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From Cancer to Crops: How Antibody Engineering is Changing Everything

The discovery of monoclonal antibodies (mAbs) in 1975 heralded a remarkable breakthrough in precision medicine, opening up many doors for personalized diagnosis and care. Just over a decade later, in 1986, the FDA approved the first mAb product to prevent kidney transplant rejection.1 Since then, advances in mAb engineering have been monumental, fundamentally changing how we approach the treatment of cancer, infectious diseases, and autoimmune conditions. Beyond healthcare, antibody technology is now essential for developing new tools to monitor the health of our environment, improve agricultural products, and build more resilient food sources.

The Evolution of Antibody Discovery and Engineering

In recent years, the discovery and design of small non-canonical antibody types, such as variable heavy domain of heavy chains (VHH), have enabled new avenues of research. These single-domain antibodies (sdAbs) offer significant benefits due to their small size, high affinity and stability, low immunogenicity, good solubility, and enhanced tissue penetration.2

Diagram of full-length antibodies and antibody fragments including heavy-chain-only antibodies, Fab, scFv, and VHH/single-domain antibodies.

As these novel antibody types were discovered and characterized, engineering methods for both monoclonal antibodies (mAbs) and VHH have also evolved tremendously. Early hybridoma techniques for the production of mAbs relied on the availability of suitable myeloma cell lines and risked low yields and genetic instability. Later hybridoma approaches minimized the number of animals needed and integrated humanization methods to reduce immunogenicity.1 Now, the gold standard method of mAb production is a recombinant approach, which reduces the need for animal involvement and increases the consistency between production lots of the antibody.

Methods for identifying the best antibody clone for the desired application have also expanded. Phage display is a method to discover high-affinity full-size mAbs or sdAbs by engineering a library of bacteriophages that display unique proteins on their surface, then exposing them to a target molecule. Phages that bind to the target are eluted and used to infect E. coli bacteria. Then the infected cells are amplified and the phages isolated to derive the target antibodies.3 Completing multiple rounds of panning further refines antibody screening.3

The cumulative effect of these advancements has resulted in a new generation of enhanced mAbs and VHH, now positioned at the leading edge of diagnostics and therapeutics.

Antibody-based Rapid Immunoassays

With these developments, antibodies have become integral to various technologies that aid in disease diagnosis, environmental monitoring, food safety, and more. Immunoassays leverage antibodies’ natural ability to bind to target molecules. Two common formats are enzyme-linked immunosorbent assays (ELISA) and lateral flow assays (LFA).

ELISA is a laboratory-based assay that uses capture antibodies specific to the desired analyte and detection antibodies labeled with an enzyme like horseradish peroxidase or alkaline phosphatase. After a substrate is added to the plate, a chromogenic reaction converts the substrate into a colored product measured by the plate reader.4

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LFAs are used as rapid point-of-care (POC) assays. Here, samples are applied and through capillary force, flow laterally to a conjugate pad labeled with antibodies. If the target analyte is present in the sample, it will bind to the detection antibodies, and a colored line will appear.

Antibodies as Biosensors

Both ELISA and LFA-based immunoassays are frequently used for detecting pathogens, drugs of abuse, hormones, and contaminants in biologic samples including blood, saliva, urine, milk, and water5. Both mAbs and VHH are well-suited for point-of-care biosensing diagnostics due to their inherent selectivity and ability to act as high-affinity recognition elements.

Single-domain antibodies, owing to their small size and ability to penetrate to otherwise inaccessible epitopes, lend themselves to use in small portable biosensors, and are currently being used in environmental and food monitoring.6 For the detection of environmental contaminants, sbAb-based biosensors demonstrated good stability. Furthermore, they have been explored for detecting biomarkers in human samples like tumor glycoproteins and viral antigens. Strategic immobilization of the antibodies onto a solid support and ensuring analytes are accessible to the antibodies are key to designing effective biosensors.7

Antibodies in Imaging

In imaging applications, antibodies have proven to be invaluable in helping researchers and clinicians diagnose malignancies. In 1998, the first mAb approved for tumor imaging used an IgG molecule with an affinity for the tumor-associated antigen TAG-72, found in many adenocarcinomas.8 The application of antibodies to diagnostic imaging has huge potential, as almost any imaging probe can be linked to an antibody.

Watch an overview of our workflow for multiplex imaging

Multiplex immunofluorescence image stained using the Activated T Cell PathPlex Panel.

Antibodies in PET, CT, and MRI

For cancer imaging and localization, modality-specific antibody probes include radioisotopes for SPECT and PET imaging, fluorophores for optical imaging, microbubbles for ultrasound, paramagnetic particles for MRI, CT contrast agents, and multimodality probes.9 ImmunoPET, in particular, has emerged as a breakthrough in diagnostic antibody applications, combining the specificity of mAb targeting with the sensitivity of PET.

Moreover, non-canonical antibody types like VHH and bispecific antibodies are being explored for imaging applications. VHH may shorten time between administration and imaging due to their small size, rapid uptake in tissues, and faster blood clearance compared to mAbs. Moreover, VHH have been shown to have high specificity to detect primary and metastatic lesions in models of PET/CT imaging of melanoma, breast, ovarian, and early and advanced pancreatic lesions.10 Bispecific antibodies–engineered antibodies that bind two antigenic epitopes–play a role in cancer imaging as well. For example, a bispecific antibody targeting EGFR and CD105 simultaneously was able to visualize small brain modules in mice that may otherwise be missed on conventional PET.11

Advances in Immunohistochemistry

Immunohistochemistry (IHC) is used in several FDA-approved companion diagnostics to help select appropriate mAb therapies. For example, pathologists use immunohistochemistry (IHC) to assess HER2-positivity in breast and ovarian cancers, leveraging monoclonal or polyclonal antibodies conjugated to an enzyme. When added to biopsied tissue samples, this combination binds to HER2 proteins in cell surfaces, generating a visible staining that reveals the proportion of HER2 proteins present in the sample.12 Patients with HER2+ breast cancer are eligible for targeted therapies against this protein.

Although IHC has earned its place as a gold standard method in cancer diagnostics, it can only detect a single marker at a time. Multiplex immunofluorescence (mIF) is a specialized type of IHC that addresses the need in personalized medicine to assess multiple biomarkers simultaneously. Using fluorescently labeled antibodies, mIF can detect up to 40 or more biomarkers simultaneously, reducing the need for tissue and enhancing the amount of diagnostic and prognostic information that can be obtained.13
Diagram of the multitude of downstream VHH engineering options including bivalent VHH, enzyme-VHH conjugates, nanoparticle-VHH conjugates, and protein-conjugated VHH.
Used under a CC-BY 4.0 License.

Antibodies for Therapeutics

Due to their ability to closely target some cancer cell surface proteins without the systemic drawbacks of standard chemotherapy, mAb therapies are widely used in cancers that express known targets like EGFR and HER2.14 Antibody therapies are also well established for treating infectious diseases and autoimmune disorders like inflammatory bowel diseases, type 1 diabetes mellitus, and multiple sclerosis.15

Bispecific Antibodies

To enhance efficacy and reduce risk of drug resistance and toxicity from combination therapies, bispecific antibodies are engineered to target two antigenic epitopes. By performing two functions in one molecule—binding tumor cells and recruiting cytotoxic immune cells, for example—bispecific antibodies can do more with less drug and potentially fewer side effects. As of 2023, seven bispecific antibodies have been approved for cancer therapy, with many more expected to come to market in the future. Bispecific antibodies can be either IgG-based or fragment-based. Antibody fragment formats carry advantages in yield, cost, and tumor penetration but require careful engineering to ensure optimal affinity and valency.16 The known advantages of VHH, including increased solubility and thermal stability, can be harnessed into bispecific or even trispecific VHH, in which two or three VHH domains are connected by a flexible peptide linker. Multispecific VHH are undergoing investigation for treatment of solid tumors and conditions like psoriatic arthritis and psoriasis.17

Antibody-Drug Conjugates

Advances in antibody engineering have enabled more precise payload targeting, allowing for highly specific therapies that deliver treatments directly to disease sites. Beyond standard mAb treatment, antibody-based therapy has expanded to include other types of drug products, such as antibody-drug conjugates (ADCs). ADCs combine a mAb with a cytotoxic payload and a linker that releases the payload once inside a tumor cell.18 As of 2023, thirteen ADC products have been FDA-approved for certain solid and hematologic cancers.19

This mechanism of payload targeting has been expanded to antibody-antibiotic conjugates (AAC), which enable highly selective delivery of antibiotics to target bacteria-infected cells and enhancing phagocytosis while minimizing off-target effects.20 While no AAC products have been FDA-approved as of the time of publication, several are in preclinical or clinical trials for major infectious diseases like Staphylococcus aureus.

CAR-T for Cancer Therapy

For the treatment of hematological malignancies, chimeric antigen receptor T-cell (CAR-T) therapy provides new second- or third-line treatment options for some patients. Six CAR-T therapies have been approved in the US and most of these use single-chain fragment variables (scFvs) as targeting domains. However, use of scFvs for CAR-T design may have limitations, such as the potential for folding instability and changes in binding affinity when engineered into a CAR using a linker. To overcome some of these issues, VHH are being explored for numerous CAR-T therapies and offer advantages in stability, low immunogenicity, binding affinity, and modularity that could lead to improvements in CAR-T efficacy.21

Beyond CAR-T

CAR therapies employing other immune cells, such as Natural Killer cells (CAR-NK), macrophages (CAR-M), and B cells (CAR-B), have been tested in numerous studies. Since most of the cytokines released by NK cells do not induce systemic inflammation, CAR-NK therapies have potential to reduce undesirable side effects of CAR therapy. CAR-NK therapies assembled with MICA-specific VHH have been recently shown to selectively kill MICA-positive lung tumor cells in mice, opening up more avenues for immunotherapy development.22

The first in-human trial testing macrophages engineered to target phagocytic activity against tumors began recruiting in 2022. In mouse xenograft models, these CAR-M cells reduced tumor burden and prolonged overall survival.23 Studies also observed that CAR-M engineering converted the macrophage phenotype from M2 to M1, cross-presented antigens to T cells, and contributed to dendritic cell maturation. Researchers have also started to investigate the possibility of designing B cells and dendritic cells against specific tumor cells, primarily integrating scFv antibodies in their construction.24

Antibodies for Agricultural and Industrial Applications

The potential of antibodies extends into other fields such as environmental monitoring. Immunosensors are favored for their high sensitivity, selectivity, and ability to assess the immunoreaction in real-time. Monitoring programs, regulatory agencies, and environmental researchers leverage antibodies to assess for the presence of herbicides and toxins in water, soil, air, and plants.25 For example, a mAb has been leveraged in an immunoassay to screen for excess levels of forchlorfenuron (CPPU) in cucumber samples—a plant growth regulator that can harm environmental and human health when used excessively.26 Additionally, antibodies are used to diagnose and monitor pathogens that affect crops and livestock. VHH have been engineered into ELISA tests for certain toxins, pathogens, and herbicides in crops like zucchini.27

Full-length rmAb or VHH can also be employed to produce pathogen-resistant plants. This method can help overcome risks associated with older pathogen resistance approaches, such as expressing a DNA sequence that disrupts a pathogenic life cycle, which is effective for viruses but runs the risk of recombination events. Since the late 1980s, engineered antibody expression has been used in numerous crops, such as tomatoes, beets, and citrus, to improve pathogen resistance and create a more secure global food supply.28 Researchers have also successfully used VHH as a component in tailored plant immune receptors to confer pathogen resistance to new species. For instance, blocking crops susceptible to broad bean mottle virus and grapevine fanleaf virus and engineered with VHH recognizing these viral targets improved resistance to a potentially widespread crop disease. VHH can also enhance delivery of bioactive compounds to insects and reduce the amount of insecticide that needs to be administered.27

Future Trends in Antibody Applications

The future of antibodies is wide open. Ongoing research into the capabilities of smaller and multispecific antibodies will unlock new classes of diagnostics and therapeutics for diseases that are not amenable to traditional mAb approaches. The potential of VHH to cross the blood-brain barrier, for example, could unlock possibilities in brain cancer, migraine, Alzheimer’s disease, and more.18 Investments into continuous drug manufacturing processes and scalability will increase yields and permit the distribution of products for chronic conditions.29 And artificial intelligence (AI) and machine learning are beginning to be integrated into the screening and discovery process.30

Fueled by these leaps, the next generation of antibody technology will continue to focus on overcoming barriers of conventional antibody design and engineering, making highly efficacious antibodies accessible to more people and for more conditions. We should expect to see improvements in precision, stability, manufacturing, and administration options.31

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References

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CC-BY 4.0 image reference: Iezzi ME, Policastro L, Werbaijh S, Podhajcer O and Canziani GA (2018). Single-Domain Antibodies and the Promise of Molecular Targeting in Cancer Imaging and Treatment. Front. Immunol. 9:273. doi: 10.3389/fimmu.2018.00273





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