When to Use Recombinant Antibodies

The term “recombinant antibody” has become synonymous with product consistency and reliability. It also hints at possibilities for antibody engineering to support a particular experimental need. This article provides a recap of some of the different types of recombinant antibodies available and comments on their value to scientific research.

Defined by sequence

“For many researchers, the term ‘recombinant antibody’ seems to invoke the idea of an antibody generated by phage display to a novel or unusual target, which they are unlikely to be working with on a daily basis,” reports Dr. Michael Fiebig, Chief Scientific Officer at Absolute Antibody. “However, it more generally refers to any antibody produced using recombinant methods. This includes antibodies that were originally developed using hybridoma technology, which have subsequently been sequenced and the antibody-coding DNA expressed in another cell line. In fact, most classic research antibodies are now available as recombinant versions, allowing researchers to benefit from the improved reproducibility recombinant production offers, while still being able to use antibodies with which they are familiar. Critically, recombinant means that an antibody is absolutely defined by a specific protein sequence, which brings a wealth of advantages to both research and clinical applications.”

Opportunities through engineering

According to Dr. Jayne Hammersley, Head of Antibody Discovery Technology (ADT) at Abcam, one of the benefits of having a defined antibody sequence is that it allows shuttling of antibody binding domains between different backbones. “A popular engineering strategy involves switching the antibody Fc region to a different species or subclass to minimize cross-reaction in a particular host system or assay,” she explains. “For example, the Fc region of a rat antibody might be switched to a mouse Fc for reduced immunogenicity during a murine in vivo study, or to a selection of different species to provide greater flexibility for co-immunostaining applications.”

Engineering additionally permits manipulation of antibody size and presentation. “Expressing smaller antibody fragments—such as scFv, Fab or F(ab)2, VHH or VH-only domain—can be advantageous to increase tissue penetration and access to obscured epitopes,” says Hammersley. “These fragments can also be used as building blocks to combine multiple antibody specificities within the same molecule, creating bi-, tri-, and multi-specific constructs for therapeutic use.”

Applications of antibody engineering

Recombinant antibodies provide a wealth of opportunities for engineering that can lead to more meaningful research. For example, during a recent study, the rat anti-mouse PD1 antibody, RMP1-14, was converted into a mouse IgG2a format and engineered to feature several Fc silencing mutations. This not only reduced its immunogenicity but also increased its anti-tumor activity. Likewise, by switching the rat IgG2a anti-mLy6G antibody 1A8 to a mouse IgG2a, researchers have been able to induce a potent neutrophil depletion in mice. This has been useful in investigating the role of the complement protein C3 in cutaneous squamous cell carcinoma (a form of non-melanoma skin cancer), where C3 has been shown to promote tumor development in a neutrophil independent manner.

Also commenting on antibody engineering to develop therapeutics, Anne Sloan, Technical Scientist at Cell Sciences, notes that chimeric antibodies featuring an antigen binding domain from one species (mouse) and a constant domain from another species (human) limit the number of foreign epitopes introduced into a patient. Humanized mouse monoclonals are likewise better tolerated in vivo and typically generate lower levels of anti-drug antibodies (ADAs) than their chimeric counterparts. “Another form of therapeutic antibody engineering yields bispecific antibodies, such as those that can bind tumor cell receptors and cytotoxic immune cells at the same time to stimulate an ADCC response,” she says. “More recently, neutralizing human antibodies to the SARS-CoV-2 Spike protein receptor binding domain have been isolated from the serum of convalescent COVID-19 patients and produced recombinantly in bulk during efforts to tackle the ongoing pandemic.”

More streamlined production

From an antibody manufacturer’s perspective, recombinant production offers several advantages. “Recombinant antibodies are encoded in DNA vectors, usually containing a constitutive promoter for expression, which are stably or transiently transfected into a suitable host cell line,” notes Hammersley. “This would usually be a suspension HEK or CHO line to maintain mammalian glycosylation, but in the case of some antibody fragments, a prokaryotic expression system may be equally successful. Because the vectors and host cell line are well defined, optimizing the process is straightforward. Also, compared with hybridoma-derived antibodies, the cell lines used for recombinant production can be grown in minimal or defined media to simplify purification requirements and maximize antibody yields.” This translates to benefits for the researcher, including provision of antibodies as a standardized formulation (e.g., 1 mg/mL in PBS), which removes various limitations governing their use. Sloan adds that recombinant production also allows for in vitro affinity maturation—the process of manipulating the antibody sequence to produce antibodies with a higher affinity and lower off-rate. “Recombinant affinity maturation can give antibodies with 10–50 times higher affinity or increased half-life than the parent antibody,” she says.

Antibody characterization remains essential

Although knowing the antibody sequence is, inarguably, beneficial, it is important not to see recombinant as a buzzword for specific. “Regardless of the means of antibody production, the most critical element in any experiment is the careful, application-driven characterization of the chosen antibody,” cautions Katie Crosby, director of IHC at Cell Signaling Technology.

“The recombinant nature of an antibody does not guarantee specificity. Rather, demonstration of antibody specificity in the application of interest typically requires using multiple testing strategies, like testing on binary or ranged expression models; leveraging orthogonal, non-antibody-based approaches like RNA expression profiling; and employing a second antibody directed against a non-overlapping epitope, to name but a few.”

When to go recombinant?

At present, deciding whether to use traditional or recombinant antibodies is largely a matter of personal preference. However, a subtle shift toward the latter appears to be on the cards. For example, Hammersley suggests that although conventional polyclonals may still be recognized to offer an advantage where some level of signal amplification is required, this is likely to change as more recombinant oligoclonal mixes reach the market. Sloan makes a similar point, noting that such products also eliminate the requirement for animal use. “Oligoclonal antibodies represent highly specific, reproducible, low background reagents, with no animal involvement in their production,” she says. “For these reasons, they are increasingly being used as substitutes for traditional polyclonals.”

“While we are seeing wider awareness of the benefits of recombinant antibodies among researchers, the choice of reagents isn’t always driven by scientific or even price considerations, but by precedents from the literature, or what a specific laboratory has always done,” observes Fiebig. “However, especially when animal studies are involved, this comes at a cost—not only to the reliability of experimental data but also to animal welfare. As a scientific community, we still have a lot of work to do to encourage the uptake of recombinant antibodies and lower the barriers for researchers to use improved tools. This may require pressure from funding bodies and publications, to question whether researchers have used the most suitable reagents for their study or simply replicated established but less refined methods.”