Genetic Tailoring With CRISPR

The development of therapeutic antibodies is well established but remains challenging and time-intensive. CRISPR gene editing, with its precision, accessibility and relatively low cost point could address some of the challenges, while also improving the functionality of therapeutic antibodies.

The development of therapeutic antibodies typically begins with identifying a disease-specific antigen. This is followed by generating antibodies that can bind to this antigen, using techniques such as hybridoma technology or phage display. Once an antibody candidate is selected, it undergoes extensive engineering to enhance its properties, including improving binding affinity, specificity, or half-life. Preclinical testing in cell-based assays and animal models follows to assess the antibody's safety and efficacy before it progresses to clinical trials.

While this pipeline is effective, it has limitations. Traditional methods of antibody production can be slow, and engineering antibodies to achieve the desired therapeutic properties often requires multiple iterations. CRISPR technology offers a more efficient approach to optimizing this process, particularly in the modification of host cells used for antibody production.

CRISPR/Cas9 technology can significantly advance the deconvolution of antibody targets. Deconvolution is the process of identifying the specific molecular targets that antibodies bind to, a critical step that has traditionally been a bottleneck in the development pipeline. Traditional phenotypic discovery methods can be effective in identifying potential therapeutic antibodies but falls short when it comes to precisely pinpointing the exact molecular targets of these antibodies. This step, known as target deconvolution, is typically labour-intensive and unreliable with conventional techniques, such as immunoprecipitation or protein overexpression.

Employing genome-wide CRISPR/Cas9 knockout libraries allows researchers to more efficiently identify the specific targets of antibodies discovered through phenotypic discovery. CRISPR/Cas9 screening has been used to successfully identify the targets of 97 percent of tested antibodies, far surpassing the success rates of traditional methods (1). This efficiency not only streamlines the development of new antibody-based therapies but also aids in uncovering novel targets, thereby broadening therapeutic options for diseases such as cancer and autoimmune disorders.

CRISPR can also add to the functionality of therapeutic antibodies. A significant application involves the site-specific alteration of antibodies to improve their ability to carry therapeutic agents (2). Traditional methods for attaching these agents rely heavily on chemical processes that can be imprecise and affect the antibody’s ability to bind to its target. However, with CRISPR, scientists can introduce specific sequences into the DNA of antibody-producing cells, such as hybridoma cells, allowing for the insertion of tags.

Sortase is an enzyme that can attach various molecules to a specific tag on the antibody, and FLAG is a short peptide sequence often used as a tag for purification or detection. By engineering these tags directly into the antibody genes, CRISPR enables the production of antibodies that can be conjugated with therapeutic agents, such as fluorescent markers or radioactive molecules, more accurately and uniformly. This method ensures that the therapeutic agents are attached only at desired locations on the antibody, preserving its ability to recognize and bind to the target antigen with high specificity. This precise modification improves the antibody’s effectiveness in targeting diseased cells or tissues, such as cancer cells, making it a more powerful tool for diagnosis and treatment in clinical settings.

When it comes to antibody production, CRISPR has yet more benefits. It can be used to engineer host cells employed for large-scale antibody production. One application in this area involves knocking out genes in CHO cells that negatively affect protein production. For example, researchers can use CRISPR to knock out the gene encoding the enzyme glutamine synthetase. This knockout enhances selection efficiency of the CHO cells expressing the desired antibodies (3).

As CRISPR continues to evolve, its integration into the field of therapeutic antibody development is poised to bring about substantial improvements in how treatments are designed and produced. Despite advances in antibody development, the emergence of resistance mechanisms in patients is a problem. CRISPR offers a way to quickly adapt antibodies to overcome these mechanisms. For instance, if a tumour evolves to express a variant of the target antigen that the original antibody no longer binds to effectively, CRISPR can be used to rapidly engineer new antibodies or modify existing ones to bind the new variant, without having to go back to the drawing board.

By enabling more precise modifications, CRISPR enhances the efficiency of antibody production and supports the creation of more effective therapies. Its ability to expedite the identification of novel therapeutic targets will also contribute to expanding the range of treatable conditions. As research progresses, the ongoing adaptation and refinement of CRISPR technologies is expected to play a critical role in the advancement of antibody-based therapies, making them more accessible and effective for a broader range of patients.