Antibody engineering encompasses various development, production strategies, and modification techniques to improve the biological properties ofmonoclonal antibodies (mAbs) as therapeutic agents. During the development stages of antibody therapeutics, engineering methods are typically applied following antibody discovery, screening, and selection efforts (Figure 1). Some of theessential properties sought after in the development of therapeutic mAbs include: low immunogenicity, high-affinity antigen binding, and high stability. As a result, many of the objectives in antibody engineering involve:
Reducing immunogenicity through efforts in chimerization, humanization, or species backbone switching of mAbs
Performance optimization through in vitro affinity maturation or generation of bivalent formats
Increasing half-life, reducing or optimizing effector functions through Fc engineering or Fc silencing
Targeting more than one receptor through efforts in engineering bispecific or multispecific antibodies
Simplifying production or increasing biological activity through generation of antibody derivatives, such as single-chain variable fragments (scFvs) or fragment antigen-binding (Fab) fragments
Murine-derived antibodies cause immunological responses in patients, thereby causing rapid elimination of these molecules and significantly limits their therapeutic potential. This response produces human anti-mouse antibodies (HAMA), which recognize the therapeutic mAb as a foreign molecule. This is often due to differences in glycosylation patterns and other post-translational modifications between human and mouse antibodies.
Consequently, engineering efforts have focused on the development of chimeric and humanized antibodies to alleviate immunogenic responses. Chimeric antibodies are engineered where murine constant regions were replaced by human constant regions, leaving mouse-derived variable regions to interact and bind the antigen (Figure 2). Engineering humanized antibodies involvescomplementarity determining region(CDR) grafting, a process that incorporates mouse-derived CDRs into human-derived framework and constant regions.
Fully human antibodies utilize fully human antibody sequences, selected from isolated human peripheral blood mononuclear cells (PBMCs) or transgenic mouse models. These engineered antibodies are associated with a lower risk of inducing an immunogenic response, thus improving their therapeutic potential.
Antibody Fragment and Bispecific Engineering
In some cases, engineering of antibodies into their minimal binding fragments is advantageous over their full-length formats. Their smaller size allows them to penetrate through tissues, reach embedded epitopes of the antigen, and are sometimes easier to produce at a large scale. Variable heavy chains and light chains can be isolated and amplified from any candidate antibody and engineered intovarious fragmented formats (Figure 2). Many forms of fragmented antibodies have been engineered for therapeutic purposes, including:
Fv fragment – composed of only the variable region from either the heavy or the light chain
VHH or nanobody – composed of the antigen binding fragment of the heavy chain only
Fab fragment – composed of the variable domain, first constant region of the heavy chain and the light chain
ScFv – composed of the variable domain from both the heavy and the light chain, connected through a linker rather than the Fc region
Diabody or bispecific fragments – composed of two different antigen binding domains, providing dual specificity
Figure 2. Types of engineered antibodies and antibody fragments
The Fc domain of an antibody mediates the activation ofeffector functions of the immune system. Complement dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP) are activated through receptor binding to the Fc fragment and induce tumour cytotoxicity and cell death.
Fc engineering involvesenhancing the effector function of therapeutic antibodies to improve their potency and therapeutic potential. Improving binding affinity and capacity of the Fc domain for their cognate Fc receptors can improve the therapeutic efficiency. Conversely, reduced effector functions may be advantageous for receptor blocking, and thus requiresFc engineering to silence effector functions.
Labeled antibodies are an essential research tool used to detect and quantify antigens in a variety of immunoassays, such as Western blots, flow cytometry, and ELISAs. Labels such as biotin, horseradish peroxidase (HRP), or fluorophores can be linked to antibodies through charged amino acids, tyrosine residues, carbohydrates or sulfhydryl groups.
Driving Antibody Engineering with Next Generation Protein Sequencing and Proteomics
The emergence of antibody engineering has expanded the use and diversity of antibody-based therapies. Engineered antibodies may experience increased complexity; as such they may benefit from in-depth characterization to ensure their reproducibility, specificity, and affinity. At Rapid Novor, we support antibody engineering efforts in all stages of development with:
De novo antibody sequencing – Obtaining the amino acid sequence of antibodies known to perform well against a target can help researchers to understand how they work, and guide the discovery and/or engineering of novel antibody constructs: species switching, humanization, fragment design, affinity maturation, and in silico design. Rapid Novor utilizes a well-establishedmass spectrometry-based serviceto sequence antibodies directly from the mass spectral data, without reference to genomic sequence information..
SPR kinetic binding analysis – SPR kinetic binding analysis will reveal properties such as specificity, affinity, and efficiency of antibody-antigen interactions to help evaluate engineering strategies and select therapeutic candidates. SPR can characterize diverse biomolecules and their interactions, read about SPR kinetic binding analysis to learn more about its applications.
HDX-MS epitope mapping – HDX-MS epitope mapping can characterize antibody-antigen protein complexes, to accurately determine linear, conformational, and structural epitopes. Read about HDX-MS epitope mapping as a powerful technique for antibody characterization.
We Have Sequenced 7000+ Antibodies and We Are Eager to Help You.
Through next generation protein sequencing, Rapid Novor enables reliable discovery and development of novel reagents, diagnostics, and therapeutics. Thanks to our Next Generation Protein Sequencing and antibody discovery services, researchers have furthered thousands of projects, patented antibody therapeutics, and developed the first recombinant polyclonal antibody diagnostics.
Talk to Our Scientists.
We Have Sequenced 6000+ Antibodies and We Are Eager to Help You.
Through next generation protein sequencing, Rapid Novor enables timely and reliable discovery and development of novel reagents, diagnostics, and therapeutics. Thanks to our Next Generation Protein Sequencing and antibody discovery services, researchers have furthered thousands of projects, patented antibody therapeutics, and ran the first recombinant polyclonal antibody diagnostics
Talk to our scientists. We have sequenced over 6000 antibodies and we are eager to help you.