Abstract
Development of antibody-based immunotherapeutics has progressed from direct tumor-targeting, with antibodies such as rituximab, to blocking of immune checkpoints to reactivate antitumor immunity. In addition, bispecific antibodies/antibody fragments are also of great interest in cancer therapy, as these constructs have the ability to redirect immune effector cells to cancer targets and, thereby, enhance therapeutic efficacy. A number of bispecific antibody formats have been reported, with the first FDA-approved bispecific antibody being blinatumomab, a so-called bispecific T cell engager (BiTE), which redirects and potently activates T cell immune responses. Recently, we described an additional novel bispecific antibody derivative, termed RTX-CD47, which was designed to inhibit the innate immune checkpoint CD47-SIRPα only on -positive cancer cells. RTX-CD47 contains two antibody fragments in tandem and has monovalent binding specificity for CD47 and . Only upon dual binding to and CD47 RTX-CD47 blocks CD47 "Don't eat me" signaling. Here, we provide a detailed protocol for the construction and functional evaluation of such a bispecific antibody derivative.
Keywords:
Antibody fragment; CD47; Cancer; Immunotherapy.
MeSH terms
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Animals
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Antibodies, Bispecific / pharmacology*
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Antibodies, Bispecific / therapeutic use
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Antigens, Differentiation / genetics
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Antigens, Differentiation / immunology
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Antigens, Differentiation / metabolism
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Antineoplastic Agents / pharmacology*
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Antineoplastic Agents / therapeutic use
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Biological Assay / instrumentation
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Biological Assay / methods*
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CD47 Antigen / genetics
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CD47 Antigen / immunology
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CD47 Antigen / metabolism
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CHO Cells
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Cell Culture Techniques / instrumentation
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Cell Culture Techniques / methods
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Cell Separation / instrumentation
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Cell Separation / methods
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Chromatography, Affinity / instrumentation
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Chromatography, Affinity / methods
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Cricetulus
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Drug Evaluation, Preclinical / instrumentation
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Drug Evaluation, Preclinical / methods
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HEK293 Cells
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Humans
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Immunotherapy / methods
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Neoplasms / drug therapy*
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Neoplasms / immunology
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Neoplasms / pathology
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Receptors, Immunologic / genetics
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Receptors, Immunologic / immunology
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Receptors, Immunologic / metabolism
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / immunology*
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Recombinant Fusion Proteins / metabolism
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T-Lymphocytes / immunology
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T-Lymphocytes / metabolism
Substances
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Antibodies, Bispecific
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Antigens, Differentiation
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Antineoplastic Agents
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CD47 Antigen
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CD47 protein, human
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Receptors, Immunologic
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Recombinant Fusion Proteins
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SIRPA protein, human