protein databases can be used to identify antibody-antigen pairs that characterize a specific cell type.
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Affiliation(s)
- B Stausbøl-Grøn
- Department of Dermatology, Marselisborg Hospital, University of Aarhus, Denmark
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Abstract
We have isolated several organ- and tumor-homing peptides by using in vivo phage display. This technology involves the screening of peptide libraries in a living animal. The peptides that result from such a selection home to specific organs or tissues because they recognize molecular 'addresses', receptors that are differentially expressed in vascular beds. Targeted delivery of chemotherapeutics, pro-apoptotic peptides and cytokines to tumors using these peptides improved therapeutic efficacy in animal models. Translation of this technology into clinical applications will form the basis for targeting therapeutic and imaging agents in the context of cancer and other diseases.
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Affiliation(s)
- M Kolonin
- The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Box 427, 77030-4095, Houston, TX, USA
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Yang LJ, Sui YF, Chen ZN. Preparation and activity of conjugate of monoclonal antibody HAb18 against hepatoma F(ab')(2) fragment and staphylococcal enterotoxin A. World J Gastroenterol 2001; 7:216-21. [PMID: 11819763 PMCID: PMC4723525 DOI: 10.3748/wjg.v7.i2.216] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/15/2000] [Revised: 11/28/2000] [Accepted: 11/30/2000] [Indexed: 02/06/2023] Open
Abstract
AIM To prepare the conjugate of staphylococcal enterotoxin A (SEA) protein which is a bacterial SAg and the F(ab')(2) fragment of mAb HAb18 against human hepatocellular carcinoma (HCC), and identify its activity in order to use SAg in the targeting therapy of HCC. METHODS MAb HAb18 was extracted from the abdominal dropsy of Balb/c mice, and was purified through chromatography column SP 40HR with Fast protein liquid chromatography (FPLC) system. The F(ab')(2) fragment of mAb HAb18 was prepared by papainic digestion method. The conjugate of mAb HAb18 F(ab')(2) fragment and SEA was prepared with chemical conjugating reagent N succinimidyl 3 (2-pyridyldithio) propionate (SPDP) and purified through chromatography column Superose 12 with FPLC system. The molecular mass and purity of each collected peak were identified with SDS-PAGE assay. The protein content was assayed by Lowry's method. The antibody activity of HAb18 F(ab')(2) against HCC in the conjugate was identified by indirect immunocytochemical ABC method, and the activity of SEA in the conjugate to activate peripheral blood mononuclear cells (PBMC) was identified with MTT assay. RESULTS The IgG mAb HAb18 was extracted, and purified successfully. Immunocytochemical staining demonstrated that it reacted with most of HHCC cells of human HCC cell line. There were two peaks in the process of purification of the prepared HAb18 F(ab')(2) SEA conjugate. SDS-PAGE assay demonstrated that the molecular mass of the first peak was about 130 ku, and the second peak was the mixture of about 45 ku and a little 100 ku proteins. The immunocytochemical staining was similar in HAb18 F(ab') (2) SEA conjugate and HAb18 F(ab') (2), i.e.the cytoplasm and/or cell membranes of most HHCC cells were positively stained. The MTT assay showed that the optical absorbance (A) value at 490 nm of HAb18 F(ab') (2) SEA conjugate was 0.182 +/- 0.012, that of negative control was 0.033 +/- 0.009, and there was significant difference between them (P < 0.05). CONCLUSION SPDP is a good protein conjugating reagent and can be used in preparing protein conjugate. The conjugate of mAb HAb18 F(ab') (2) fragment and SEA protein was prepared successfully in present study and can be used in the experimental study of HCC targeting therapy with the conjugate of SAg and anti HCC mAbs or their fragments.
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Affiliation(s)
- L J Yang
- Department of Pathology, Fourth Military Medical University, Xi'an 710032, Shaanxi Province,China.
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Roovers RC, van der Linden E, de Bruïne AP, Arends JW, Hoogenboom HR. Identification of colon tumour-associated antigens by phage antibody selections on primary colorectal carcinoma. Eur J Cancer 2001; 37:542-9. [PMID: 11267865 DOI: 10.1016/s0959-8049(00)00432-9] [Citation(s) in RCA: 20] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023]
Abstract
Immunotargeting of solid tumours using antibodies has become a valuable tool for the detection of cancer metastases and the treatment of minimal residual disease. However, only few tumour antigens useful for targeting have been described to date. To identify cell-surface targets on colorectal carcinoma (CRC), we selected a large, human phage antibody repertoire on freshly isolated colon tumour cells. Two antibodies were identified that reacted with epithelial cell-restricted cell-surface antigens, whereas one clone preferentially reacted with stromal cells. These antigens are tumour-associated antigens, as shown by their uniform expression in tumours of different patients and of different differentiation stages and by their limited expression on normal tissues. The pattern of reactivity in immunohistochemistry (IHC) and enzyme-linked immunosorbent assay (ELISA) suggests that these antigens are different from previously identified tumour-associated antigens (e.g. Ep-CAM or c-ERB-2). This phage antibody-based method may lead to the cloning of novel tumour antigens that are useful for the immunotargeting of solid tumours.
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Affiliation(s)
- R C Roovers
- Department of Pathology, University of Maastricht, Maastricht, The Netherlands
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