ABSTRACT
Multispecific antibodies, often composed of three to five polypeptide chains, have become increasingly relevant in the development of biotherapeutics. These molecules have mechanisms of action that include redirecting T cells to tumors and blocking multiple pathogenic mediators simultaneously. One of the major challenges for asymmetric multispecific antibodies is generating a high proportion of the correctly paired antibody during production. To understand the causes and effects of chain mispairing impurities in a difficult to express multispecific hetero-IgG, we investigated consequences of individual and pairwise chain expression in mammalian transient expression hosts. We found that one of the two light chains (LC) was not secretion competent when transfected individually or cotransfected with the noncognate heavy chain (HC). Overexpression of this secretion impaired LC reduced cell growth while inducing endoplasmic reticulum stress and CCAAT/enhancer-binding protein homologous protein (CHOP) expression. The majority of this LC was observed as monomer with incomplete intrachain disulfide bonds when expressed individually. Russell bodies (RB) were induced when this LC was co-expressed with the cognate HC. Moreover, one HC paired promiscuously with noncognate LC. These results identify the causes for the low product quality observed from stable cell lines expressing this heteroIgG and suggest mitigation strategies to improve overall process productivity of the correctly paired multispecific antibody. The approach described here provides a general strategy for identifying the molecular and cellular liabilities associated with difficult to express multispecific antibodies.
Subject(s)
Antibodies, Bispecific , Gene Expression , Protein Engineering , Animals , Antibodies, Bispecific/biosynthesis , Antibodies, Bispecific/genetics , CHO Cells , Cricetulus , Goats , HEK293 Cells , Humans , Immunoglobulin Light Chains/biosynthesis , Immunoglobulin Light Chains/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/geneticsABSTRACT
AGS-16C3F is an antibody-drug conjugate (ADC) against ectonucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3) containing the mcMMAF linker-payload currently in development for treatment of metastatic renal cell carcinoma. AGS-16C3F and other ADCs have been reported to cause ocular toxicity in patients by unknown mechanisms. To investigate this toxicity, we developed an in vitro assay using human corneal epithelial cells (HCEC) and show that HCECs internalized AGS-16C3F and other ADCs by macropinocytosis, causing inhibition of cell proliferation. We observed the same mechanism for target-independent internalization of AGS-16C3F in fibroblasts and human umbilical vein endothelial cells (HUVEC). Macropinocytosis-mediated intake of macromolecules is facilitated by the presence of positive charges or hydrophobic residues on the surface of the macromolecule. Modification of AGS-16C3F, either by attachment of poly-glutamate peptides, mutation of residue K16 to D on AGS-16C3F [AGS-16C3F(K16D)], or decreasing the overall hydrophobicity via attachment of polyethylene glycol moieties, significantly reduced cytotoxicity against HCECs and other primary cells. Rabbits treated with AGS-16C3F showed significant ocular toxicity, whereas those treated with AGS-16C3F(K16D) presented with less severe and delayed toxicities. Both molecules displayed similar antitumor activity in a mouse xenograft model. These findings establish a mechanism of action for target-independent toxicities of AGS-16C3F and ADCs in general, and provide methods to ameliorate these toxicities.Significance: These findings reveal a mechanism for nonreceptor-mediated toxicities of antibody drug conjugates and potential solutions to alleviate these toxicities. Cancer Res; 78(8); 2115-26. ©2018 AACR.
Subject(s)
Antibodies, Monoclonal, Humanized/pharmacology , Epithelium, Corneal/drug effects , Immunoconjugates/toxicity , Pinocytosis/drug effects , Amino Acid Sequence , Animals , Antibodies, Monoclonal, Humanized/therapeutic use , Cells, Cultured , Human Umbilical Vein Endothelial Cells , Humans , Macaca fascicularis , Male , Models, Animal , Rabbits , Sequence Homology, Amino AcidABSTRACT
Tumor necrosis factor (TNF) is a key regulator of inflammatory responses and has been implicated in many pathological conditions. We used structure-based design to engineer variant TNF proteins that rapidly form heterotrimers with native TNF to give complexes that neither bind to nor stimulate signaling through TNF receptors. Thus, TNF is inactivated by sequestration. Dominant-negative TNFs represent a possible approach to anti-inflammatory biotherapeutics, and experiments in animal models show that the strategy can attenuate TNF-mediated pathology. Similar rational design could be used to engineer inhibitors of additional TNF superfamily cytokines as well as other multimeric ligands.
Subject(s)
Protein Engineering , Signal Transduction , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/pharmacology , Amino Acid Substitution , Animals , Antigens, CD/metabolism , Apoptosis , Arthritis, Experimental/drug therapy , Biopolymers , Caspases/metabolism , Cell Line , Cell Nucleus/metabolism , Computer Simulation , Disease Progression , Enzyme-Linked Immunosorbent Assay , Female , Galactosamine/pharmacology , HeLa Cells , Humans , Liver/drug effects , NF-kappa B/metabolism , Point Mutation , Rats , Receptors, Tumor Necrosis Factor/metabolism , Receptors, Tumor Necrosis Factor, Type I , Receptors, Tumor Necrosis Factor, Type II , Transcription Factor RelA , Transcription, Genetic , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Highlights of the 9th (Biennial) International Congress on TNF-Related Cytokines (30 October-2 November 2002; The Manchester Grand Hyatt, San Diego, CA, USA.
Subject(s)
Membrane Glycoproteins , Membrane Proteins , Receptors, Tumor Necrosis Factor , Tumor Necrosis Factor-alpha , Animals , Apoptosis Regulatory Proteins , B-Cell Activation Factor Receptor , Humans , Membrane Glycoproteins/metabolism , Membrane Glycoproteins/therapeutic use , Receptors, Tumor Necrosis Factor/metabolism , Receptors, Tumor Necrosis Factor/therapeutic use , TNF-Related Apoptosis-Inducing Ligand , Tumor Necrosis Factor-alpha/metabolism , Tumor Necrosis Factor-alpha/therapeutic useABSTRACT
This unit presents protocols for sperm isolation using two different methods, amplification of simple sequence-length polymorphisms (SSLP) and/or single nucleotide polymorphisms (SNP) from single cells or whole genome-amplified single cells using primer extension preamplification (PEP), and discusses the statistical analysis of sperm-typing recombination data. Newer methods for studying recombination over very short distances (a few kilobases) using total sperm DNA and allele-specific PCR are also discussed.