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1
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Goldberg BS, Ackerman ME. Underappreciated layers of antibody-mediated immune synapse architecture and dynamics. mBio 2025; 16:e0190024. [PMID: 39660921 PMCID: PMC11708040 DOI: 10.1128/mbio.01900-24] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2024] Open
Abstract
The biologic activities of antibody drugs are dictated by structure-function relationships-emerging from the kind, composition, and degree of interactions with a target antigen and with soluble and cellular antibody receptors of the innate immune system. These activities are canonically understood to be both modular: antigen recognition is driven by the heterodimeric antigen-binding fragment, and innate immune recruitment by the homodimeric constant/crystallizable fragment. The model that treats these domains with a high degree of independence has served the field well but is not without limitations. Here, we consider how new insights, particularly from structural studies, complicate the model of neat biophysical separation between these domains and shape our understanding of antibody effector functions. The emerging model endeavors to explain the phenotypic impact of both antibody intrinsic characteristics and extrinsic features-fitting them within a spatiotemporal paradigm that better accounts for observed antibody activities. In this review, we will use insights from recent models of classical complement complexes and T cell immune synapse formation to explore how structural differences in antibody-mediated immune synapses may relate to their functional diversity.
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Affiliation(s)
| | - Margaret E. Ackerman
- Thayer School of Engineering, Dartmouth College, Hanover, New Hampshire, USA
- Geisel School of Medicine, Dartmouth College, Hanover, New Hampshire, USA
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2
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Liao X, Qi T, Zhou J, Liu C, Cao Y. Optimizing Clinical Translation of Bispecific T-cell Engagers through Context Unification with a Quantitative Systems Pharmacology Model. Clin Pharmacol Ther 2024; 116:415-425. [PMID: 38751031 PMCID: PMC11251864 DOI: 10.1002/cpt.3302] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/04/2024] [Accepted: 04/30/2024] [Indexed: 07/17/2024]
Abstract
Bispecific T-cell engagers (bsTCEs) have emerged as a promising class of cancer immunotherapy. BsTCEs enable physical connections between T cells and tumor cells to enhance T-cell activity against cancer. Despite several marketing approvals, the development of bsTCEs remains challenging, especially at early clinical translational stages. The intricate design of bsTCEs makes their pharmacologic effects and safety profiles highly dependent on patient's immunological and tumor conditions. Such context-dependent pharmacology introduces considerable uncertainty into translational efforts. In this study, we developed a Quantitative Systems Pharmacology (QSP) model, through context unification, that can facilitate the translation of bsTCEs preclinical data into clinical activity. Through characterizing the formation dynamics of immunological synapse (IS) induced by bsTCEs, this model unifies a broad range of contexts related to target affinity, tumor characteristics, and immunological conditions. After rigorous calibration using both experimental and clinical data, the model enables consistent translation of drug potency observed under diverse experimental conditions into predictable exposure-response relationships in patients. Moreover, the model can help identify optimal target-binding affinities and minimum efficacious concentrations across different clinical contexts. This QSP approach holds significant promise for the future development of bsTCEs.
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Affiliation(s)
- Xiaozhi Liao
- Division of Pharmacotherapy and Experimental Therapeutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, NC, USA
| | - Timothy Qi
- Division of Pharmacotherapy and Experimental Therapeutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, NC, USA
| | - Jiawei Zhou
- Division of Pharmacotherapy and Experimental Therapeutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, NC, USA
| | - Can Liu
- Division of Pharmacotherapy and Experimental Therapeutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, NC, USA
| | - Yanguang Cao
- Division of Pharmacotherapy and Experimental Therapeutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, NC, USA
- Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
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3
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Zhang B, Shi J, Shi X, Xu X, Gao L, Li S, Liu M, Gao M, Jin S, Zhou J, Fan D, Wang F, Ji Z, Bian Z, Song Y, Tian W, Zheng Y, Xu L, Li W. Development and evaluation of a human CD47/HER2 bispecific antibody for Trastuzumab-resistant breast cancer immunotherapy. Drug Resist Updat 2024; 74:101068. [PMID: 38402670 DOI: 10.1016/j.drup.2024.101068] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2023] [Revised: 01/28/2024] [Accepted: 02/10/2024] [Indexed: 02/27/2024]
Abstract
The treatment for trastuzumab-resistant breast cancer (BC) remains a challenge in clinical settings. It was known that CD47 is preferentially upregulated in HER2+ BC cells, which is correlated with drug resistance to trastuzumab. Here, we developed a novel anti-CD47/HER2 bispecific antibody (BsAb) against trastuzumab-resistant BC, named IMM2902. IMM2902 demonstrated high binding affinity, blocking activity, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and internalization degradation effects against both trastuzumab-sensitive and trastuzumab-resistant BC cells in vitro. The in vivo experimental data indicated that IMM2902 was more effective than their respective controls in inhibiting tumor growth in a trastuzumab-sensitive BT474 mouse model, a trastuzumab-resistant HCC1954 mouse model, two trastuzumab-resistant patient-derived xenograft (PDX) mouse models and a cord blood (CB)-humanized HCC1954 mouse model. Through spatial transcriptome assays, multiplex immunofluorescence (mIFC) and in vitro assays, our findings provided evidence that IMM2902 effectively stimulates macrophages to generate C-X-C motif chemokine ligand (CXCL) 9 and CXCL10, thereby facilitating the recruitment of T cells and NK cells to the tumor site. Moreover, IMM2902 demonstrated a high safety profile regarding anemia and non-specific cytokines release. Collectively, our results highlighted a novel therapeutic approach for the treatment of HER2+ BCs and this approach exhibits significant anti-tumor efficacy without causing off-target toxicity in trastuzumab-resistant BC cells.
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Affiliation(s)
- Binglei Zhang
- Department of Hematology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450052, China
| | - Jianxiang Shi
- Henan Institute of Medical and Pharmaceutical Sciences, Academy of Medical Science, Zhengzhou University, Zhengzhou, Henan 450052, China
| | - Xiaojing Shi
- Academy of Medical Sciences, Zhengzhou University, Zhengzhou, Henan 450052, China
| | - Xiaolu Xu
- Academy of Medical Sciences, Zhengzhou University, Zhengzhou, Henan 450052, China
| | - Le Gao
- Department of Hematology, The Affiliated Cancer Hospital of Zhengzhou University & Henan Cancer Hospital, Zhengzhou, Henan 450008, China; Academy of Medical Sciences, Zhengzhou University, Zhengzhou, Henan 450052, China
| | - Song Li
- ImmuneOnco Biopharmaceuticals (Shanghai) Inc, Shanghai 201203, China
| | - Mengmeng Liu
- Department of Hematology, The Affiliated Cancer Hospital of Zhengzhou University & Henan Cancer Hospital, Zhengzhou, Henan 450008, China; Academy of Medical Sciences, Zhengzhou University, Zhengzhou, Henan 450052, China
| | - Mengya Gao
- Department of Hematology, The Affiliated Cancer Hospital of Zhengzhou University & Henan Cancer Hospital, Zhengzhou, Henan 450008, China; Academy of Medical Sciences, Zhengzhou University, Zhengzhou, Henan 450052, China
| | - Shuiling Jin
- Department of Oncology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450052, China
| | - Jian Zhou
- Department of Hematology, The Affiliated Cancer Hospital of Zhengzhou University & Henan Cancer Hospital, Zhengzhou, Henan 450008, China
| | - Dandan Fan
- Henan Institute of Medical and Pharmaceutical Sciences, Academy of Medical Science, Zhengzhou University, Zhengzhou, Henan 450052, China
| | - Fang Wang
- Department of Hematology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450052, China
| | - Zhenyu Ji
- Henan Institute of Medical and Pharmaceutical Sciences, Academy of Medical Science, Zhengzhou University, Zhengzhou, Henan 450052, China
| | - Zhilei Bian
- Department of Hematology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450052, China
| | - Yongping Song
- Department of Hematology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450052, China
| | - Wenzhi Tian
- ImmuneOnco Biopharmaceuticals (Shanghai) Inc, Shanghai 201203, China
| | - Yichao Zheng
- School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, Henan 450008, China.
| | - Linping Xu
- Department of Research and Foreign Affairs, The Affiliated Cancer Hospital of Zhengzhou University & Henan Cancer Hospital, Zhengzhou 450008, China.
| | - Wei Li
- Department of Hematology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450052, China.
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4
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The Generation of Dual-Targeting Fusion Protein PD-L1/CD47 for the Inhibition of Triple-Negative Breast Cancer. Biomedicines 2022; 10:biomedicines10081843. [PMID: 36009390 PMCID: PMC9405206 DOI: 10.3390/biomedicines10081843] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/23/2022] [Revised: 07/19/2022] [Accepted: 07/21/2022] [Indexed: 11/16/2022] Open
Abstract
Triple-negative breast cancer (TNBC) is a highly aggressive subset of breast cancer with limited therapeutic options. However, its immune evasion mechanisms, characterized by the over-expression of the immune checkpoint molecules PD-L1 and CD47, can be targeted in order to facilitate cancer elimination by cells of innate and adaptive immunity. In this paper, we describe the design, preparation, and evaluation of three novel dual-targeting fusion proteins that were based on the structure frame of prototype IAB (innate and adaptive dependent bispecific fusion protein) and the “Orcutt-type IgG-scFv” molecular model. Three molecules with different spatial conformations were designed to improve antigen–antibody affinity by the addition of Ag–Ab binding sites from the variable region sequences of the anti-PD-L1 monoclonal antibody (mAb) atezolizumab and CV1, a high-affinity receptor of CD47. The results showed that the best-performing among the three proteins designed in this study was protein Pro3; its CV1 N-terminus and Fc domain C-terminus were not sterically hindered. Pro3 was better at boosting T cell proliferation and the engulfment of macrophages than the IAB prototype and, at the same time, retained a level of ADCC activity similar to that of IAB. Through improved design, the novel constructed dual-targeting immunomodulatory protein Pro3 was superior at activating the anti-tumor immune response and has thus shown potential for use in clinical applications.
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5
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Dillon PM, Tushir-Singh J, Lum LG. Bispecific antibodies for the treatment of breast cancer. Expert Opin Biol Ther 2021; 22:1017-1027. [PMID: 33896311 PMCID: PMC8576064 DOI: 10.1080/14712598.2021.1922665] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
INTRODUCTION There are more than two dozen bispecific antibodies (BsAbs) in development with a variety of designs which are relevant to breast cancer. The field of BsAbs for breast cancer includes agents that co-direct immune recognition of the cancer cell, target unique cancer antigens, and target the microenvironment. BsAbs are being developed for use as antibody-drug conjugates and as homing signals for engineered T-cells. AREAS COVERED This review covers potential targets for bispecific antibodies, agents in pre-clinical development, agents in clinical trials, combinatorial therapies, and future directions. EXPERT OPINION There is no BsAb approval expected for breast cancer in the near term, but late-stage trials are underway. Future BsAb roles in breast cancer are possible given unmet needs in estrogen receptor+ disease, residual disease, and de-escalating chemotherapy use. The HER2+ space shows hints of success for BsAbs, but is already crowded. Areas of unmet need still exist.
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Affiliation(s)
- Patrick M Dillon
- Division of Hematology/Oncology, University of Virginia Charlottesville, VA, USA
| | - Jogender Tushir-Singh
- Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, VA, USA
| | - Lawrence G Lum
- Division of Hematology/Oncology, University of Virginia Charlottesville, VA, USA
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6
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Swope N, Chung WK, Cao M, Motabar D, Liu D, Ahuja S, Handlogten M. Impact of enzymatic reduction on bivalent bispecific antibody fragmentation and loss of product purity upon reoxidation. Biotechnol Bioeng 2020; 117:1063-1071. [PMID: 31930476 PMCID: PMC10947566 DOI: 10.1002/bit.27264] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/03/2019] [Revised: 12/06/2019] [Accepted: 01/06/2020] [Indexed: 11/11/2022]
Abstract
Antibody disulfide bond (DSB) reduction during manufacturing processes is a widely observed phenomenon attributed to host cell reductases present in harvest cell culture fluid. Enzyme-induced antibody reduction leads to product fragments and aggregates that increase the impurity burden on the purification process. The impact of reduction on bivalent bispecific antibodies (BisAbs), which are increasingly entering the clinic, has yet to be investigated. We focused on the reduction and reoxidation properties of a homologous library of bivalent BisAb formats that possess additional single-chain Fv (scFv) fragments with engineered DSBs. Despite all BisAbs having similar susceptibilities to enzymatic reduction, fragmentation pathways were dependent on the scFv-fusion site. Reduced molecules were allowed to reoxidize with and without low pH viral inactivation treatment. Both reoxidation studies demonstrated that multiple, complex BisAb species formed as a result of DSB mispairing. Furthermore, aggregate levels increased for all molecules when no low pH treatment was applied. Combined, our results show that complex DSB mispairing occurs during downstream processes while aggregate formation is dependent on sample treatment. These results are applicable to other novel monoclonal antibody-like formats containing engineered DSBs, thus highlighting the need to prevent reduction of novel protein therapeutics to avoid diminished product quality during manufacturing.
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Affiliation(s)
- Nicole Swope
- Department of Chemistry, University of Virginia, Charlottesville, VA, USA
| | - Wai Keen Chung
- Purification Process Sciences, AstraZeneca, Gaithersburg, MD, USA
| | - Mingyan Cao
- Analytical Sciences, AstraZeneca, Gaithersburg, USA
| | - Dana Motabar
- Purification Process Sciences, AstraZeneca, Gaithersburg, MD, USA
| | - Dengfeng Liu
- Analytical Sciences, AstraZeneca, Gaithersburg, USA
| | - Sanjeev Ahuja
- Cell Culture and Fermentation Sciences, AstraZeneca, Gaithersburg, MD, USA
| | - Michael Handlogten
- Cell Culture and Fermentation Sciences, AstraZeneca, Gaithersburg, MD, USA
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7
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Zhao Y, Li Y, Wu X, Li L, Liu J, Wang Y, Liu Y, Li Q, Wang Z. Identification of anti-CD16a single domain antibodies and their application in bispecific antibodies. Cancer Biol Ther 2019; 21:72-80. [PMID: 31564196 DOI: 10.1080/15384047.2019.1665953] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
CD16a (FcγRIIIa) mediates the antibody dependent cellular cytotoxicity (ADCC) and is important for anti-tumor activities of many therapeutic antibodies. Bispecific antibody targeting natural killer (NK) cells has been studied for cancer therapy. In this work, anti-CD16a single-domain antibodies were identified from hCD16a immunized camel. Bispecific antibodies are then constructed by fusing these single domain antibodies with an anti-CEA single domain antibody. These bispecific antibodies can recruite NK cells to kill CEA-positive tumor cells, and inhibit tumor growth in vivo, suggesting that these anti-CD16a single domain antibodies are powerful tools to engaging NK cells for cancer therapy.
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Affiliation(s)
- Yining Zhao
- School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, China.,Center for Cellular & Structural Biology, Sun Yat-Sen University, Guangzhou, China
| | - Yumei Li
- School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, China.,Center for Cellular & Structural Biology, Sun Yat-Sen University, Guangzhou, China
| | - Xiaoqiong Wu
- School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, China.,Center for Cellular & Structural Biology, Sun Yat-Sen University, Guangzhou, China
| | - Li Li
- School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, China.,Center for Cellular & Structural Biology, Sun Yat-Sen University, Guangzhou, China
| | - Jiayu Liu
- School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, China.,Center for Cellular & Structural Biology, Sun Yat-Sen University, Guangzhou, China
| | - Yanlan Wang
- School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, China.,Center for Cellular & Structural Biology, Sun Yat-Sen University, Guangzhou, China
| | - Yue Liu
- School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, China.,Center for Cellular & Structural Biology, Sun Yat-Sen University, Guangzhou, China
| | - Qing Li
- School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, China.,Center for Cellular & Structural Biology, Sun Yat-Sen University, Guangzhou, China
| | - Zhong Wang
- School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, China.,Center for Cellular & Structural Biology, Sun Yat-Sen University, Guangzhou, China
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8
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Nayyar G, Chu Y, Cairo MS. Overcoming Resistance to Natural Killer Cell Based Immunotherapies for Solid Tumors. Front Oncol 2019; 9:51. [PMID: 30805309 PMCID: PMC6378304 DOI: 10.3389/fonc.2019.00051] [Citation(s) in RCA: 112] [Impact Index Per Article: 18.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2018] [Accepted: 01/18/2019] [Indexed: 12/22/2022] Open
Abstract
Despite advances in the diagnostic and therapeutic modalities, the prognosis of several solid tumor malignancies remains poor. Different factors associated with solid tumors including a varied genetic signature, complex molecular signaling pathways, defective cross talk between the tumor cells and immune cells, hypoxic and immunosuppressive effects of tumor microenvironment result in a treatment resistant and metastatic phenotype. Over the past several years, immunotherapy has emerged as an attractive therapeutic option against multiple malignancies. The unique ability of natural killer (NK) cells to target cancer cells without antigen specificity makes them an ideal candidate for use against solid tumors. However, the outcomes of adoptive NK cell infusions into patients with solid tumors have been disappointing. Extensive studies have been done to investigate different strategies to improve the NK cell function, trafficking and tumor targeting. Use of cytokines and cytokine analogs has been well described and utilized to enhance the proliferation, stimulation and persistence of NK cells. Other techniques like blocking the human leukocyte antigen-killer cell receptors (KIR) interactions with anti-KIR monoclonal antibodies, preventing CD16 receptor shedding, increasing the expression of activating NK cell receptors like NKG2D, and use of immunocytokines and immune checkpoint inhibitors can enhance NK cell mediated cytotoxicity. Using genetically modified NK cells with chimeric antigen receptors and bispecific and trispecific NK cell engagers, NK cells can be effectively redirected to the tumor cells improving their cytotoxic potential. In this review, we have described these strategies and highlighted the need to further optimize these strategies to improve the clinical outcome of NK cell based immunotherapy against solid tumors.
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Affiliation(s)
- Gaurav Nayyar
- Department of Pediatrics, New York Medical College, Valhalla, NY, United States
| | - Yaya Chu
- Department of Pediatrics, New York Medical College, Valhalla, NY, United States
| | - Mitchell S Cairo
- Department of Pediatrics, New York Medical College, Valhalla, NY, United States.,Department of Cell Biology & Anatomy, New York Medical College, Valhalla, NY, United States.,Department of Microbiology & Immunology, New York Medical College, Valhalla, NY, United States.,Department of Medicine, New York Medical College, Valhalla, NY, United States.,Department of Pathology, New York Medical College, Valhalla, NY, United States
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9
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Abstract
Bispecific antibodies have moved from being an academic curiosity with therapeutic promise to reality, with two molecules being currently commercialized (Hemlibra® and Blincyto®) and many more in clinical trials. The success of bispecific antibodies is mainly due to the continuously growing number of mechanisms of actions (MOA) they enable that are not accessible to monoclonal antibodies. One of the earliest MOA of bispecific antibodies and currently the one with the largest number of clinical trials is the redirecting of the cytotoxic activity of T-cells for oncology applications, now extending its use in infective diseases. The use of bispecific antibodies for crossing the blood-brain barrier is another important application because of its potential to advance the therapeutic options for neurological diseases. Another noteworthy application due to its growing trend is enabling a more tissue-specific delivery or activity of antibodies. The different molecular solutions to the initial hurdles that limited the development of bispecific antibodies have led to the current diverse set of bispecific or multispecific antibody formats that can be grouped into three main categories: IgG-like formats, antibody fragment-based formats, or appended IgG formats. The expanded applications of bispecific antibodies come at the price of additional challenges for clinical development. The rising complexity in their structure may increase the risk of immunogenicity and the multiple antigen specificity complicates the selection of relevant species for safety assessment.
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Affiliation(s)
- Bushra Husain
- Protein Chemistry Department, Genentech Inc., South San Francisco, CA, 94080, USA
| | - Diego Ellerman
- Protein Chemistry Department, Genentech Inc., South San Francisco, CA, 94080, USA.
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10
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Brinkmann U, Kontermann RE. The making of bispecific antibodies. MAbs 2017; 9:182-212. [PMID: 28071970 PMCID: PMC5297537 DOI: 10.1080/19420862.2016.1268307] [Citation(s) in RCA: 660] [Impact Index Per Article: 82.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/12/2016] [Revised: 11/18/2016] [Accepted: 11/29/2016] [Indexed: 12/12/2022] Open
Abstract
During the past two decades we have seen a phenomenal evolution of bispecific antibodies for therapeutic applications. The 'zoo' of bispecific antibodies is populated by many different species, comprising around 100 different formats, including small molecules composed solely of the antigen-binding sites of two antibodies, molecules with an IgG structure, and large complex molecules composed of different antigen-binding moieties often combined with dimerization modules. The application of sophisticated molecular design and genetic engineering has solved many of the technical problems associated with the formation of bispecific antibodies such as stability, solubility and other parameters that confer drug properties. These parameters may be summarized under the term 'developability'. In addition, different 'target product profiles', i.e., desired features of the bispecific antibody to be generated, mandates the need for access to a diverse panel of formats. These may vary in size, arrangement, valencies, flexibility and geometry of their binding modules, as well as in their distribution and pharmacokinetic properties. There is not 'one best format' for generating bispecific antibodies, and no single format is suitable for all, or even most of, the desired applications. Instead, the bispecific formats collectively serve as a valuable source of diversity that can be applied to the development of therapeutics for various indications. Here, a comprehensive overview of the different bispecific antibody formats is provided.
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Affiliation(s)
- Ulrich Brinkmann
- Roche Pharma Research and Early Development (pRED), Roche Innovation Center Munich, Im Nonnenwald, Penzberg, Germany
| | - Roland E. Kontermann
- Institute of Cell Biology and Immunology, University of Stuttgart, Allmandring, Stuttgart, Germany
- Stuttgart Research Center Systems Biology, University of Stuttgart, Nobelstraße, Stuttgart, Germany
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11
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Chen BM, Al-Aghbar MA, Lee CH, Chang TC, Su YC, Li YC, Chang SE, Chen CC, Chung TH, Liao YC, Lee CH, Roffler SR. The Affinity of Elongated Membrane-Tethered Ligands Determines Potency of T Cell Receptor Triggering. Front Immunol 2017; 8:793. [PMID: 28740495 PMCID: PMC5502409 DOI: 10.3389/fimmu.2017.00793] [Citation(s) in RCA: 24] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2017] [Accepted: 06/22/2017] [Indexed: 01/17/2023] Open
Abstract
T lymphocytes are important mediators of adoptive immunity but the mechanism of T cell receptor (TCR) triggering remains uncertain. The interspatial distance between engaged T cells and antigen-presenting cells (APCs) is believed to be important for topological rearrangement of membrane tyrosine phosphatases and initiation of TCR signaling. We investigated the relationship between ligand topology and affinity by generating a series of artificial APCs that express membrane-tethered anti-CD3 scFv with different affinities (OKT3, BC3, and 2C11) in addition to recombinant class I and II pMHC molecules. The dimensions of membrane-tethered anti-CD3 and pMHC molecules were progressively increased by insertion of different extracellular domains. In agreement with previous studies, elongation of pMHC molecules or low-affinity anti-CD3 scFv caused progressive loss of T cell activation. However, elongation of high-affinity ligands (BC3 and OKT3 scFv) did not abolish TCR phosphorylation and T cell activation. Mutation of key amino acids in OKT3 to reduce binding affinity to CD3 resulted in restoration of topological dependence on T cell activation. Our results show that high-affinity TCR ligands can effectively induce TCR triggering even at large interspatial distances between T cells and APCs.
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Affiliation(s)
- Bing-Mae Chen
- Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan
| | - Mohammad Ameen Al-Aghbar
- Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan.,Taiwan International Graduate Program in Molecular Medicine, National Yang-Ming University and Academia Sinica, Taipei, Taiwan.,Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei, Taiwan
| | - Chien-Hsin Lee
- Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan
| | - Tien-Ching Chang
- Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan.,Taiwan International Graduate Program in Molecular Medicine, National Yang-Ming University and Academia Sinica, Taipei, Taiwan.,Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei, Taiwan
| | - Yu-Cheng Su
- Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan
| | - Ya-Chen Li
- Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan
| | - Shih-En Chang
- Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan
| | - Chin-Chuan Chen
- Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan
| | - Tsai-Hua Chung
- Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan
| | - Yuan-Chun Liao
- Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan
| | - Chau-Hwang Lee
- Research Center for Applied Sciences, Academia Sinica, Taipei, Taiwan.,Institute of Biophotonics, National Yang-Ming University, Taipei, Taiwan.,Department of Physics, National Taiwan University, Taipei, Taiwan
| | - Steve R Roffler
- Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan.,Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan
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12
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Liu H, Pan Y, Meng S, Zhang W, Zhou F. Current treatment options of T cell-associated immunotherapy in multiple myeloma. Clin Exp Med 2017; 17:431-439. [PMID: 28120217 DOI: 10.1007/s10238-017-0450-9] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/12/2016] [Accepted: 12/15/2016] [Indexed: 11/29/2022]
Abstract
Multiple myeloma (MM) is a complex disease and is presently an incurable malignant plasma cell tumor. Although the introduction of proteasome inhibitor and the immunomodulators markedly improved the effect of myeloma therapy, most patients still suffer from relapse even with an initially effective therapy. Accumulating evidence suggests that immunotherapy is a promising option in treating MM. And T cell plays crucial role through inducing sustained immune response in vivo in the immunotherapy of tumors. In this article, we will discuss progress of several T cell-based immunotherapies with insight into how they eradicate myeloma cells and their disadvantages.
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Affiliation(s)
- Hailing Liu
- Department of Clinical Hematology, Second Affiliated Hospital, Medical School of Xi'an Jiaotong University, Xi'an, 710004, China
| | - Yunbao Pan
- Department of Pathology, Affiliated Hospital, Wuxi Medical School, Jiangnan University, Wuxi, 214062, Jiangsu, China
| | - Shan Meng
- Department of Clinical Hematology, Second Affiliated Hospital, Medical School of Xi'an Jiaotong University, Xi'an, 710004, China
| | - Wanggang Zhang
- Department of Clinical Hematology, Second Affiliated Hospital, Medical School of Xi'an Jiaotong University, Xi'an, 710004, China
| | - Fuling Zhou
- Department of Clinical Hematology, Second Affiliated Hospital, Medical School of Xi'an Jiaotong University, Xi'an, 710004, China. .,Department of Clinical Hematology, Zhongnan Hospital, Wuhan University, No. 169 Donghu Road, Wuchang District, Wuhan, 430071, Hubei, China.
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13
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Yao VJ, D'Angelo S, Butler KS, Theron C, Smith TL, Marchiò S, Gelovani JG, Sidman RL, Dobroff AS, Brinker CJ, Bradbury ARM, Arap W, Pasqualini R. Ligand-targeted theranostic nanomedicines against cancer. J Control Release 2016; 240:267-286. [PMID: 26772878 PMCID: PMC5444905 DOI: 10.1016/j.jconrel.2016.01.002] [Citation(s) in RCA: 125] [Impact Index Per Article: 13.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2015] [Revised: 12/17/2015] [Accepted: 01/02/2016] [Indexed: 02/06/2023]
Abstract
Nanomedicines have significant potential for cancer treatment. Although the majority of nanomedicines currently tested in clinical trials utilize simple, biocompatible liposome-based nanocarriers, their widespread use is limited by non-specificity and low target site concentration and thus, do not provide a substantial clinical advantage over conventional, systemic chemotherapy. In the past 20years, we have identified specific receptors expressed on the surfaces of tumor endothelial and perivascular cells, tumor cells, the extracellular matrix and stromal cells using combinatorial peptide libraries displayed on bacteriophage. These studies corroborate the notion that unique receptor proteins such as IL-11Rα, GRP78, EphA5, among others, are differentially overexpressed in tumors and present opportunities to deliver tumor-specific therapeutic drugs. By using peptides that bind to tumor-specific cell-surface receptors, therapeutic agents such as apoptotic peptides, suicide genes, imaging dyes or chemotherapeutics can be precisely and systemically delivered to reduce tumor growth in vivo, without harming healthy cells. Given the clinical applicability of peptide-based therapeutics, targeted delivery of nanocarriers loaded with therapeutic cargos seems plausible. We propose a modular design of a functionalized protocell in which a tumor-targeting moiety, such as a peptide or recombinant human antibody single chain variable fragment (scFv), is conjugated to a lipid bilayer surrounding a silica-based nanocarrier core containing a protected therapeutic cargo. The functionalized protocell can be tailored to a specific cancer subtype and treatment regimen by exchanging the tumor-targeting moiety and/or therapeutic cargo or used in combination to create unique, theranostic agents. In this review, we summarize the identification of tumor-specific receptors through combinatorial phage display technology and the use of antibody display selection to identify recombinant human scFvs against these tumor-specific receptors. We compare the characteristics of different types of simple and complex nanocarriers, and discuss potential types of therapeutic cargos and conjugation strategies. The modular design of functionalized protocells may improve the efficacy and safety of nanomedicines for future cancer therapy.
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Affiliation(s)
- Virginia J Yao
- University of New Mexico Comprehensive Cancer Center, Albuquerque, NM 87131; Division of Molecular Medicine, Department of Internal Medicine, University of New Mexico School of Medicine, Albuquerque, NM 87131
| | - Sara D'Angelo
- University of New Mexico Comprehensive Cancer Center, Albuquerque, NM 87131; Division of Molecular Medicine, Department of Internal Medicine, University of New Mexico School of Medicine, Albuquerque, NM 87131
| | - Kimberly S Butler
- Department of Chemical and Biological Engineering, University of New Mexico, Albuquerque, NM 87131
| | - Christophe Theron
- Department of Chemical and Biological Engineering, University of New Mexico, Albuquerque, NM 87131
| | - Tracey L Smith
- University of New Mexico Comprehensive Cancer Center, Albuquerque, NM 87131; Division of Molecular Medicine, Department of Internal Medicine, University of New Mexico School of Medicine, Albuquerque, NM 87131
| | - Serena Marchiò
- University of New Mexico Comprehensive Cancer Center, Albuquerque, NM 87131; Division of Molecular Medicine, Department of Internal Medicine, University of New Mexico School of Medicine, Albuquerque, NM 87131; Department of Oncology, University of Turin, Candiolo, 10060, Italy
| | - Juri G Gelovani
- Department of Biomedical Engineering, College of Engineering and School of Medicine, Wayne State University, Detroit, MI 48201
| | - Richard L Sidman
- Department of Neurology, Harvard Medical School, Beth Israel Deaconess Medical Center, Boston, MA 02215
| | - Andrey S Dobroff
- University of New Mexico Comprehensive Cancer Center, Albuquerque, NM 87131; Division of Molecular Medicine, Department of Internal Medicine, University of New Mexico School of Medicine, Albuquerque, NM 87131
| | - C Jeffrey Brinker
- Department of Chemical and Biological Engineering, University of New Mexico, Albuquerque, NM 87131; Center for Micro-Engineered Materials, University of New Mexico, Albuquerque, NM 87131; Cancer Research and Treatment Center, Department of Molecular Genetics and Microbiology, University of New Mexico Health Sciences Center, University of New Mexico, Albuquerque, NM 87131; Self-Assembled Materials Department, Sandia National Laboratories, Albuquerque, NM 87185
| | - Andrew R M Bradbury
- Bioscience Division, Los Alamos National Laboratories, Los Alamos, NM, 87545
| | - Wadih Arap
- University of New Mexico Comprehensive Cancer Center, Albuquerque, NM 87131; Division of Hematology/Oncology, Department of Internal Medicine, University of New Mexico School of Medicine, Albuquerque, NM 87131.
| | - Renata Pasqualini
- University of New Mexico Comprehensive Cancer Center, Albuquerque, NM 87131; Division of Molecular Medicine, Department of Internal Medicine, University of New Mexico School of Medicine, Albuquerque, NM 87131.
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14
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Abstract
PURPOSE OF REVIEW Adoptive T-cell therapy has become one of the most exciting fields of cancer therapy in the past few years. In this article, we describe a method which combines adoptive T-cell therapy with antibody therapy by arming T cells from cord blood, normal patients, and cancer patients with bispecific antibodies capable of binding to tumor-associated antigens on one side of the bispecific antibody construct and T cells on another side of the construct. This approach redirects T cells against tumor cells in a non-MHC-restricted manner. RECENT FINDINGS Various methods for manipulating the immune system including check-point inhibitors, chimeric antigen receptor T cells, and bispecific antibodies have shown promising activity in treating both hematological malignancies and solid tumors with excellent success. In recent studies, activated T cells armed with bispecific antibodies have shown good preclinical activity, safety, and promising efficacy in the clinical trials. SUMMARY Activated T cells armed with bispecific antibodies represent a promising treatment for cancer immunotherapy.
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15
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Asano R, Shimomura I, Konno S, Ito A, Masakari Y, Orimo R, Taki S, Arai K, Ogata H, Okada M, Furumoto S, Onitsuka M, Omasa T, Hayashi H, Katayose Y, Unno M, Kudo T, Umetsu M, Kumagai I. Rearranging the domain order of a diabody-based IgG-like bispecific antibody enhances its antitumor activity and improves its degradation resistance and pharmacokinetics. MAbs 2014; 6:1243-54. [PMID: 25517309 PMCID: PMC4623410 DOI: 10.4161/mabs.29445] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022] Open
Abstract
One approach to creating more beneficial therapeutic antibodies is to develop bispecific antibodies (bsAbs), particularly IgG-like formats with tetravalency, which may provide several advantages such as multivalent binding to each target antigen. Although the effects of configuration and antibody-fragment type on the function of IgG-like bsAbs have been studied, there have been only a few detailed studies of the influence of the variable fragment domain order. Here, we prepared four types of hEx3-scDb-Fc, IgG-like bsAbs, built from a single-chain hEx3-Db (humanized bispecific diabody [bsDb] that targets epidermal growth factor receptor and CD3), to investigate the influence of domain order and fusion manner on the function of a bsDb with an Fc fusion format. Higher cytotoxicities were observed with hEx3-scDb-Fcs with a variable light domain (VL)-variable heavy domain (VH) order (hEx3-scDb-Fc-LHs) compared with a VH-VL order, indicating that differences in the Fc fusion manner do not affect bsDb activity. In addition, flow cytometry suggested that the higher cytotoxicities of hEx3-scDb-Fc-LH may be attributable to structural superiority in cross-linking. Interestingly, enhanced degradation resistance and prolonged in vivo half-life were also observed with hEx3-scDb-Fc-LH. hEx3-scDb-Fc-LH and its IgG2 variant exhibited intense in vivo antitumor effects, suggesting that Fc-mediated effector functions are dispensable for effective anti-tumor activities, which may cause fewer side effects. Our results show that merely rearranging the domain order of IgG-like bsAbs can enhance not only their antitumor activity, but also their degradation resistance and in vivo half-life, and that hEx3-scDb-Fc-LHs are potent candidates for next-generation therapeutic antibodies.
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Key Words
- ADCC, antibody-dependent cell-mediated cytotoxicity
- AUC, area-under-the-curve
- CD3
- EGFR, epidermal growth factor receptor
- FITC-CD3ϵγ, fluorescein isothiocyanate-labeled CD3ϵγ; DVD-IgTM, dual variable domain immunoglobulin
- FITC-sEGFR, FITC-labeled sEGFR
- Fv, variable fragment
- ICR, imprinting control region
- IgG-like bispecific antibody
- MTS, 3-(4, 5-dimethylthiazole-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt
- PBMCs, peripheral blood mononuclear cells
- PBS, phosphate-buffered saline
- SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis
- SPR, surface plasmon resonance
- SUV, standardized uptake value
- T-LAK cells, lymphokine-activated killer cells with the T-cell phenotype
- VH, variable heavy domain
- VL, variable light domain
- antibody engineering
- bispecific diabody
- bsAb, bispecific antibody
- bsDb, bispecific diabody
- cancer immunotherapy
- effective domain order
- epidermal growth factor receptor
- sEGFR, soluble EGFR
- scDb, single-chain diabody
- scFv, single-chain Fv
- taFv, tandem scFv
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Affiliation(s)
- Ryutaro Asano
- a Department of Biomolecular Engineering ; Graduate School of Engineering; Tohoku University ; Sendai , Japan
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16
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Reusch U, Burkhardt C, Fucek I, Le Gall F, Le Gall M, Hoffmann K, Knackmuss SHJ, Kiprijanov S, Little M, Zhukovsky EA. A novel tetravalent bispecific TandAb (CD30/CD16A) efficiently recruits NK cells for the lysis of CD30+ tumor cells. MAbs 2014; 6:728-39. [PMID: 24670809 PMCID: PMC4011917 DOI: 10.4161/mabs.28591] [Citation(s) in RCA: 115] [Impact Index Per Article: 10.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022] Open
Abstract
To improve recruitment and activation of natural killer (NK) cells to lyse tumor cells, we isolated a human anti-CD16A antibody with similar affinity for the CD16A 158F/V allotypes, but no binding to the CD16B isoform. Using CD16A-targeting Fv domains, we constructed a tetravalent bispecific CD30/CD16A tandem diabody (TandAb®) consisting solely of Fv domains. This TandAb has two binding sites for CD16A and two for CD30, the antigen identifying Hodgkin lymphoma cells. The binding and cytotoxicity of the TandAb were compared with antibodies with identical anti-CD30 domains: (1) a native IgG, (2) an IgG optimized for binding to Fc receptors, and (3) a bivalent bispecific CD30/CD16A diabody. Due to its CD16A-bivalency and reduced koff, the TandAb was retained longer on the surface of NK cells than the IgGs or the diabody. This contributed to the higher potency and efficacy of the TandAb relative to those of the other anti-CD30 antibodies. TandAb cytotoxicity was independent of the CD16A allotype, whereas the anti-CD30 IgGs were substantially less cytotoxic when NK cells with low affinity CD16A allotype were employed. TandAb activation of NK cells was strictly dependent on the presence of CD30+ target cells. Therefore, the CD30/CD16A TandAb may represent a promising therapeutic for the treatment of Hodgkin’s lymphoma; further, anti-CD16A TandAbs may function as potent immunotherapeutics that specifically recruit NK cells to destroy cancer cells.
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Affiliation(s)
- Uwe Reusch
- Affimed Therapeutics AG; Heidelberg, Germany
| | | | - Ivica Fucek
- Affimed Therapeutics AG; Heidelberg, Germany
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17
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Bispecific small molecule-antibody conjugate targeting prostate cancer. Proc Natl Acad Sci U S A 2013; 110:17796-801. [PMID: 24127589 DOI: 10.1073/pnas.1316026110] [Citation(s) in RCA: 69] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Bispecific antibodies, which simultaneously target CD3 on T cells and tumor-associated antigens to recruit cytotoxic T cells to cancer cells, are a promising new approach to the treatment of hormone-refractory prostate cancer. Here we report a site-specific, semisynthetic method for the production of bispecific antibody-like therapeutics in which a derivative of the prostate-specific membrane antigen-binding small molecule DUPA was selectively conjugated to a mutant αCD3 Fab containing the unnatural amino acid, p-acetylphenylalanine, at a defined site. Homogeneous conjugates were generated in excellent yields and had good solubility. The efficacy of the conjugate was optimized by modifying the linker structure, relative binding orientation, and stoichiometry of the ligand. The optimized conjugate showed potent and selective in vitro activity (EC50 ~ 100 pM), good serum half-life, and potent in vivo activity in prophylactic and treatment xenograft mouse models. This semisynthetic approach is likely to be applicable to the generation of additional bispecific agents using drug-like ligands selective for other cell-surface receptors.
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18
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A tale of two specificities: bispecific antibodies for therapeutic and diagnostic applications. Trends Biotechnol 2013; 31:621-32. [PMID: 24094861 PMCID: PMC7114091 DOI: 10.1016/j.tibtech.2013.08.007] [Citation(s) in RCA: 131] [Impact Index Per Article: 10.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2013] [Revised: 08/01/2013] [Accepted: 08/27/2013] [Indexed: 12/20/2022]
Abstract
Recombinant DNA technologies are leading the rapid expansion of bispecific antibody formats. The therapeutic potential of bispecific antibodies is being realized through creative design. Bispecific antibodies are potentially underutilized reagents for diagnostics. Artificial manipulation of antibody genes has facilitated the production of several unique recombinant antibody formats, which have highly important therapeutic and biotechnological applications. Although bispecific antibodies (bsAbs) are not new, they are coming to the forefront as our knowledge of the potential efficacy of antibody-based therapeutics expands. The next generation of bsAbs is developing due to significant improvements in recombinant antibody technologies. This review focuses on recent advances with a particular focus on improvements in format and design that are contributing to the resurgence of bsAbs, and in particular, on innovative structures applicable to next generation point-of-care (POC) devices with applicability to low resource environments.
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19
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Asano R, Kumagai T, Nagai K, Taki S, Shimomura I, Arai K, Ogata H, Okada M, Hayasaka F, Sanada H, Nakanishi T, Karvonen T, Hayashi H, Katayose Y, Unno M, Kudo T, Umetsu M, Kumagai I. Domain order of a bispecific diabody dramatically enhances its antitumor activity beyond structural format conversion: the case of the hEx3 diabody. Protein Eng Des Sel 2013; 26:359-67. [DOI: 10.1093/protein/gzt009] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/14/2022] Open
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20
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Cai Z, Fu T, Nagai Y, Lam L, Yee M, Zhu Z, Zhang H. scFv-based "Grababody" as a general strategy to improve recruitment of immune effector cells to antibody-targeted tumors. Cancer Res 2013; 73:2619-27. [PMID: 23396586 DOI: 10.1158/0008-5472.can-12-3920] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
Recruitment of immune cells to tumor cells targeted by a therapeutic antibody can heighten the antitumor efficacy of the antibody. For example, p185(her2/neu)-targeting antibodies not only downregulate the p185(her2/neu) kinase (ERBB2) but also trigger complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) through the antibody Fc region. Here, we describe a generalized strategy to improve immune cell recruitment to targeted cancer cells, using a modified scFv antibody we call a "Grababody" that binds the target protein and endogenous immunoglobulins. The model system we used to illustrate the use of this platform recognizes p185(her2/neu) and includes an IgG binding domain. The recombinant scFv Grababody that was created recruited circulating human IgGs and attracted immune cells carrying Fc receptors to tumor cells that expressed p185(her2/neu). The presence of the IgG binding domain significantly enhanced CDC and ADCC activity and improved antitumor activity in vivo. Our results illustrate a novel general approach to improve antibody-like proteins for therapeutic applications.
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Affiliation(s)
- Zheng Cai
- Department of Pathology and Lab Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA
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21
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Mohseni Nodehi S, Repp R, Kellner C, Bräutigam J, Staudinger M, Schub N, Peipp M, Gramatzki M, Humpe A. Enhanced ADCC activity of affinity maturated and Fc-engineered mini-antibodies directed against the AML stem cell antigen CD96. PLoS One 2012; 7:e42426. [PMID: 22879978 PMCID: PMC3411760 DOI: 10.1371/journal.pone.0042426] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2011] [Accepted: 07/09/2012] [Indexed: 12/23/2022] Open
Abstract
CD96, a cell surface antigen recently described to be preferentially expressed on acute myeloid leukemia (AML) leukemic stem cells (LSC) may represent an interesting target structure for the development of antibody-based therapeutic approaches. The v-regions from the CD96-specific hybridoma TH-111 were isolated and used to generate a CD96-specific single chain fragment of the variable regions (scFv). An affinity maturated variant resulting in 4-fold enhanced CD96-binding was generated by random mutagenesis and stringent selection using phage display. The affinity maturated scFv CD96-S32F was used to generate bivalent mini-antibodies by genetically fusing an IgG1 wild type Fc region or a variant with enhanced CD16a binding. Antibody dependent cell-mediated cytotoxicity (ADCC) experiments revealed that Fc engineering was essential to trigger significant effector cell-mediated lysis when the wild type scFv was used. The mini-antibody variant generated by fusing the affinity-maturated scFv with the optimized Fc variant demonstrated the highest ADCC activity (2.3-fold enhancement in efficacy). In conclusion, our data provide proof of concept that CD96 could serve as a target structure for effector cell-mediated lysis and demonstrate that both enhancing affinity for CD96 and for CD16a resulted in mini-antibodies with the highest cytolytic potential.
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MESH Headings
- Amino Acid Sequence
- Antibody Affinity/immunology
- Antibody Specificity/immunology
- Antibody-Dependent Cell Cytotoxicity/immunology
- Antigens, CD/chemistry
- Antigens, CD/immunology
- Antigens, Neoplasm/immunology
- Cell Line, Tumor
- Dose-Response Relationship, Immunologic
- Humans
- Hybridomas
- Immunoglobulin G/immunology
- Leukemia, Myeloid, Acute/immunology
- Leukemia, Myeloid, Acute/pathology
- Models, Molecular
- Molecular Sequence Data
- Mutant Proteins/chemistry
- Mutant Proteins/metabolism
- Mutation/genetics
- Neoplastic Stem Cells/immunology
- Protein Binding
- Protein Engineering
- Protein Structure, Tertiary
- Receptors, Fc/immunology
- Sequence Alignment
- Single-Chain Antibodies/immunology
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Affiliation(s)
- Sahar Mohseni Nodehi
- Division of Stem Cell Transplantation and Immunotherapy, Department of Medicine II, Zoological Institute, Christian-Albrechts-University, Kiel, Germany
| | - Roland Repp
- Division of Stem Cell Transplantation and Immunotherapy, Department of Medicine II, Zoological Institute, Christian-Albrechts-University, Kiel, Germany
| | - Christian Kellner
- Division of Stem Cell Transplantation and Immunotherapy, Department of Medicine II, Zoological Institute, Christian-Albrechts-University, Kiel, Germany
| | - Joachim Bräutigam
- Department of Structural Biology, Zoological Institute, Christian-Albrechts-University, Kiel, Germany
| | - Matthias Staudinger
- Division of Stem Cell Transplantation and Immunotherapy, Department of Medicine II, Zoological Institute, Christian-Albrechts-University, Kiel, Germany
| | - Natalie Schub
- Division of Stem Cell Transplantation and Immunotherapy, Department of Medicine II, Zoological Institute, Christian-Albrechts-University, Kiel, Germany
| | - Matthias Peipp
- Division of Stem Cell Transplantation and Immunotherapy, Department of Medicine II, Zoological Institute, Christian-Albrechts-University, Kiel, Germany
| | - Martin Gramatzki
- Division of Stem Cell Transplantation and Immunotherapy, Department of Medicine II, Zoological Institute, Christian-Albrechts-University, Kiel, Germany
| | - Andreas Humpe
- Division of Stem Cell Transplantation and Immunotherapy, Department of Medicine II, Zoological Institute, Christian-Albrechts-University, Kiel, Germany
- * E-mail:
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22
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Abstract
Bispecific antibodies (BiAbs) offer a unique opportunity to redirect immune effector cells to kill cancer cells. BiAbs combine the benefits of different binding specificities of two monoclonal antibodies (mAbs) into a single construct. This unique feature of BiAbs enables approaches that are not possible with single mAbs. Advances in antibody engineering and antigen profiling of malignant cells have led to the development of a number of BiAb formats and their combinations for redirecting effector cells to tumor targets. There have been significant advances in the design and application of BiAbs for intravenous and local injection.The initial barrier of cytokine storm has been partially overcome by more recent constructs that have improved clinical effectiveness without dose-limiting toxicities. Since the recent revival of BiAbs, there has been multiple, ongoing, phase I/II and III trials, and some promising clinical outcomes have been reported in completed clinical studies. This review focuses on arming T cells with BiAbs to create the 'poor man's cytotoxic lymphocyte'.
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Affiliation(s)
- Lawrence G Lum
- Department of Oncology, Wayne State University and Barbara Ann Karmanos Cancer Center, Detroit, MI, USA
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23
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Glorius P, Baerenwaldt A, Kellner C, Staudinger M, Dechant M, Stauch M, Beurskens FJ, Parren PWHI, Winkel JGJVD, Valerius T, Humpe A, Repp R, Gramatzki M, Nimmerjahn F, Peipp M. The novel tribody [(CD20)2xCD16] efficiently triggers effector cell-mediated lysis of malignant B cells. Leukemia 2012; 27:190-201. [DOI: 10.1038/leu.2012.150] [Citation(s) in RCA: 43] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
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Abstract
The advent of HER2-directed therapies has significantly improved the outlook for patients with HER2-positive early stage breast cancer. However, a significant proportion of these patients still relapse and die of breast cancer. Trials to define, refine and optimize the use of the two approved HER2-targeted agents (trastuzumab and lapatinib) in patients with HER2-positive early stage breast cancer are ongoing. In addition, promising new approaches are being developed including monoclonal antibodies and small-molecule tyrosine kinase inhibitors targeting HER2 or other HER family members, antibodies linked to cytotoxic moieties or modified to improve their immunological function, immunostimulatory peptides, and targeting the PI3K and IGF-1R pathways. Improved understanding of the HER2 signaling pathway, its relationship with other signaling pathways and mechanisms of resistance has also led to the development of rational combination therapies and to a greater insight into treatment response in patients with HER2-positive breast cancer. Based on promising results with new agents in HER2-positive advanced-stage disease, a series of large trials in the adjuvant and neoadjuvant settings are planned or ongoing. This Review focuses on current treatment for patients with HER2-positive breast cancer and aims to update practicing clinicians on likely future developments in the treatment for this disease according to ongoing clinical trials and translational research.
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25
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Bagley RG, Rouleau C, Weber W, Mehraein K, Smale R, Curiel M, Callahan M, Roy A, Boutin P, St Martin T, Nacht M, Teicher BA. Tumor endothelial marker 7 (TEM-7): a novel target for antiangiogenic therapy. Microvasc Res 2011; 82:253-62. [PMID: 21958527 DOI: 10.1016/j.mvr.2011.09.004] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2011] [Revised: 08/10/2011] [Accepted: 09/09/2011] [Indexed: 12/25/2022]
Abstract
Antiangiogenesis has been validated as a therapeutic strategy to treat cancer, however, a need remains to identify new targets and therapies for specific diseases and to improve clinical benefit from antiangiogenic agents. Tumor endothelial marker 7 (TEM-7) was investigated as a possible target for therapeutic antiangiogenic intervention in cancer. TEM-7 expression was assessed by in situ hybridization or by immunohistochemistry (IHC) in 130 formalin-fixed paraffin-embedded (FFPE) and 410 frozen human clinical specimens of cancer plus 301 normal tissue samples. In vitro TEM-7 expression was evaluated in 4 human endothelial cell models and in 32 human cancer cell lines by RT-PCR and flow cytometry. An anti-TEM-7 antibody was tested in vitro on human SKOV3 ovarian and MDA-MB-231 breast carcinoma cells that expressed TEM-7 in antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis assays. In frozen tumor tissues, TEM-7 mRNA and protein was detected in all but one of the cancer types tested and was infrequently expressed in normal frozen tissues. In FFPE tumor tissues, TEM-7 protein was detected by IHC in colon, breast, lung, bladder, ovarian and endometrial cancers and in sarcomas. TEM-7 protein was not detected in head and neck, prostate or liver cancers. TEM-7 expression was restricted to the vasculature and was absent from tumor cells. In vitro, TEM-7 was not detected in human microvascular endothelial cells (HMVEC) or human umbilical vein endothelial cells (HUVEC) but was induced in endothelial precursor/progenitor cells (EPC) in the presence of the mitogen phorbol ester PMA. An anti-TEM-7 antibody mediated ADCC and phagocytosis in SKOV3 and MDA-MB-231 cell lines infected with an adenovirus expressing TEM-7. These data demonstrate that TEM-7 is a vascular protein associated with angiogenic states. TEM-7 is a novel and attractive target for antiangiogenic therapy.
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Affiliation(s)
- Rebecca G Bagley
- Genzyme Corporation, 49 New York Ave., Framingham, MA 01701, USA.
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26
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Villa A, Lovato V, Bujak E, Wulhfard S, Pasche N, Neri D. A novel synthetic naïve human antibody library allows the isolation of antibodies against a new epitope of oncofetal fibronectin. MAbs 2011; 3:264-72. [PMID: 21487243 DOI: 10.4161/mabs.3.3.15616] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022] Open
Abstract
Human monoclonal antibodies (mAbs) can routinely be isolated from phage display libraries against virtually any protein available in sufficient purity and quantity, but library design can influence epitope coverage on the target antigen. Here we describe the construction of a novel synthetic human antibody phage display library that incorporates hydrophilic or charged residues at position 52 of the CDR2 loop of the variable heavy chain domain, instead of the serine residue found in the corresponding germline gene. The novel library was used to isolate human mAbs to various antigens, including the alternatively-spliced EDA domain of fibronectin, a marker of tumor angiogenesis. In particular, the mAb 2H7 was proven to bind to a novel epitope on EDA, which does not overlap with the one recognized by the clinical-stage F8 antibody. F8 and 2H7 were used for the construction of chelating recombinant antibodies (CRAbs), whose tumor-targeting properties were assessed in vivo in biodistribution studies in mice bearing F9 teratocarcinoma, revealing a preferential accumulation at the tumor site.
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27
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Schoonooghe S, Burvenich I, Vervoort L, De Vos F, Mertens N, Grooten J. PH1-derived bivalent bibodies and trivalent tribodies bind differentially to shed and tumour cell-associated MUC1. Protein Eng Des Sel 2010; 23:721-8. [PMID: 20616115 DOI: 10.1093/protein/gzq044] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
Most adenocarcinomas express altered MUC1 as a tumour-associated antigen. Due to suboptimal glycosylation in tumour-associated MUC1, the apomucin core is exposed, revealing new epitopes for antibody-directed immunotherapy. The human PH1 Fab binds specifically to this MUC1 apomucin. We describe the engineering and functional characterization of bi- and trivalent recombinant antibody derivatives from the PH1 Fab. Bi- and tribodies were made using the disulfide-stabilized Fab fragment as a heterodimerization scaffold with PH1 single-chain variable fragments fused to either one or both Fab-chain C-termini. Immunoassays revealed 27- and 165-fold improved dissociation constants (K(D) = 30 and 5 nM) of the PH1 bi- and tribodies compared with the parental Fab (K(D) = 820 nM). Unexpectedly, major differences were seen in the ability of the antibody constructs to bind shed and tumour cell-tethered MUC1. While the tribody did not discriminate between both MUC1 forms, the bibody demonstrated preferential interaction with membrane-bound MUC1 compared with shed MUC1. This preferential recognition of membrane-bound MUC1, along with the high serum stability of the bibody, its intermediate size and efficient internalization by MUC1(+) cells, makes the human PH1-derived bibody a valuable candidate as a cancer-targeting therapeutic.
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Affiliation(s)
- Steve Schoonooghe
- Molecular Immunology Laboratory, Department of Molecular Biology, Ghent University, Technologiepark 927, B-9052 Zwijnaarde, Belgium
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Orcutt KD, Ackerman ME, Cieslewicz M, Quiroz E, Slusarczyk AL, Frangioni JV, Wittrup KD. A modular IgG-scFv bispecific antibody topology. Protein Eng Des Sel 2009; 23:221-8. [PMID: 20019028 DOI: 10.1093/protein/gzp077] [Citation(s) in RCA: 74] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/14/2022] Open
Abstract
Here we present a bispecific antibody (bsAb) format in which a disulfide-stabilized scFv is fused to the C-terminus of the light chain of an IgG to create an IgG-scFv bifunctional antibody. When expressed in mammalian cells and purified by one-step protein A chromatography, the bsAb retains parental affinities of each binding domain, exhibits IgG-like stability and demonstrates in vivo IgG-like tumor targeting and blood clearance. The extension of the C-terminus of the light chain of an IgG with an scFv or even a smaller peptide does appear to disrupt disulfide bond formation between the light and heavy chains; however, this does not appear to affect binding, stability or in vivo properties of the IgG. Thus, we demonstrate here that the light chain of an IgG can be extended with an scFv without affecting IgG function and stability. This format serves as a standardized platform for the construction of functional bsAbs.
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Affiliation(s)
- Kelly Davis Orcutt
- Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
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A novel CD19-directed recombinant bispecific antibody derivative with enhanced immune effector functions for human leukemic cells. J Immunother 2009; 31:871-84. [PMID: 18833000 DOI: 10.1097/cji.0b013e318186c8b4] [Citation(s) in RCA: 51] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
A novel bispecific antibody-derived recombinant protein targeting leukemias and lymphomas was designed, a single-chain Fv triple body (sctb) consisting of 1 polypeptide chain with 3 scFvs connected in tandem. The distal scFvs were specific for the tumor antigen CD19, and the central scFv for the trigger molecule CD16 (FcgammaRIII) on natural killer (NK) cells and macrophages. We had previously built a disulphide stabilized (ds) bsscFv [19 x 16] with monovalent binding for CD19 from ds components. The sctb ds[19 x 16 x 19] also used ds components and displayed 3-fold greater avidity for CD19 than the bsscFv (KD = 13 vs. 42 nM), whereas both had equal affinity for CD16 (KD = 58 nM). Plasma half-lives in mice were 4 and 2 hours for the sctb and the bsscFv, respectively. In antibody-dependent cellular cytotoxicity reactions with human mononuclear cells as effectors, the sctb promoted equal lysis of leukemic cell lines and primary cells from leukemia and lymphoma patients at 10-fold to 40-fold lower concentrations than the bsscFv. This new format may also be applicable to a variety of other tumor antigens and effector molecules. With half-maximum effective concentrations (EC50) in the low picomolar range, the sctb ds[19 x 16 x 19] is an attractive candidate for further preclinical evaluation.
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Ko K, Brodzik R, Steplewski Z. Production of Antibodies in Plants: Approaches and Perspectives. Curr Top Microbiol Immunol 2009; 332:55-78. [DOI: 10.1007/978-3-540-70868-1_4] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/24/2023]
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Lum LG, Al-Kadhimi Z. Development and prospects for bispecific antibody-based therapeutics in cancer and other applications. Expert Opin Drug Discov 2008; 3:1081-97. [PMID: 23506181 DOI: 10.1517/17460441.3.9.1081] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
BACKGROUND Progress in molecular biology for the production of bispecific antibodies (BiAbs) and the expanding knowledge on receptors on malignant and normal target cells provide the basis for developing new strategies using antibody- and/or receptor-based platform technology for the treatment of cancer and other diseases. OBJECTIVE This review provides a preclinical and clinical perspective on application of bispecific antibodies for the treatment of solid tumors, hematologic malignancies and other diseases. METHODS This review focuses on BiAb-based immunotherapy, clinical trials, alternative strategies, the challenges of technology and future applications. RESULTS/CONCLUSION The successful application of a particular BiAb will depend on a thorough evaluation of the expected functional application and thoughtful engineering of structure, affinity and number of binding sites based on the desired function.
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Affiliation(s)
- Lawrence G Lum
- Professor of Medicine and Professor of Immunology and Microbiology, Scientific Director of BMT and Immunotherapy Program, Wayne State University, Member Barbara Ann Karmanos Cancer Institute, Hudson-Webber Cancer Research Center, 4100 John R., 7th Floor, Rm 740.1, Detroit, Michigan, MI 48201, USA +1 313 576 8326 ; +1 313 576 8939 ;
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Turatti F, Figini M, Balladore E, Alberti P, Casalini P, Marks JD, Canevari S, Mezzanzanica D. Redirected activity of human antitumor chimeric immune receptors is governed by antigen and receptor expression levels and affinity of interaction. J Immunother 2007; 30:684-93. [PMID: 17893561 DOI: 10.1097/cji.0b013e3180de5d90] [Citation(s) in RCA: 65] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
Novel Ab-based immunotherapeutic strategies have exploited T-cell receptor-like chimeric immune receptors (CIR) expressed on the surface of transduced human peripheral blood mononuclear cell (PBMC) to redirect potent non-major histocompatibility complex-dependent cytotoxicity to tumor cells expressing a tumor-associated antigens. We transduced human PBMC with 2 fully human CIRs that trigger through the zeta-chain of CD3 and contain either one of two human scFv specific for the same epitope on the extracellular domain of HER2 but with distinctly different affinities (KD 1616 and 1 nM) for this antigen. Potent direct CIR-mediated killing and in vitro tumor growth inhibition mediated by transduced PBMC were observed against targets expressing different levels of HER2. High-affinity CIR showed stronger ability to bind Ag and retain binding than low-affinity CIR. When lytic potential of the 2 CIRs was evaluated, their efficiency was comparable under conditions of high CIR and Ag expression, whereas low-affinity CIR was more efficient than high-affinity CIR in conditions of limiting Ag and CIR expression levels. When tumor growth inhibition was evaluated, Ag and CIR levels, rather than CIR affinity appeared relevant. Ag-driven CIR activation resulted in the production of soluble factors mediating efficient bystander effect. By carefully defining CIR surface expression and increasing affinity for a specific target antigen, it may be possible to selectively exclude CIR-mediated activity against targets expressing low levels of antigen, as normal cells. On the contrary, low antigen-expressing tumor variants could be eliminated by decreasing CIR affinity. Tuning CIR expression and affinity might help in discriminating different biologic contexts.
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MESH Headings
- Antibody Affinity
- Antigens, Neoplasm/immunology
- Cell Line
- Cell Line, Tumor
- Cytotoxicity, Immunologic
- Humans
- Immunoglobulin Variable Region/immunology
- Leukocytes, Mononuclear/immunology
- Receptor, ErbB-2/genetics
- Receptor, ErbB-2/immunology
- Receptors, Immunologic/genetics
- Receptors, Immunologic/immunology
- Recombinant Fusion Proteins/genetics
- Recombinant Fusion Proteins/immunology
- T-Lymphocytes, Cytotoxic/immunology
- Transduction, Genetic
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Affiliation(s)
- Fabio Turatti
- Molecular Therapies Unit, Department of Experimental Oncology, Istituto Nazionale Tumori, Milan, Italy
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Tang Y, Lou J, Alpaugh RK, Robinson MK, Marks JD, Weiner LM. Regulation of antibody-dependent cellular cytotoxicity by IgG intrinsic and apparent affinity for target antigen. THE JOURNAL OF IMMUNOLOGY 2007; 179:2815-23. [PMID: 17709495 DOI: 10.4049/jimmunol.179.5.2815] [Citation(s) in RCA: 95] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/28/2023]
Abstract
Unconjugated mAbs have emerged as useful cancer therapeutics. Ab-dependent cellular cytotoxicity (ADCC) is believed to be a major antitumor mechanism of some anticancer Abs. However, the factors that regulate the magnitude of ADCC are incompletely understood. In this study, we described the relationship between Ab affinity and ADCC. A series of human IgG1 isotype Abs was created from the anti-HER2/neu (also named c-erbB2) C6.5 single-chain Fv (scFv) and its affinity mutants. The scFv affinities range from 10(-7) to 10(-11) M, and the IgG Abs retain the affinities of the scFv from which they were derived. The apparent affinity of the Abs ranged from nearly 10(-10) M (the lowest affinity variant) to almost 10(-11) M (the other variants). The IgG molecules were tested for their ability to elicit ADCC in vitro against three tumor cell lines with differing levels of HER2/neu expression using unactivated human PBMC from healthy donors as the effector cells. The results demonstrated that both the apparent affinity and intrinsic affinity of the Abs studied regulate ADCC. High-affinity tumor Ag binding by the IgGs led to the most efficient and powerful ADCC. Tumor cells expressing high levels of HER2/neu are more susceptible to the ADCC triggered by Abs than the cells expressing lower amounts of HER2/neu. These findings justify the examination of high affinity Abs for ADCC promotion. Because high affinity may impair in vivo tumor targeting, a careful examination of Ab structure to function relationships is required to develop optimized therapeutic unconjugated Abs.
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Affiliation(s)
- Yong Tang
- Department of Medical Oncology, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA
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34
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Weiner LM. Building better magic bullets--improving unconjugated monoclonal antibody therapy for cancer. Nat Rev Cancer 2007; 7:701-6. [PMID: 17721434 DOI: 10.1038/nrc2209] [Citation(s) in RCA: 56] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/10/2023]
Abstract
The potential of monoclonal antibodies to effectively treat cancer is beginning to be widely acknowledged. Advances in antibody engineering make it possible to produce various recombinant proteins that exploit the specificity of the antibody-combining site to manipulate tumour-related signalling, and to stimulate anti-tumour immune responses. Future advances in the field will rely on the improved identification of functional antibody targets to perturb cancer-relevant signalling, and by the improved selection of tumours that can be effectively treated. These advances will be complemented by the use of antibodies that induce clinically meaningful host-protective immune responses. But, can we afford this progress?
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Affiliation(s)
- Louis M Weiner
- Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19,111, USA.
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35
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Capobianco JA, Shih WY, Shih WH. 3-mercaptopropyltrimethoxysilane as insulating coating and surface for protein immobilization for piezoelectric microcantilever sensors. THE REVIEW OF SCIENTIFIC INSTRUMENTS 2007; 78:046106. [PMID: 17477697 DOI: 10.1063/1.2727466] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/15/2023]
Abstract
We have examined coating (PbMg(13)Nb(23)O(3))(0.63)-(PbTiO(3))(0.37) (PMN-PT)/tin and lead zirconate titanate (PZT)/glass piezoelectric microcantilever sensor (PEMS) with 3-mercaptopropyl-trimethoxysilane (MPS) by a simple solution method to electrically insulate the PEMS for in-water applications. In contrast to earlier methytrimethoxysilane insulation coating, the MPS coating also facilitated receptor immobilization on the sensor surface via bonding of its sulhydryl group to a bifunctional linker, sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate. We showed that a MPS coating of 21 nm in thickness is sufficient to electrically insulate and provide immobilization surface to the PEMS for in-liquid electrical self-excitation and self-sensing. The in-phosphate buffered saline solution resonance spectra were stable with Q values ranging from 41 to 55. The mass detection sensitivities were determined to be 5x10(-11) and 8x10(-12) gHz for the MPS-insulated PZT-glass and PMN-PT/tin PEMSs, respectively.
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Affiliation(s)
- Joseph A Capobianco
- Department of Materials Science and Engineering, Drexel University, Philadelphia, PA 19104, USA.
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36
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Nielsen UB, Kirpotin DB, Pickering EM, Drummond DC, Marks JD. A novel assay for monitoring internalization of nanocarrier coupled antibodies. BMC Immunol 2006; 7:24. [PMID: 17014727 PMCID: PMC1633733 DOI: 10.1186/1471-2172-7-24] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/06/2006] [Accepted: 10/02/2006] [Indexed: 01/16/2023] Open
Abstract
Background Discovery of tumor-selective antibodies or antibody fragments is a promising approach for delivering therapeutic agents to antigen over-expressing cancers. Therefore it is important to develop methods for the identification of target- and function specific antibodies for effective drug delivery. Here we describe a highly selective and sensitive method for characterizing the internalizing potential of multivalently displayed antibodies or ligands conjugated to liposomes into tumor cells. The assay requires minute amounts of histidine-tagged ligand and relies on the non-covalent coupling of these antibodies to fluorescent liposomes containing a metal ion-chelating lipid. Following incubation of cells with antibody-conjugated liposomes, surface bound liposomes are gently removed and the remaining internalized liposomes are quantitated based on fluorescence in a high throughput manner. We have termed this methodology "Chelated Ligand Internalization Assay", or CLIA. Results The specificity of the assay was demonstrated with different antibodies to the ErbB-2 and EGF receptors. Antibody-uptake correlated with receptor expression levels in tumor cell lines with a range of receptor expression. Furthermore, Ni-NTA liposomes containing doxorubicin were used to screen for the ability of antibodies to confer target-specific cytotoxicity. Using an anti-ErbB2 single chain Fv (scFv) (F5) antibody, cytotoxicity could be conferred to ErbB2-overexpressing cells; however, a poly(ethylene glycol)-linked lipid (DSPE-PEG-NTA-Ni) was necessary to allow for efficient loading of the drug and to reduce nonspecific drug leakage during the course of the assay. Conclusion The CLIA method we describe here represents a rapid, sensitive and robust assay for the identification and characterization of tumor-specific antibodies capable of high drug-delivery efficiency when conjugated to liposomal nanocarriers.
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Affiliation(s)
- Ulrik B Nielsen
- Departments of Anesthesia and Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA 94110 (UBN, JDM, EMP), USA
- Merrimack Pharmaceuticals, Inc., One Kendall Square, Cambridge, MA 02139, USA
| | | | - Edward M Pickering
- Departments of Anesthesia and Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA 94110 (UBN, JDM, EMP), USA
| | - Daryl C Drummond
- Hermes Biosciences, South San Francisco, CA 94080 (DCD, DK), USA
| | - James D Marks
- Departments of Anesthesia and Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA 94110 (UBN, JDM, EMP), USA
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Germain C, Larbouret C, Cesson V, Donda A, Held W, Mach JP, Pèlegrin A, Robert B. MHC class I-related chain A conjugated to antitumor antibodies can sensitize tumor cells to specific lysis by natural killer cells. Clin Cancer Res 2006; 11:7516-22. [PMID: 16243826 DOI: 10.1158/1078-0432.ccr-05-0872] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
PURPOSE As a first step for the development of a new cancer immunotherapy strategy, we evaluated whether antibody-mediated coating by MHC class I-related chain A (MICA) could sensitize tumor cells to lysis by natural killer (NK) cells. EXPERIMENTAL DESIGN Recombinant MICA (rMICA) was chemically conjugated to Fab' fragments from monoclonal antibodies specific for tumor-associated antigens, such as carcinoembryonic antigen, HER2, or CD20. RESULTS Flow cytometry analysis showed an efficient coating of MICA-negative human cancer cell lines with the Fab-rMICA conjugates. This was strictly dependent on the expression of the appropriate tumor-associated antigens in the target cells. Importantly, preincubation of the tumor cells with the appropriate Fab-rMICA conjugate resulted in NK cell-mediated tumor cell lysis. Antibody blocking of the NKG2D receptor in NK cells prevented conjugate-mediated tumor cell lysis. CONCLUSIONS These results open the way to the development of immunotherapy strategies based on antibody-mediated targeting of MICA.
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Affiliation(s)
- Claire Germain
- INSERM, EMI0227, Centre de Recherche en Cancérologie de Montpellier, Centre Régional de Lutte contre le Cancer Val d'Aurelle-Paul Lamarque, Montpellier, France
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Chowdhury PS, Wu H. Tailor-made antibody therapeutics. Methods 2005; 36:11-24. [PMID: 15848071 DOI: 10.1016/j.ymeth.2005.01.002] [Citation(s) in RCA: 51] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2004] [Revised: 01/17/2005] [Accepted: 01/17/2005] [Indexed: 01/16/2023] Open
Abstract
Therapeutic antibodies represent one of the fastest growing areas of the pharmaceutical industry. There are currently 18 monoclonal antibodies in the market that have been approved by the FDA and over 150 in clinical developments. Driven by innovation and technological developments, scientists have gone beyond the traditional antibody molecules. Antibodies have been engineered in a variety of ways to meet the challenges posed by different biological settings. Described in this review is an abridged account of the different ways antibodies have been tailored to make them efficient drug molecules.
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Affiliation(s)
- Partha S Chowdhury
- Department of Antibody Discovery and Protein Engineering, MedImmune, Inc., One MedImmune Way, Gaithersburg, MD 20878, USA.
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39
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Affiliation(s)
- Peter Kufer
- Micromet AG, Staffelseestrasse 2, 81477 Munich, Germany.
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40
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Abstract
Bispecific antibodies can serve as mediators to retarget effector mechanisms to disease-associated sites. Studies over the past two decades have revealed the potentials but also the limitations of conventional bispecific antibodies. The development of recombinant antibody formats has opened up the possibility of generating bispecific molecules with improved properties. This review summarizes recent developments in the field of recombinant bispecific antibodies and discusses further requirements for clinical development.
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Affiliation(s)
- Roland E Kontermann
- Institute of Cell Biology and Immunology, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany.
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41
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Lum LG, Davol PA. Retargeting T cells and immune effector cells with bispecific antibodies. CANCER CHEMOTHERAPY AND BIOLOGICAL RESPONSE MODIFIERS ANNUAL 2005; 22:273-91. [PMID: 16110617 DOI: 10.1016/s0921-4410(04)22013-0] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/01/2022]
Abstract
The development of BiAbs for therapeutic applications in cancer shows promise. As our understanding of effector cell receptor biology for triggering of cytotoxic functions improves and the behavior of TAA and the targeting antibody engagement is elucidated, customized BiAb reagents can be engineered to optimize in vivo or ex vivo arming of T cells for targeting tumors. Additionally, other variables that require consideration in the equation for successful T cell immunotherapy include: the type of effector cells, their state of activation, the type of effector receptor being activated or tareeted. the presence of Tregs, the affinity of the anti-effector cell antibody and the anti-TAA antibody, the type of BiAb (mouse, humanized, or human), the number of binding sites for the T cells or TAA, the presence or absence of decoy antigen, whether the TAA modulates after being engaged by antibody, the type of tumor, the tumor burden, and last, but not least, the amount of 'immunologic' space available for the adoptively transferred cells to expand and function.
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Affiliation(s)
- Lawrence G Lum
- Immunotherapy Program, Adele R. Deof Cancer Center, Roger Williams Hospital, Providence, RI 02908, USA.
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Shahied LS, Tang Y, Alpaugh RK, Somer R, Greenspon D, Weiner LM. Bispecific Minibodies Targeting HER2/neu and CD16 Exhibit Improved Tumor Lysis When Placed in a Divalent Tumor Antigen Binding Format. J Biol Chem 2004; 279:53907-14. [PMID: 15471859 DOI: 10.1074/jbc.m407888200] [Citation(s) in RCA: 85] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
Unconjugated monoclonal antibodies have emerged as important therapeutic agents for selected malignancies. One mechanism by which antibodies can exert cytotoxic effects is antibody-dependent cellular cytotoxicity (ADCC). In an effort to increase the efficiency of ADCC at tumor sites, we have focused on the construction of bispecific antibodies specific for the tumor antigen HER2/neu and the Fc gamma RIII-activating receptor (CD16) found on NK cells, mononuclear phagocytes, and neutrophils. Here, we describe the production of bispecific minibodies in two distinct binding formats. The parent minibody was constructed such that the IgG1 C(H)3 constant domain serves as the oligomerization domain and is attached to an anti-CD16 and an anti-HER2/ neu single-chain Fv via 19- and 29-amino acid linkers, respectively. This molecule can be expressed in mammalian cells from a dicistronic vector and has been purified using sequential affinity purification techniques. Analysis by surface plasmon resonance shows that the bispecific minibody can bind to HER2/neu and CD16, both individually and simultaneously. Furthermore, cytotoxicity studies show that the minibody can induce significant tumor cell lysis at a concentration as low as 20 nm. A trimeric, bispecific minibody (TriBi) that binds dimerically to HER2/neu and monomerically to CD16 induces equivalent cytotoxicity at lower antibody concentrations than either the parent minibody or the corresponding single-chain dimer. Both minibody constructs are stable in mouse and human serum for up to 72 h at 37 degrees C. These minibodies have the potential to target solid tumors and promote tumor lysis by natural killer cells and mononuclear phagocytes.
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MESH Headings
- Animals
- Antibodies, Bispecific/administration & dosage
- Antibodies, Bispecific/genetics
- Antibodies, Bispecific/immunology
- Antibodies, Monoclonal/administration & dosage
- Antibodies, Monoclonal/genetics
- Antibodies, Monoclonal/immunology
- Antibody Specificity
- Antibody-Dependent Cell Cytotoxicity
- Antigens, Neoplasm/immunology
- Binding Sites, Antibody
- Blood
- COS Cells
- Chlorocebus aethiops
- Cloning, Molecular
- Drug Stability
- Embryo, Mammalian
- Female
- Gene Expression
- Humans
- Immunoglobulin G/chemistry
- Immunoglobulin G/immunology
- Kidney
- Mice
- Neoplasms/immunology
- Ovarian Neoplasms
- Receptor, ErbB-2/immunology
- Receptors, IgG/immunology
- Transfection
- Tumor Cells, Cultured
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Affiliation(s)
- Lillian S Shahied
- Department of Medical Oncology, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA
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Zhang J, Liu YF, Yang SJ, Qiao Q, Cheng H, Zhang CS, Ma FC, Guo HZ. Primary targeting of recombinant Fv-immunotoxin hscFv 25-mTNFα against hepatocellular carcinoma. World J Gastroenterol 2004; 10:1872-5. [PMID: 15222026 PMCID: PMC4572220 DOI: 10.3748/wjg.v10.i13.1872] [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: 12/15/2022] Open
Abstract
AIM: To obtain human recombinant Fv-immunotoxin hscFv25-mTNFα (mutant human TNFα fused to human scFv25) against hepatocellular carcinoma (HCC).
METHODS: Two relevant sites of enzymatic digestion were added to mTNFα by PCR. mTNFα was linked to the 3’ end of hscFv25 in pGEX4T-1 vector. This anti-HCC recombinant Fv-immunotoxin hscFv25-mTNFα was expressed in Escherichia coli and purified from inclusions. After purified by glutathione-S-transferase affinity chromatography and thrombin digestion, it was identified by electrophoresis and Western blot. And then, the purified recombinant Fv-immunotoxin was injected into nude mice with HCC xenografts through their tail veins. mTNFα protein and PBS were used as control at the same time. After treated for two weeks, nude mice were executed. The bulk and weight of tumors were observed. The tumor tissues were stained by immunohistochemical method with TNFα antibody.
RESULTS: The expression ratio of recombinant Fv-immunotoxin hscFv25-mTNFα was 12% of bacterial protein. The result of tumor restraining trials of hscFv25-mTNFα showed 2/5 complete remission and 3/5 partial remission. mTNFα restraining trials showed 5/5 partial remission. The therapeutic result of hscFv25-mTNFα was better than that of mTNFα (F = 8.70, P < 0.05). The hscFv25-mTNFα remedial tumor tissues were positive for TNFα by immunohistochemical staining. The positive granules mainly existed in the cytoplasm of tumor cell.
CONCLUSION: Recombinant Fv-immunotoxin hscFv25-mTNFα has better therapeutic effect than mTNFα. It can inhibit the cellular growth of HCC and has some potential of clinical application.
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Affiliation(s)
- Jing Zhang
- Department of Pathology, Xijing Hospital, Fourth Military Medical University, Xi'an 710033, Shaanxi Province, China.
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Bruenke J, Fischer B, Barbin K, Schreiter K, Wachter Y, Mahr K, Titgemeyer F, Niederweis M, Peipp M, Zunino SJ, Repp R, Valerius T, Fey GH. A recombinant bispecific single-chain Fv antibody against HLA class II and FcgammaRIII (CD16) triggers effective lysis of lymphoma cells. Br J Haematol 2004; 125:167-79. [PMID: 15059139 DOI: 10.1111/j.1365-2141.2004.04893.x] [Citation(s) in RCA: 44] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
Bispecific antibodies offer the possibility of improving effector-cell recruitment for antibody therapy. For this purpose, a recombinant bispecific single-chain Fv antibody (bsscFv), directed against FcgammaRIII (CD16) and human leucocyte antigen (HLA) class II, was constructed and tested in functional assays. RNA from the hybridomas 3G8 and F3.3, reacting with CD16 and HLA class II, respectively, was used to generate phage display libraries. From these libraries, reactive phages were isolated and the bsscFv was constructed by connecting both single-chain Fv components through a 20 amino acid flexible linker. After expression in SF21 insect cells and chromatographic purification, the bsscFv bound specifically and simultaneously to both antigens. The affinities of the anti-CD16 and the anti-HLA class II scFv components of the bsscFv were 8.6 x 10(-8) mol/l and 13.7 x 10(-8) mol/l, respectively, which was approximately sevenfold lower than the F(ab) fragments of the parental antibodies. In antibody-dependent cellular cytotoxicity experiments with human mononuclear cells as effectors, the bsscFv-mediated specific lysis of both HLA class II-positive, malignant human B-lymphoid cell lines and primary cells from patients with chronic B-cell lymphocytic leukaemia. Optimal lysis was obtained at bsscFv concentrations of approximately 400 ng/ml, similar to the concentration required for maximum lysis by the corresponding chemically linked bispecific antibody. Thus, this recombinant bsscFv-antibody is an efficient molecule for effector-cell mediated lysis of malignant human B-lymphoid cells.
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Affiliation(s)
- Joerg Bruenke
- University of Erlangen-Nuremburg, Staudtstrasse 5, D-91058 Erlangen, Germany
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45
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Roskos LK, Davis CG, Schwab GM. The clinical pharmacology of therapeutic monoclonal antibodies. Drug Dev Res 2004. [DOI: 10.1002/ddr.10346] [Citation(s) in RCA: 74] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023]
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Kipriyanov SM, Moldenhauer G, Braunagel M, Reusch U, Cochlovius B, Le Gall F, Kouprianova OA, Von der Lieth CW, Little M. Effect of domain order on the activity of bacterially produced bispecific single-chain Fv antibodies. J Mol Biol 2003; 330:99-111. [PMID: 12818205 DOI: 10.1016/s0022-2836(03)00526-6] [Citation(s) in RCA: 46] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Bispecific single-chain Fv antibodies comprise four covalently linked immunoglobulin variable (VH and VL) domains of two different specificities. Depending on the order of the VH and VL domains and on the length of peptides separating them, the single-chain molecule either forms two single-chain Fv (scFv) modules from the adjacent domains of the same specificity, a so-called scFv-scFv tandem [(scFv)(2)], or folds head-to-tail with the formation of a diabody-like structure, a so-called bispecific single-chain diabody (scBsDb). We generated a number of four-domain constructs composed of the same VH and VL domains specific either for human CD19 or CD3, but arranged in different orders. When expressed in bacteria, all (scFv)(2) variants appeared to be only half-functional, binding to CD19 and demonstrating no CD3-binding activity. Only the diabody-like scBsDb could bind both antigens. Comparison of the scBsDb with a structurally similar non-covalent dimer (diabody) demonstrated a stabilizing effect of the linker in the middle of the scBsDb molecule. We demonstrated that the mechanism of inactivation of CD19xCD3 diabody under physiological conditions is initiated by a dissociation of the weaker (anti-CD3) VH/VL interface followed by domain swapping with the formation of non-active homodimers. The instability of one homodimer makes the process of diabody dissociation/reassociation irreversible, thus gradually decreasing the fraction of active molecules. The structural parameters influencing the formation of functional bispecific single-chain antibodies are indicated and ways of making relatively stable bispecific molecules are proposed.
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Affiliation(s)
- Sergey M Kipriyanov
- Recombinant Antibody Research Group, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany.
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Wimberger P, Xiang W, Mayr D, Diebold J, Dreier T, Baeuerle PA, Kimmig R. Efficient tumor cell lysis by autologous, tumor-resident T lymphocytes in primary ovarian cancer samples by an EP-CAM-/CD3-bispecific antibody. Int J Cancer 2003; 105:241-8. [PMID: 12673686 DOI: 10.1002/ijc.11056] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
The epithelial cell adhesion molecule (Ep-CAM) is expressed on the surface of most human carcinomas, including ovarian, breast, lung, prostate and colorectal carcinoma. Ep-CAM was shown to be a valid target for monoclonal antibody-based therapies. We have investigated whether an Ep-CAM-/CD3-bispecific single-chain antibody called bscEp-CAM x CD3 is effective in tumor cell elimination within the cellular microenvironment of primary ovarian cancer tissue. The ex vivo elimination of ovarian cancer cells in tumor preparations from 21 patients was monitored by flow cytometry using Ep-CAM/CA-125 double-labeling or Ep-CAM single-labeling combined with propidium iodide uptake of cells. Methodology was established by the ovarian cancer cell line OvCAR. A total of 17 (81%) patient samples showed a dose-dependent tumor cell elimination by bscEp-CAM x CD3. High and specific tumor cell lysis was seen at bscEp-CAM x CD3 concentrations as low as 1 ng/ml, at very low effector:target ratios and in the absence of T cell costimulation. The high efficacy of the bispecific antibody may be due to the non-restricted activation of tumor-resident cytotoxic T lymphocytes. In clinical trials, the ex vivo data with the T cell-recruiting bispecific antibody bscEp-CAM x CD3 may translate into a high response rate and efficacy of tumor cell elimination.
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Affiliation(s)
- Pauline Wimberger
- Department of Gynecology and Obstetrics, University of Essen, Essen, Germany.
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Zhang J, Liu YF, Yang SJ, Qiao Q, Zhang SZ, Cheng H. Targeting therapeutic of humanized scFv25 and the fusion to mutant TNFα against hepatocellular carcinoma: a preliminary study. Shijie Huaren Xiaohua Zazhi 2003; 11:285-288. [DOI: 10.11569/wcjd.v11.i3.285] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To explore the cytotoxic effects of humanized scFv25 and the fusion to mutant TNFα on HCC xenografts in nude mice.
METHODS: The mice with HCC xenografts were injected the purified recombinant immunotoxin through tail vain and executed after two weeks. The bulk and weight of tumor were observed. Expression of TNFα was detected by immunohistochemical staining in the tumor tissues.
RESULTS: The tumor regression trials of hscFv25-mTNFα showed 5/5 effective, with 2/5 completely remission and 3/5 partly remission. The therapeutic result of hscFv25-mTNFα was better than that of mutant TNFα (Xh2 = 6.62, P < 0.05). The HCC tissue treated by hscFv25-mTNFα expressed TNFα, and the positive granules were mainly existed in cytoplasm.
CONCLUSION: Recombinant immunotoxin the hscFV25-mTNFα can regress the growth of HCC with a great potential.
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Abstract
We have assembled references of 700 articles published in 2001 that describe work performed using commercially available optical biosensors. To illustrate the technology's diversity, the citation list is divided into reviews, methods and specific applications, as well as instrument type. We noted marked improvements in the utilization of biosensors and the presentation of kinetic data over previous years. These advances reflect a maturing of the technology, which has become a standard method for characterizing biomolecular interactions.
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Affiliation(s)
- Rebecca L Rich
- Center for Biomolecular Interaction Analysis, University of Utah, Salt Lake City, UT 84132, USA
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Dreier T, Lorenczewski G, Brandl C, Hoffmann P, Syring U, Hanakam F, Kufer P, Riethmuller G, Bargou R, Baeuerle PA. Extremely potent, rapid and costimulation-independent cytotoxic T-cell response against lymphoma cells catalyzed by a single-chain bispecific antibody. Int J Cancer 2002; 100:690-7. [PMID: 12209608 DOI: 10.1002/ijc.10557] [Citation(s) in RCA: 257] [Impact Index Per Article: 11.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Abstract
A recent study reported on an anti-CD19/anti-CD3 single-chain bispecific antibody (bscCD19xCD3) exhibiting high activity against human B lymphoma cell lines (Löffler et al., Blood 2000;95:2098-103). In the present study, we have explored in detail the in vitro efficacy, T-cell donor variability, binding characteristics, specificity, kinetics and interleukin-2 (IL-2) dependence of bscCD19xCD3. We found that a majority of human donor T cells tested (n = 86) gave half-maximal B-lymphoma cell lysis (ED(50)) within a range of 10-50 pg/ml bscCD19xCD3, corresponding to sub-picomolar concentrations of the bispecific antibody. Under identical experimental conditions, the anti-CD20 monoclonal antibody rituximab had an at least 100,000-fold lower in vitro efficacy. The extreme potency of bscCD19xCD3 was in sharp contrast to the relatively low affinity of the anti-CD3 and anti-CD19 single-chain Fv portions in K(D) ranges of 10(-7) and 10(-9) M, respectively. Cell lysis by bscCD19xCD3 was predominantly mediated by the population of CD8/CD45RO-positive T cells. Both immortalized CD4- and CD8-positive human T-cell clones were highly active effector cells as well. Cell lysis by bscCD19xCD3 was rapid and specific. The respective parental monoclonal antibodies inhibited cell lysis and CD19-negative cells were not harmed by T cells in the presence of high amounts of bscCD19xCD3. The potent T-cell stimulus IL-2 could not markedly augment the activity of bscCD19xCD3-stimulated T cells. In conclusion, bscCD19xCD3 could redirect unstimulated cytotoxic T cells against CD19-positive cells in an unexpectedly potent, rapid and specific fashion.
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Affiliation(s)
- Torsten Dreier
- Micromet AG, Am Klopferspitz 19, 82152 Martinsried, Germany
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