Dr. Clara Brando - Associate Director, WuXi AppTec
When it comes to modern medicine, one size does not fit all. A therapy that treats one person’s asthma, cancer or depression might do very little for another person with the same condition. Meanwhile, one medication’s side effects may be minimal for one patient but severe for another. To treat disease more effectively, researchers have long been working toward a more customized approach. Precision medicine incorporates genetics, environment, and lifestyle to devise a treatment that supports health by treating disease within the context of the individual.
Precision medicine has become a research priority in the United States and worldwide. The U.S. National Institutes of Health’s All of Us Research Program advances precision medicine through its proprietary health database that contains a million participants. But despite investments and advancements in precision medicine, the field has yet to fully understand and unlock treatments tailored to a person’s unique biology and pathology. As researchers shift their attention from genomics to proteomics, they are moving ever closer to the promise of precision medicine.
Protein biomarkers may be a single protein or a panel of multiple proteins that help diagnose illness, inform prognosis and monitor a patient’s biological response to treatment. Protein biomarkers are valuable indicators of disease because protein is easier to measure than the complex biological events they indicate. Numerous protein biomarkers are discovered by using genomic and proteomic approaches. The genomic approach allows the identification of gene modulation, while proteomic techniques pinpoint the expression of large proteins. Together, these techniques enable the identification of hallmark proteins whose concentration is modulated during disease progression and treatment.
Once identified, biomarkers of a given disease are used to follow its progression in patients and understand the individual’s reaction to medications, thus directing the compilation of personalized treatment.
Protein Biomarkers in Drug Development
Protein biomarkers play several valuable roles at various stages of drug development. For industry researchers, protein biomarkers can accelerate drug development by identifying promising pathways and potentially adverse reactions early in the process. These discoveries will ultimately improve efficacy and safety and increase the likelihood of successful regulatory applications and approvals.
Bioanalysis of Biomarkers
For a biomarker to be a diagnostic/prognostic tool, it needs to be detected and quantified in a robust, reliable manner. During clinical trials for new drugs, biomarker evaluation is one of the parameters used to evaluate efficacy, safety and dosage. Thus, suitable methods for biomarker monitoring are a primary concern in drug development.
Oncology boasts some of the most successful examples of using protein biomarkers for diagnosis and prognosis. The Human Epidermal Growth Factor Receptor (HER2) overexpresses in certain types of breast cancer. The combination of trastuzumab (Herceptin) and pertuzumab (Perjeta) has successfully controlled HER2 cancer-forming cells. Unlike traditional chemotherapy, this targeted treatment travels through the bloodstream to find additional cancer-forming cells wherever they are hiding in the patient. The targeted therapy is only effective in proteins that overexpress HER2—which can be as many as 30% of breast cancer tumors— but its effectiveness as an indicator makes a patient’s HER2 status a valuable biomarker for guiding precision medicine.
Identifying useful biomarkers can also help researchers better understand a disease’s pathophysiology and a drug’s mode of action. That understanding may open additional avenues for developers as cancer from multiple tissues of origin can share biomarkers. For example, solid tumors expressing high Tumor Mutational Burden (TMB) responded to treatment with pembrolizumab (Keytruda), an antibody that blocks the T-cell inhibitory receptor PDL-1. Keytruda was initially developed to treat melanoma but is now being used to combat various types of cancer in children and adults—especially tumors with multiple genetic mutations.
Protein biomarkers also support safety in preclinical programs by helping researchers refine dosage levels and evaluate a drug’s toxicological threshold. In doing so, protein biomarkers can help reduce side effects by identifying patients more susceptible to adverse events. For example, drug-induced nephrotoxicity is a common problem that affects as much as 60 percent of patients in clinical medicine. It’s a costly condition that often requires multiple therapeutic interventions, including hospitalization. The emergence of renal safety biomarkers can improve the identification of conditions that may cause renal injury.
Biomarkers may also improve safety at the time of diagnosis. Researchers have found that testing for protein biomarkers in ocular fluid from patients with uveal melanoma can predict metastatic risk earlier and more safely than conducting biopsies.
Streamlining Your Biomarker Validation Program
Drug developers and sponsors face several challenges in moving the field forward despite the great potential for precision medicine. For example, costs are a significant hurdle. Identifying and validating biomarkers can be a lengthy and complex process that generates substantial amounts of data and requires a large, experienced team.
There are also challenges inherent in the biomarker validation process. In some cases, it can be difficult to collect a suitable amount of the biological matrix. For example, children or people with severe illnesses (i.e., oncology patients) cannot spare large amounts of blood, plasma or serum, and their disease may interfere with the matrix and sacrifice assay accuracy. In such cases, scientists may use surrogate matrices, but not using the patient’s own biological material inherently limits their precision.
Some of the most common methods used in protein biomarker discovery and validation include:
- ELISA: Enzyme-Linked Immunosorbent Assays (ELISA) are some of the most common methods used in large-scale protein biomarker analysis. They provide high throughput, sensitivity, and selectivity and can be less expensive than other assays. A disadvantage of ELISA assays is that they can demonstrate the “matrix effect,” or interference from foreign substances present in biological fluid. Validating ELISA assays comes down to identifying and eliminating interference caused by using recombinant proteins and endogenous analytes during multiple dilutions.
- MSD multiplex/Luminex: Meso-Scale Discovery (MSD) multiplex immunoassays maintain the sensitivity of ELISAs while targeting several related analytes in a single sample. They can also handle complex biological matrices without multiple dilutions. Used primarily to detect cancer, the MSD multiplex platform measures phosphoproteins and intracellular signaling proteins. It can also detect biomarkers related to angiogenic (i.e., cardiovascular) and apoptosis (i.e., cell death).Using a quantitative assay to validate a multiplex with numerous analytes is challenging and may require less strict criteria. For this reason, exploratory studies should be conducted to identify fewer analytes that can be individually validated.
- Simoa HD-X: Quanterix and the Simoa Joint Laboratory feature the ultra-sensitive Simoa HD-X analyzer, detecting proteins with 1,000 times more sensitivity than the ELISA and MSD multiplex platforms. Researchers calibrate multiplex assays to test three or four biomarkers simultaneously, while Simoa HD-X can test up to 10. Simoa HD-X can detect biomarkers related to neurologic, cardiac and oncologic conditions with groundbreaking sensitivity.
- Flow cytometry: Flow cytometry includes evaluating cell markers (e.g., receptor expression) and monitoring cell phenotypes. It allows for simultaneous evaluation for multiple cellular parameters, can be used for high throughput testing and does not suffer from matrix interference. However, flow cytometry requires cell handling/culture capabilities using highly technical equipment and experienced personnel. Validating the precision and methodology for these assays is not as strictly defined as for pharmacokinetic assays, but common industry practices should be followed to achieve standardization.
- Elispot: These assays are used to monitor the production of chemokines by cells ex vivo or in vitro for immune modulators and vaccines. Elispot assays are extremely sensitive and allow high throughput testing, but they require cell culture capabilities and take several days to complete. Elispot assays are most often used in gene therapy to evaluate the number of cells responding to viral vectors and vaccine development to identify antigen-specific cells. Multiple protocols exist to validate these assays.
These platforms are an essential part of biomarker discovery and validation, but their efficacy will depend on several factors, including matrix, sample size, timeline and available resources. These assays require experienced technicians and/or proprietary techniques and machinery in many cases. Given the multitude of options, developers and sponsors must find laboratory testing partners with the skills and experience most appropriate to achieve the study’s goals.
Validating Biomarkers: A Regulatory Perspective
Measuring the body’s response to therapeutic intervention is critical to drug development. A new generation of reliable biomarkers could allow developers to learn about a drug’s toxicity and efficacy earlier in the development timeline, accelerating the creation of new therapies at lower costs.
But biomarker - quantitation is still a bit of a gray area. The U.S. FDA does not regulate protein biomarker assays. Further, researchers do not often share biomarker development data with the broader scientific community. But the U.S. FDA’s Biomarker Qualification Program works with external stakeholders to develop and evaluate biomarkers to address that challenge. Qualified biomarkers within the program enjoy formal regulatory recognition, and the resulting data are made publicly available for use in other drug development programs. When the U.S. FDA qualifies a biomarker, it is designated for a specific Context of Use (CoU). The regulatory body defines the biomarker’s purpose and manner of use, but drug developers can provide additional data to expand its CoU as they conduct further studies.
Whether a sponsor chooses to participate in the biomarker qualification process, validation data strongly supports subsequent Investigative New Drug (IND), New Drug Applications (NDA) or Biologic License Application (BLA) submissions. While not required, regulators may look favorably on biomarker data used to measure a drug’s efficacy and reliability as an indicator of disease. Experienced laboratory testing partners can help developers choose the most effective pathway for validating protein biomarkers and help build a case for biomarkers that will support regulatory approval.
The Future of Protein Biomarkers
Whether they are used to monitor high blood pressure, regulate kidney function or find previously undetectable cancer, biomarkers are critical tools in preventing and treating disease. But patients are not all created equal. Precision medicine can improve health on a much grander scale, and protein biomarkers will be an essential means to making medical treatment both more personalized and more precise.
To date, the field of oncology has seen the most significant achievements in precision medicine. But protein biomarkers promise game-changing impacts across other areas of medicine, including autoimmunity, cardiovascular disease and infectious disease. Studies incorporating these emerging methods will be successful as precision medicine remains a medical priority.
The bottom line on protein biomarkers is that identifying and validating them can be complex and costly. Working with an experienced laboratory partner with expertise in various assay platforms, validation pathways and regulatory dynamics can mean the difference between your program’s success or failure.
Clara Brando received her Pharma Doctor and her Ph.D. in immunology/Immuno-pathology from the University of Turin (Italy) in 1990. She received post-doctoral training at the Laboratory of Immunology at the National Institute of Allergic and Infectious Disease (NIAID) at NIH from 1990-94. During this time, Brando was trained in Cellular immunology with emphasis on T cell response. Brando has served as a senior scientist at Temple University, Wistar Institute and The Walter Reed Institute for Research, working on autoimmunity, immunity to cancer, and vaccines to infectious agents. Brando’s research focuses on the generation of cell-based and ligand binding assays to assess the cellular and humoral immune response to vaccines and therapeutics. Brando has developed various novel flow-cytometry, Elispots and Elisa assays to investigate T and B cell response.
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