A new approach to produce IgG4-like bispecific antibodies.
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ABSTRACT:
SUBMITTER: Zhao C
PROVIDER: S-EPMC8452627 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:We describe a generic approach to assemble correctly two heavy and two light chains, derived from two existing antibodies, to form human bivalent bispecific IgG antibodies without use of artificial linkers. Based on the knobs-into-holes technology that enables heterodimerization of the heavy chains, correct association of the light chains and their cognate heavy chains is achieved by exchange of heavy-chain and light-chain domains within the antigen binding fragment (Fab) of one half of the bispecific antibody. This "crossover" retains the antigen-binding affinity but makes the two arms so different that light-chain mispairing can no longer occur. Applying the three possible "CrossMab" formats, we generated bispecific antibodies against angiopoietin-2 (Ang-2) and vascular endothelial growth factor A (VEGF-A) and show that they can be produced by standard techniques, exhibit stabilities comparable to natural antibodies, and bind both targets simultaneously with unaltered affinity. Because of its superior side-product profile, the CrossMab(CH1-CL) was selected for in vivo profiling and showed potent antiangiogenic and antitumoral activity.
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Project description:The development of bispecific antibodies has attracted substantial interest, and many different formats have been described. Those specifically containing an Fc part are mostly tetravalent, such as stabilized IgG-scFv fusions or dual-variable domain (DVD) IgGs. However, although they exhibit IgG-like properties and technical developability, these formats differ in size and geometry from classical IgG antibodies. Thus, considerable efforts focus on bispecific heterodimeric IgG antibodies that more closely mimic natural IgG molecules. The inherent chain association problem encountered when producing bispecific heterodimeric IgG antibodies can be overcome by several methods. While technologies like knobs-into-holes (KiH) combined with a common light chain or the CrossMab technology enforce the correct chain association, other approaches, e.g., the dual-acting Fab (DAF) IgGs, do not rely on a heterodimeric Fc part. This review discusses the state of the art in bispecific heterodimeric IgG antibodies, with an emphasis on recent progress.
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Project description:Some antibodies exhibit elevated viscosity at high concentrations, making them poorly suited for therapeutic applications requiring administration by injection such as subcutaneous or ocular delivery. Here we studied an anti-IL-13/IL-17 bispecific IgG4 antibody, which has anomalously high viscosity compared to its parent monospecific antibodies. The viscosity of the bispecific IgG4 in solution was decreased by only ~30% in the presence of NaCl, suggesting electrostatic interactions are insufficient to fully explain the drivers of viscosity. Intriguingly, addition of arginine-HCl reduced the viscosity of the bispecific IgG4 by ~50% to its parent IgG level. These data suggest that beyond electrostatics, additional types of interactions such as cation-π and/or π-π may contribute to high viscosity more significantly than previously understood. Molecular dynamics simulations of antibody fragments in the mixed solution of free arginine and explicit water were conducted to identify hotspots involved in self-interactions. Exposed surface aromatic amino acids displayed an increased number of contacts with arginine. Mutagenesis of the majority of aromatic residues pinpointed by molecular dynamics simulations effectively decreased the solution's viscosity when tested experimentally. This mutational method to reduce the viscosity of a bispecific antibody was extended to a monospecific anti-GCGR IgG1 antibody with elevated viscosity. In all cases, point mutants were readily identified that both reduced viscosity and retained antigen-binding affinity. These studies demonstrate a new approach to mitigate high viscosity of some antibodies by mutagenesis of surface-exposed aromatic residues on complementarity-determining regions that may facilitate some clinical applications.
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Project description:Many types of cancers are prevalent in India and worldwide. Monoclonal antibodies (MAbs) are one of the major types of cancer therapeutics, which have included MAbs of hybridoma, chimeric, humanized, or human origin. MAbs are mostly generated currently by direct cloning from B cells. Bispecific antibodies (BAbs), as the name suggests, have two different antigen-binding domains in a single molecule and thus have dual functionality/specificity combined in a single antibody. In addition to the detection of two different antigenic molecules, the dual functionality of BAbs can be utilized to mount T-cell-mediated killing of tumor cells wherein one Fv binds to the tumor-specific antigen and the another recruits T cells to the site of action. Breast cancer and prostate cancer are among the most prevalent cancers in women and men, respectively. Biomarkers such as HER2 and ER/PR are expressed in breast cancer, while overexpression of hepsin and prostate-specific membrane antigen is observed in prostate cancer. Developing BAbs against these biomarkers may be a potent therapeutic option to target breast and prostate cancer, respectively. Therefore, an efficient method using recombinant DNA technology and mammalian cell culture platform is required to generate BAbs against specific diseases as biomarkers as well as for the generation of antibody-based therapeutics.
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Project description:Monovalent bispecific antibodies (BsAbs) are projected to have broad clinical applications due to their ability to bind two different targets simultaneously. Although they can be produced using recombinant technologies, the correct pairing of heavy and light chains is a significant manufacturing problem. Various approaches exploit mutations or linkers to favor the formation of the desired BsAb, but a format using a single common light chain has the advantage that no other modification to the antibody is required. This strategy reduces the number of formed molecules to three (the BsAb and the two parent mAbs), but the separation of the BsAb from the two monovalent parent molecules still poses a potentially difficult purification challenge. Current methods employ ion exchange chromatography and linear salt gradients, but are only successful if the difference in the observed isoelectric points (pIs) of two parent molecules is relatively large. Here, we describe the use of highly linear pH gradients for the facile purification of common light chain BsAbs. The method is effective at separating molecules with differences in pI as little as 0.10, and differing in their sequence by only a single charged amino acid. We also demonstrate that purification resins validated for manufacturing are compatible with this approach.
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Project description:Many methods have been developed to produce bispecific antibodies (BsAbs) for industrial application. However, huge challenges still remain in synthesizing whole length BsAbs, including their assembly, stability, immunogenicity, and pharmacodynamics. Here we present for first time a generic technology platform of generating bispecific IgG antibodies, "Bispecific Antibody by Protein Trans-splicing (BAPTS)". Different from published methods, we assembled two parental antibody fragments in the hinge region by the protein trans-splicing reaction of a split intein to generate BsAbs without heavy/heavy and light/heavy chain mispairing. Utilizing this simple and efficient approach, there have been several BsAbs (CD3×HER2, CD3×EGFR, EGFR×HER2) synthesized to demonstrate its broad applicability. Correctly paired mAb arms were assembled to form BsAbs that were purified through protein A affinity chromatography to demonstrate industrial applicability at large scale. Further, the products were characterized through physical-biochemistry properties and biological activities to confirm expected quality of the products from "BAPTS". More importantly, correct pairing was confirmed by mass spectrum. Proof-of-concept studies with CD3×HER2 BsAb (T-cell recruitment) demonstrated superior bioactivity compared with trastuzumab. The results of undetectable mispairing and high biological activity have indicated that this method has the potential to be utilized to manufacture BsAbs with high efficiency at industrial scale.
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