medical research projects

77 interesting medical research topics for 2024

Last updated

25 November 2023

Reviewed by

Brittany Ferri, PhD, OTR/L

Medical research is the gateway to improved patient care and expanding our available treatment options. However, finding a relevant and compelling research topic can be challenging.

Use this article as a jumping-off point to select an interesting medical research topic for your next paper or clinical study.

• How to choose a medical research topic

When choosing a research topic , it’s essential to consider a couple of things. What topics interest you? What unanswered questions do you want to address? 

During the decision-making and brainstorming process, here are a few helpful tips to help you pick the right medical research topic:

Focus on a particular field of study

The best medical research is specific to a particular area. Generalized studies are often too broad to produce meaningful results, so we advise picking a specific niche early in the process. 

Maybe a certain topic interests you, or your industry knowledge reveals areas of need.

Look into commonly researched topics

Once you’ve chosen your research field, do some preliminary research. What have other academics done in their papers and projects? 

From this list, you can focus on specific topics that interest you without accidentally creating a copycat project. This groundwork will also help you uncover any literature gaps—those may be beneficial areas for research.

Get curious and ask questions

Now you can get curious. Ask questions that start with why, how, or what. These questions are the starting point of your project design and will act as your guiding light throughout the process. 

For example: 

What impact does pollution have on children’s lung function in inner-city neighborhoods? 

Why is pollution-based asthma on the rise? 

How can we address pollution-induced asthma in young children? 

• 77 medical research topics worth exploring in 2023

Need some research inspiration for your upcoming paper or clinical study? We’ve compiled a list of 77 topical and in-demand medical research ideas. Let’s take a look. 

• Exciting new medical research topics

If you want to study cutting-edge topics, here are some exciting options:

COVID-19 and long COVID symptoms

Since 2020, COVID-19 has been a hot-button topic in medicine, along with the long-term symptoms in those with a history of COVID-19. 

Examples of COVID-19-related research topics worth exploring include:

The long-term impact of COVID-19 on cardiac and respiratory health

COVID-19 vaccination rates

The evolution of COVID-19 symptoms over time

New variants and strains of the COVID-19 virus

Changes in social behavior and public health regulations amid COVID-19

Vaccinations

Finding ways to cure or reduce the disease burden of chronic infectious diseases is a crucial research area. Vaccination is a powerful option and a great topic to research. 

Examples of vaccination-related research topics include:

mRNA vaccines for viral infections

Biomaterial vaccination capabilities

Vaccination rates based on location, ethnicity, or age

Public opinion about vaccination safety 

Artificial tissues fabrication

With the need for donor organs increasing, finding ways to fabricate artificial bioactive tissues (and possibly organs) is a popular research area. 

Examples of artificial tissue-related research topics you can study include:

The viability of artificially printed tissues

Tissue substrate and building block material studies

The ethics and efficacy of artificial tissue creation

• Medical research topics for medical students

For many medical students, research is a big driver for entering healthcare. If you’re a medical student looking for a research topic, here are some great ideas to work from:

Sleep disorders

Poor sleep quality is a growing problem, and it can significantly impact a person’s overall health. 

Examples of sleep disorder-related research topics include:

How stress affects sleep quality

The prevalence and impact of insomnia on patients with mental health conditions

Possible triggers for sleep disorder development

The impact of poor sleep quality on psychological and physical health

How melatonin supplements impact sleep quality

Alzheimer’s and dementia 

Cognitive conditions like dementia and Alzheimer’s disease are on the rise worldwide. They currently have no cure. As a result, research about these topics is in high demand. 

Examples of dementia-related research topics you could explore include:

The prevalence of Alzheimer’s disease in a chosen population

Early onset symptoms of dementia

Possible triggers or causes of cognitive decline with age

Treatment options for dementia-like conditions

The mental and physical burden of caregiving for patients with dementia

• Lifestyle habits and public health

Modern lifestyles have profoundly impacted the average person’s daily habits, and plenty of interesting topics explore its effects. 

Examples of lifestyle and public health-related research topics include:

The nutritional intake of college students

The impact of chronic work stress on overall health

The rise of upper back and neck pain from laptop use

Prevalence and cause of repetitive strain injuries (RSI)

• Controversial medical research paper topics

Medical research is a hotbed of controversial topics, content, and areas of study. 

If you want to explore a more niche (and attention-grabbing) concept, here are some controversial medical research topics worth looking into:

The benefits and risks of medical cannabis

Depending on where you live, the legalization and use of cannabis for medical conditions is controversial for the general public and healthcare providers.

Examples of medical cannabis-related research topics that might grab your attention include:

The legalization process of medical cannabis

The impact of cannabis use on developmental milestones in youth users

Cannabis and mental health diagnoses

CBD’s impact on chronic pain

Prevalence of cannabis use in young people

The impact of maternal cannabis use on fetal development 

Understanding how THC impacts cognitive function

Human genetics

The Human Genome Project identified, mapped, and sequenced all human DNA genes. Its completion in 2003 opened up a world of exciting and controversial studies in human genetics.

Examples of human genetics-related research topics worth delving into include:

Medical genetics and the incidence of genetic-based health disorders

Behavioral genetics differences between identical twins

Genetic risk factors for neurodegenerative disorders

Machine learning technologies for genetic research

Sexual health studies

Human sexuality and sexual health are important (yet often stigmatized) medical topics that need new research and analysis.

As a diverse field ranging from sexual orientation studies to sexual pathophysiology, examples of sexual health-related research topics include:

The incidence of sexually transmitted infections within a chosen population

Mental health conditions within the LGBTQIA+ community

The impact of untreated sexually transmitted infections

Access to safe sex resources (condoms, dental dams, etc.) in rural areas

• Health and wellness research topics

Human wellness and health are trendy topics in modern medicine as more people are interested in finding natural ways to live healthier lifestyles. 

If this field of study interests you, here are some big topics in the wellness space:

Gluten sensitivity

Gluten allergies and intolerances have risen over the past few decades. If you’re interested in exploring this topic, your options range in severity from mild gastrointestinal symptoms to full-blown anaphylaxis. 

Some examples of gluten sensitivity-related research topics include:

The pathophysiology and incidence of Celiac disease

Early onset symptoms of gluten intolerance

The prevalence of gluten allergies within a set population

Gluten allergies and the incidence of other gastrointestinal health conditions

Pollution and lung health

Living in large urban cities means regular exposure to high levels of pollutants. 

As more people become interested in protecting their lung health, examples of impactful lung health and pollution-related research topics include:

The extent of pollution in densely packed urban areas

The prevalence of pollution-based asthma in a set population

Lung capacity and function in young people

The benefits and risks of steroid therapy for asthma

Pollution risks based on geographical location

Plant-based diets

Plant-based diets like vegan and paleo diets are emerging trends in healthcare due to their limited supporting research. 

If you’re interested in learning more about the potential benefits or risks of holistic, diet-based medicine, examples of plant-based diet research topics to explore include:

Vegan and plant-based diets as part of disease management

Potential risks and benefits of specific plant-based diets

Plant-based diets and their impact on body mass index

The effect of diet and lifestyle on chronic disease management

Health supplements

Supplements are a multi-billion dollar industry. Many health-conscious people take supplements, including vitamins, minerals, herbal medicine, and more. 

Examples of health supplement-related research topics worth investigating include:

Omega-3 fish oil safety and efficacy for cardiac patients

The benefits and risks of regular vitamin D supplementation

Health supplementation regulation and product quality

The impact of social influencer marketing on consumer supplement practices

Analyzing added ingredients in protein powders

• Healthcare research topics

Working within the healthcare industry means you have insider knowledge and opportunity. Maybe you’d like to research the overall system, administration, and inherent biases that disrupt access to quality care. 

While these topics are essential to explore, it is important to note that these studies usually require approval and oversight from an Institutional Review Board (IRB). This ensures the study is ethical and does not harm any subjects. 

For this reason, the IRB sets protocols that require additional planning, so consider this when mapping out your study’s timeline. 

Here are some examples of trending healthcare research areas worth pursuing:

The pros and cons of electronic health records

The rise of electronic healthcare charting and records has forever changed how medical professionals and patients interact with their health data. 

Examples of electronic health record-related research topics include:

The number of medication errors reported during a software switch

Nurse sentiment analysis of electronic charting practices

Ethical and legal studies into encrypting and storing personal health data

Inequities within healthcare access

Many barriers inhibit people from accessing the quality medical care they need. These issues result in health disparities and injustices. 

Examples of research topics about health inequities include:

The impact of social determinants of health in a set population

Early and late-stage cancer stage diagnosis in urban vs. rural populations

Affordability of life-saving medications

Health insurance limitations and their impact on overall health

Diagnostic and treatment rates across ethnicities

People who belong to an ethnic minority are more likely to experience barriers and restrictions when trying to receive quality medical care. This is due to systemic healthcare racism and bias. 

As a result, diagnostic and treatment rates in minority populations are a hot-button field of research. Examples of ethnicity-based research topics include:

Cancer biopsy rates in BIPOC women

The prevalence of diabetes in Indigenous communities

Access inequalities in women’s health preventative screenings

The prevalence of undiagnosed hypertension in Black populations

• Pharmaceutical research topics

Large pharmaceutical companies are incredibly interested in investing in research to learn more about potential cures and treatments for diseases. 

If you’re interested in building a career in pharmaceutical research, here are a few examples of in-demand research topics:

Cancer treatment options

Clinical research is in high demand as pharmaceutical companies explore novel cancer treatment options outside of chemotherapy and radiation. 

Examples of cancer treatment-related research topics include:

Stem cell therapy for cancer

Oncogenic gene dysregulation and its impact on disease

Cancer-causing viral agents and their risks

Treatment efficacy based on early vs. late-stage cancer diagnosis

Cancer vaccines and targeted therapies

Immunotherapy for cancer

Pain medication alternatives

Historically, opioid medications were the primary treatment for short- and long-term pain. But, with the opioid epidemic getting worse, the need for alternative pain medications has never been more urgent. 

Examples of pain medication-related research topics include:

Opioid withdrawal symptoms and risks

Early signs of pain medication misuse

Anti-inflammatory medications for pain control

• Identify trends in your medical research with Dovetail

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Medical research involves research in a wide range of fields, such as biology, chemistry, pharmacology and toxicology with the goal of developing new medicines or medical procedures or improving the application of those already available. It can be viewed as encompassing preclinical research (for example, in cellular systems and animal models) and clinical research (for example, clinical trials).

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Refining neoadjuvant immunotherapy for resectable lung cancer

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Genomics reveal unknown mutation-promoting agents at global sites

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Home » 500+ Medical Research Topic Ideas

500+ Medical Research Topic Ideas

Table of Contents

Medical research plays a crucial role in advancing healthcare and improving human health. It involves the scientific study of various aspects of medicine and health, including the causes, prevention, diagnosis, and treatment of diseases. Medical research is a dynamic and ever-evolving field, with new discoveries and breakthroughs happening all the time. It encompasses a wide range of disciplines, from basic science to clinical research, and involves collaboration between scientists, doctors, and other healthcare professionals. In this article, we will explore some exciting new and latest medical research topic ideas that are currently trending in the field. These Research Topics cover a variety of areas, including genetics, infectious diseases, mental health, and more.

Medical Research Topic Ideas

Medical Research Topic Ideas are as follows:

• The efficacy of mindfulness meditation in reducing symptoms of depression and anxiety
• The effects of vitamin D supplementation on bone health in postmenopausal women
• The impact of social media on body image and eating disorders in adolescents
• The effectiveness of telemedicine in improving access to healthcare in rural communities
• The benefits and risks of long-term use of statins for cholesterol management
• The role of gut microbiota in the development of autoimmune diseases
• The potential of gene therapy for the treatment of genetic disorders
• The relationship between sleep disorders and cardiovascular disease
• The use of artificial intelligence in diagnosing and treating cancer
• The effect of exercise on cognitive function in older adults
• The impact of environmental factors on the development of asthma in children
• The effectiveness of cognitive-behavioral therapy for the treatment of PTSD in veterans
• The potential benefits of psychedelic-assisted therapy for the treatment of mental illness
• The relationship between diet and risk of developing type 2 diabetes
• The role of epigenetics in the development of psychiatric disorders
• The impact of COVID-19 on mental health and well-being
• The effectiveness of mindfulness-based stress reduction in improving quality of life in cancer patients
• The impact of childhood trauma on the development of mental illness in adulthood
• The benefits and risks of hormone replacement therapy for menopausal women
• The effect of music therapy on reducing symptoms of dementia in older adults
• The relationship between gut microbiota and obesity
• The impact of socioeconomic status on health outcomes
• The effectiveness of acupuncture in treating chronic pain
• The use of stem cells in regenerative medicine
• The impact of air pollution on respiratory health
• The potential of nanotechnology in drug delivery
• The relationship between social support and mental health
• The effectiveness of mindfulness-based interventions for addiction treatment
• The role of inflammation in the development of Alzheimer’s disease
• The use of virtual reality in pain management
• The impact of exercise on mental health in adolescents
• The effectiveness of group therapy for the treatment of substance abuse
• The relationship between sleep and weight management
• The benefits and risks of using medical marijuana for chronic pain management
• The role of the immune system in the development of autoimmune diseases
• The effectiveness of cognitive rehabilitation therapy for traumatic brain injury patients
• The impact of maternal stress on fetal development
• The relationship between physical activity and cardiovascular health
• The potential of gene editing for the treatment of genetic disorders
• The effectiveness of mindfulness-based interventions for reducing symptoms of postpartum depression.
• The impact of social media on mental health
• Investigating the use of virtual reality in pain management
• The effectiveness of mindfulness-based interventions for depression
• Exploring the relationship between sleep and anxiety
• Examining the efficacy of telemedicine in delivering mental health care
• Investigating the impact of environmental factors on the development of cancer
• The effect of exercise on cognitive function in elderly individuals
• Examining the potential benefits of psychedelic-assisted therapy for PTSD
• The relationship between diet and cardiovascular disease
• Investigating the impact of air pollution on respiratory health
• Examining the effects of social isolation on mental and physical health
• The use of machine learning in diagnosing medical conditions
• Investigating the effectiveness of acupuncture in pain management
• The impact of childhood trauma on mental and physical health outcomes in adulthood
• Examining the relationship between stress and autoimmune diseases
• The effect of music therapy on mental health outcomes
• Investigating the impact of gender on healthcare outcomes
• Examining the relationship between sleep apnea and cardiovascular disease
• The effectiveness of mindfulness-based interventions for chronic pain
• Investigating the potential benefits of medical marijuana for chronic pain management
• Examining the impact of climate change on infectious disease transmission
• The use of robotics in surgery
• Investigating the relationship between alcohol consumption and cancer risk
• The effect of meditation on blood pressure control
• Examining the impact of social determinants of health on healthcare outcomes
• The role of genetics in the development of mental health conditions
• Investigating the efficacy of cognitive-behavioral therapy for anxiety disorders
• Examining the relationship between inflammation and depression
• The impact of shift work on sleep and circadian rhythms
• Investigating the potential benefits of probiotics in gut health
• Examining the relationship between diet and mental health outcomes
• The effectiveness of art therapy for individuals with dementia
• Investigating the relationship between chronic pain and mental health outcomes
• The impact of artificial intelligence on medical diagnosis and treatment
• Examining the effectiveness of exercise in treating depression
• Investigating the relationship between inflammation and cardiovascular disease
• The effect of aromatherapy on anxiety and stress
• Examining the impact of social support on mental health outcomes
• The effectiveness of hypnotherapy in pain management.
• The role of gut microbiota in immune system modulation
• Effects of intermittent fasting on insulin sensitivity in obese individuals
• Impact of smartphone usage on sleep quality and quantity
• The potential therapeutic effects of CBD on anxiety disorders
• Association between shift work and cardiovascular disease
• Efficacy and safety of psychedelic-assisted psychotherapy in treating depression
• The relationship between stress and autoimmune diseases
• Novel therapies for Alzheimer’s disease
• The effects of high-intensity interval training on metabolic syndrome
• The role of epigenetics in the development of cancer
• The effectiveness of virtual reality in pain management
• The effects of social media on body image and eating disorders
• The association between air pollution and respiratory diseases
• Effects of mindfulness meditation on stress and anxiety in healthcare workers
• The potential benefits of ketogenic diet in treating epilepsy
• The relationship between sleep apnea and cardiovascular disease
• The impact of climate change on infectious disease outbreaks
• The effectiveness of exercise in preventing falls in the elderly
• The effects of blue light exposure on circadian rhythm and sleep quality
• The association between alcohol consumption and liver disease
• The effectiveness of cognitive-behavioral therapy in treating obsessive-compulsive disorder
• The role of gut-brain axis in mental health disorders
• The association between chronic inflammation and cancer
• The efficacy and safety of probiotics in treating irritable bowel syndrome
• The effects of social isolation on mental health in the elderly
• The impact of exercise on cognitive function in Parkinson’s disease patients
• The association between vitamin D deficiency and autoimmune diseases
• The potential therapeutic effects of music therapy in dementia patients
• The effects of second-hand smoke on cardiovascular health
• The association between maternal smoking and infant health outcomes
• The role of microbiome in the development of allergies
• The association between sleep duration and obesity
• The effects of blue light-blocking glasses on sleep quality and quantity
• The potential therapeutic effects of ketamine in treating depression
• The association between gut dysbiosis and inflammatory bowel disease
• The effectiveness of cognitive rehabilitation therapy in traumatic brain injury patients
• The impact of early childhood stress on adult mental health
• The role of inflammation in the development of type 2 diabetes
• The potential benefits of plant-based diets in preventing chronic diseases.
• The effects of exercise on cognitive function in aging adults
• The association between sleep disorders and cardiovascular disease
• The potential therapeutic effects of psilocybin in treating addiction
• The role of gut microbiota in the development of autism spectrum disorder
• The effectiveness of mindfulness-based interventions in treating depression
• The effects of air pollution on cognitive function
• The association between maternal mental health and child development
• The potential therapeutic effects of cannabis in treating chronic pain
• The role of diet in the prevention and management of diabetes
• The effects of social support on mental health in cancer patients
• The association between shift work and mental health disorders
• The efficacy of antiviral therapies in treating COVID-19
• The effects of exercise on bone health in postmenopausal women
• The association between sleep disorders and obesity
• The potential therapeutic effects of mindfulness meditation in treating anxiety disorders
• The role of gut microbiota in the development of metabolic disorders
• The effectiveness of virtual reality therapy in treating phobias
• The association between social support and immune system function
• The impact of early life stress on adult cardiovascular health
• The potential benefits of intermittent fasting in cancer prevention
• The effects of air pollution on pregnancy outcomes
• The association between maternal obesity and child health outcomes
• The efficacy of cognitive-behavioral therapy in treating post-traumatic stress disorder
• The effects of sedentary behavior on metabolic health
• The potential therapeutic effects of omega-3 fatty acids in treating depression
• The role of microbiome in the development of obesity
• The association between social isolation and cognitive decline in older adults
• The impact of environmental toxins on child development
• The potential benefits of plant-based diets in treating metabolic disorders
• The effects of sleep deprivation on cognitive function
• The association between maternal stress and fetal development
• The efficacy of pharmacological interventions in treating anxiety disorders
• The effects of air pollution on respiratory health in children
• The association between social support and cardiovascular health
• The potential therapeutic effects of mindfulness meditation in treating chronic pain
• The role of diet in the prevention and management of cardiovascular disease
• The effects of exercise on mental health in children and adolescents
• The association between social support and cancer survival rates
• The impact of environmental factors on epigenetic modifications and disease susceptibility.
• The effects of exercise on immune function
• The association between maternal obesity and infant health outcomes
• The impact of air pollution on cognitive function in children
• The association between sleep deprivation and mental health disorders
• The effectiveness of virtual reality in rehabilitation after stroke
• The role of the microbiome in the development of obesity
• The impact of noise pollution on cardiovascular health
• The association between depression and cardiovascular disease
• The association between periodontal disease and cardiovascular health
• The impact of social support on mental health outcomes in cancer patients
• The potential therapeutic effects of melatonin in treating sleep disorders
• The association between air pollution and cognitive decline in older adults
• The effectiveness of group therapy in treating social anxiety disorder
• The impact of exercise on bone health in postmenopausal women
• The association between alcohol consumption and breast cancer risk
• The effects of blue light exposure on melatonin secretion and sleep quality
• The potential therapeutic effects of stem cells in treating Parkinson’s disease
• The role of inflammation in the development of depression
• The association between gut dysbiosis and depression
• The effectiveness of music therapy in reducing anxiety in cancer patients
• The impact of social media on mental health in adolescents
• The potential therapeutic effects of ketamine in treating post-traumatic stress disorder
• The association between vitamin D deficiency and cardiovascular disease
• The effects of chronic stress on immune function
• The potential benefits of Mediterranean diet in preventing cardiovascular disease
• The impact of noise pollution on sleep quality and quantity
• The association between sedentary behavior and depression
• The effects of air pollution on fetal development and pregnancy outcomes
• The potential therapeutic effects of acupuncture in treating anxiety disorders
• The role of microbiome in the development of multiple sclerosis
• The effectiveness of mindfulness-based stress reduction in treating chronic pain
• The impact of artificial sweeteners on metabolic health
• The association between sleep duration and cardiovascular disease
• The effects of social isolation on immune function in older adults
• The potential therapeutic effects of omega-3 fatty acids in treating depression.
• The effects of exercise on cognitive function in older adults
• The association between maternal mental health and infant development
• The potential therapeutic effects of probiotics in treating depression
• The impact of air pollution on lung health in children
• The association between sleep quality and academic performance in adolescents
• The effectiveness of cognitive-behavioral therapy in treating insomnia
• The role of gut microbiota in the development of metabolic syndrome
• The potential therapeutic effects of ayahuasca in treating addiction
• The impact of green space on mental health in urban areas
• The association between sedentary behavior and cardiometabolic risk factors
• The effects of blue light on mood and cognitive performance in shift workers
• The potential benefits of vegan diets in preventing chronic diseases
• The impact of social support on mental health in older adults
• The association between air pollution and lung cancer risk
• The effects of exercise on mental health in cancer survivors
• The potential therapeutic effects of ketamine in treating bipolar disorder
• The role of the microbiome in the development of rheumatoid arthritis
• The association between maternal nutrition and fetal development
• The effects of sleep deprivation on immune function
• The potential benefits of mindfulness meditation in managing chronic pain
• The impact of noise pollution on sleep-disordered breathing
• The association between sedentary behavior and breast cancer risk
• The effects of blue light exposure on retinal health
• The potential therapeutic effects of deep brain stimulation in treating depression
• The role of gut microbiota in the development of non-alcoholic fatty liver disease
• The association between air pollution and neurodegenerative diseases
• The effects of social support on immune function in cancer patients
• The potential therapeutic effects of acupuncture in treating migraines
• The impact of light pollution on sleep quality and quantity
• The association between sedentary behavior and type 2 diabetes risk
• The effects of mindfulness meditation on cognitive function in older adults
• The potential benefits of the DASH diet in preventing hypertension
• The impact of social media on body dissatisfaction and eating disorders in adolescents
• The association between air pollution and kidney disease
• The effects of chronic stress on cardiovascular health
• The potential therapeutic effects of gene therapy in treating inherited diseases
• The role of microbiome in the development of atopic dermatitis
• The association between maternal smoking and childhood obesity
• The effects of blue light exposure on visual function and eye health
• The potential therapeutic effects of electroconvulsive therapy in treating depression.

Healthcare Research Topics for College Students

• The impact of healthcare policies on patient outcomes
• The effectiveness of telemedicine in improving access to healthcare
• The role of cultural competency in healthcare delivery
• The impact of social determinants of health on healthcare outcomes
• The effectiveness of different types of healthcare interventions
• The role of genetics in predicting and preventing chronic diseases
• The impact of the opioid epidemic on healthcare delivery
• The effectiveness of alternative medicine in managing chronic conditions
• The role of technology in improving patient safety
• The impact of healthcare provider burnout on patient care
• The effectiveness of different healthcare models in managing chronic diseases
• The role of patient education in improving healthcare outcomes
• The impact of healthcare disparities on access to care and health outcomes
• The effectiveness of healthcare systems in responding to public health emergencies
• The role of nutrition in disease prevention and management
• The impact of healthcare policy on healthcare costs and spending
• The effectiveness of mental health interventions in improving overall health outcomes
• The role of healthcare systems in addressing health disparities
• The impact of healthcare data analytics on clinical decision making
• The effectiveness of healthcare interventions in reducing healthcare-associated infections
• The role of patient-centered care in improving healthcare outcomes
• The impact of healthcare regulations on patient safety
• The effectiveness of vaccination programs in preventing infectious diseases
• The role of healthcare systems in promoting healthy lifestyle behaviors
• The impact of chronic diseases on healthcare costs and quality of life
• The effectiveness of preventative healthcare in improving health outcomes
• The role of healthcare technology in improving healthcare delivery
• The impact of healthcare funding on healthcare outcomes
• The effectiveness of healthcare interventions in managing chronic pain
• The role of healthcare providers in promoting health equity.

Community Medicine Research Topics for Medical Students

• The impact of community-based interventions on reducing the burden of non-communicable diseases in low-income communities.
• The effectiveness of vaccination campaigns in preventing infectious diseases in marginalized communities.
• The relationship between air pollution and respiratory health in urban communities.
• The prevalence and risk factors of substance abuse among homeless populations.
• The impact of social determinants of health on health outcomes in rural communities.
• The role of community health workers in improving maternal and child health outcomes in low-resource settings.
• The association between food insecurity and obesity in low-income populations.
• The prevalence and risk factors of mental health disorders among adolescents in urban communities.
• The effectiveness of school-based health promotion programs in improving health behaviors among children and adolescents.
• The role of community-based participatory research in addressing health disparities in underserved populations.
• The impact of social support networks on mental health outcomes among elderly populations.
• The relationship between access to healthcare services and health outcomes in rural communities.
• The effectiveness of smoking cessation interventions in reducing the burden of tobacco-related diseases.
• The prevalence and risk factors of sexually transmitted infections among young adults in urban communities.
• The role of community-based organizations in promoting healthy behaviors and preventing chronic diseases.
• The impact of climate change on the incidence and distribution of infectious diseases.
• The prevalence and risk factors of intimate partner violence among women in low-income communities.
• The effectiveness of health education programs in improving health literacy and health outcomes in underserved populations.
• The relationship between social support and adherence to treatment among patients with chronic diseases.
• The prevalence and risk factors of hypertension and diabetes in urban communities.
• The impact of community-based interventions on reducing healthcare costs and improving health outcomes.
• The role of mobile health technologies in improving access to healthcare services in rural communities.
• The prevalence and risk factors of obesity among children and adolescents in low-income communities.
• The effectiveness of community-based interventions in promoting healthy behaviors among pregnant women.
• The impact of housing conditions on health outcomes in marginalized communities.
• The relationship between access to healthy food and health outcomes in urban communities.
• The prevalence and risk factors of depression among elderly populations in rural communities.
• The role of social media in promoting healthy behaviors and preventing diseases among young adults.
• The effectiveness of telemedicine in improving access to healthcare services in underserved populations.
• The prevalence and risk factors of infectious diseases among migrant populations in urban areas.

Surgery Research Topics for Medical Students

• The efficacy and safety of minimally invasive surgery for various conditions
• Comparison of laparoscopic and open surgery for common procedures
• The impact of surgeon experience on surgical outcomes
• Analysis of postoperative complications and their management
• The role of robotics in surgery
• Investigating the use of artificial intelligence in surgery
• The effectiveness of non-pharmacological pain management techniques after surgery
• The effect of preoperative anxiety on postoperative recovery
• Evaluation of different surgical approaches for breast cancer treatment
• The benefits and risks of surgical treatment for obesity
• Investigating the use of stem cells in tissue repair following surgery
• The influence of nutrition on postoperative recovery and wound healing
• Analysis of the psychological impact of surgery on patients
• The effect of different anesthesia methods on postoperative outcomes
• Comparison of outcomes between day surgery and inpatient surgery
• Evaluation of the use of surgical checklists in improving patient safety
• The impact of age on surgical outcomes and recovery
• Investigating the use of 3D printing in surgical planning and implant design
• The benefits and risks of bariatric surgery in patients with diabetes
• The role of surgery in the treatment of chronic pain
• The efficacy of arthroscopic surgery for joint conditions
• The use of lasers in surgery
• Investigating the use of virtual reality in surgical training and education
• The effect of preoperative counseling on patient satisfaction and outcomes
• The impact of comorbidities on surgical outcomes
• Analysis of the economic impact of different surgical approaches
• Investigating the use of telemedicine in surgical consultations and follow-up care
• The effectiveness of surgical treatment for endometriosis
• Comparison of outcomes between single-incision and multiport laparoscopic surgery
• The use of robotics in urologic surgery.

Research Projects for Undergraduate Medical Students

• Investigating the role of genetics in the development of cancer
• Analyzing the effectiveness of different types of pain management strategies in postoperative patients
• Evaluating the impact of diet and exercise on obesity-related health outcomes
• Examining the relationship between sleep quality and mental health in medical students
• Investigating the efficacy of different types of antibiotics in treating common bacterial infections
• Analyzing the impact of electronic medical record systems on patient care
• Evaluating the effectiveness of different types of vaccines in preventing infectious diseases
• Examining the relationship between maternal nutrition and fetal development
• Investigating the use of telemedicine in delivering healthcare services to rural populations
• Analyzing the impact of smoking on lung function and respiratory health
• Evaluating the effectiveness of different types of rehabilitation programs for stroke patients
• Examining the relationship between physical activity and cardiovascular health
• Investigating the use of stem cells in treating various medical conditions
• Analyzing the impact of stress on mental and physical health outcomes
• Evaluating the effectiveness of different types of medical interventions in managing chronic pain
• Examining the relationship between social support and mental health outcomes in patients with chronic illnesses
• Investigating the use of mindfulness-based interventions in reducing anxiety and depression
• Analyzing the impact of environmental factors on health outcomes in urban populations
• Evaluating the effectiveness of different types of cancer treatments, such as chemotherapy, radiation therapy, and surgery
• Examining the relationship between nutrition and mental health in older adults
• Investigating the use of mobile health technologies in promoting healthy behaviors
• Analyzing the impact of air pollution on respiratory health in children
• Evaluating the effectiveness of different types of treatments for substance use disorders
• Examining the relationship between socioeconomic status and health outcomes
• Investigating the use of music therapy in managing pain and anxiety in hospitalized patients
• Analyzing the impact of social media on mental health outcomes in adolescents
• Evaluating the effectiveness of different types of interventions in managing symptoms of depression and anxiety in cancer patients
• Examining the relationship between sleep and cognitive function in older adults
• Investigating the use of animal-assisted therapy in promoting physical and mental health
• Analyzing the impact of climate change on health outcomes in vulnerable populations

About the author

Muhammad Hassan

Researcher, Academic Writer, Web developer

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The HMS scholarly project requirement is designed to provide every Harvard medical student with an opportunity to pursue a mentored scholarly experience analyzing a medical or health-related question, issue or problem in depth. Students can approach issues through a wide range of disciplines in the biomedical sciences, clinical sciences, humanities, arts or other field. The process of discovery is as important as the outcome. Students and their mentors are partners in this process.

Timeline (earlier submission is encouraged for all dates)

• May 1 of penultimate year (February 7 for global health projects) : Scholarly project concept and mentor information to OSE
• August 1 of graduating year: Scholarly project proposal and mentor agreement to OSE
• March 1 of graduating year : Scholarly report to OSE   

Current MD students 

Access the internal Scholarly Project site for more information on the scholarly project requirement. Harvard Key is required. 

View a sampling of scholarly project titles from past students

Scholarly Project

Objectives of the scholarly project, for all pathways students, the learning objectives of the scholarly project are to:.

• Engage in original scholarly work addressing a question in medicine/health, using approaches from a range of scientific or social science fields
• Work closely with a faculty mentor on a scholarly project in a partnership that is mutually beneficial
• Inspire curiosity, develop critical thinking skills, and identify analytical tools useful for the future physician-scholar
• Craft a scientific plan through a written proposal that will be vetted by HMS faculty
• Review and reinforce (via quizzes) basic concepts concerning responsible conduct of research and IRB processes
• Write a report on scholarly work which will be critiqued by HMS faculty

Some Pathways students will also have the following experiences, depending on the nature of their projects:

• Review and reinforce (via quizzes) basic concepts concerning global health professionalism (for those working on global health projects)
• Prepare a submission to local IRB offices on the proposed human subjects or animal research
• Learn statistical tools and analysis techniques through practical experience
• Prepare for the challenges of working and living abroad
• Write a manuscript for publication and learn about publication requirements, processes  and protecting one’s intellectual property

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Md students may find scholarly projects through:.

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MD Students may request up to four months of optional SIM500 credit for full-time or part-time work on their approved scholarly project. The OSE faculty director must approve this credit application. HMS students should visit the internal HMS site for more information . Harvard Key is required to access. Contact Molly Hannon  with any questions. 

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A robust Faculty Committee on Scholarship in Medicine advises students pursuing their scholarly projects. Cannon, Castle, Hinton and Peabody Societies each have two designated Scholars in Medicine (SIM) Fellows who are the principal student advisors and have expertise in basic, clinical, outcomes and global health research. They help with mentor and project identification and review student funding proposals, scholarly project proposals and scholarly project reports. Additional faculty are available to advise students who work in the following specialty areas:

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Students’ passion projects address big issues in healthcare

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When graduate student Charlotte Hayes began her Ph.D. training in a lab studying brain tumors that affect young children, she was devastated to realize one reality about funding for biomedical research. "Compared to adult cancers, there isn't a lot of money allotted to pediatric cancers, let alone rare brain tumors," she says.

A first-year Ph.D. student at Mayo Clinic Graduate School of Biomedical Sciences , Hayes is also personally knowledgeable about the harsh reality of cancer, having endured surgeries and rounds of chemotherapy for her own cancer, which turned up in her 20s. "During my own ordeal with treatment," she says, "I had a moment where I thought, 'A 5-year-old has to deal with this?' That was absolutely unacceptable to me."

Never one to be told a problem is too big to tackle, she decided to take steps to improve research funding and created a foundation to raise money for pediatric brain tumor research. Hayes is among students from across Mayo Clinic College of Medicine and Science whose energy and vision have led them to devote their spare hours to solving big biomedical problems — and the schools encourage their enthusiasm. Their passion projects are making a mark in medical research and education.  

‘I wanted to do something about it’

Hayes' research in the lab of David Daniels, M.D., Ph.D. , studies tumors known as diffuse midline gliomas (DMGs). For children diagnosed with the disease, the prognosis is grim. Spending time in the lab, Hayes realized, "We don't have a lack of ideas, and it's not that we're stuck. The issue is that research is expensive, and we'll never make a dent without funding."

She adds, "I didn't want to sit around and complain about it. I wanted to do something about it."

Hayes, who has been blind since early childhood, had an undergraduate degree in business that prepared her for fundraising. Through online courses she learned web design to create the foundation's website. She read additional medical journal articles about DMG and reached out to families whose children have been affected by it. And she dove into more than 100 pages of government paperwork required to launch a 503(c)(3).

The foundation she established, KIDS MATER TOO, raises money that will go directly to DMG research projects – at Mayo and elsewhere. (The name of the foundation is not a misspelling; it's a play on words about the protective layers that cover the brain, like the dura mater.) The allocation of the funds is determined by a scientific review board, using a double-blind peer-review process to ensure objectivity.

"Charlotte's proactive and altruistic stance demonstrates the positive transformative power one individual can have on society," says Luis Lujan, Ph.D. , associate dean of student affairs. "Her efforts reflect the commitment of our students to affect positive change, which is in line with the values of Mayo Clinic and the Graduate School of Biomedical Sciences."

Hayes also has gotten other graduate students involved in her group's administration. "At this stage," says Hayes, "I'm focused on establishing meaningful relationships with donors, spreading awareness about the disease and continuing my own research. It's been time consuming and tiring, but I would do it all again."

Motivating others to succeed

Back when he was applying to medical school, JR Smith came across a statistic that made an impression on him. "I read that there were more Black men applying and matriculating to medical school in the 1970s than there are today," he says.

One factor contributing to the drop-off, he believed, was an ongoing lack of visual representation of Black men in medicine. He felt he could change that. As he progressed through the medical school application process, he documented his own journey on YouTube.

"I decided to share the strategies that I found helpful to succeed as a premedical and medical student," says Smith, now a fourth-year student at Mayo Clinic Alix School of Medicine . "My hope was that students who resonated with me would be motivated to pursue medicine as a career and have the tools necessary to succeed."  

His YouTube channel, Evolving Medic, provides motivation for students pursuing careers in medicine, with strategies and other productivity tips they can use to excel inside and outside their academic responsibilities. (He chooses and describes those that are evidence-based and that he himself has used.) The need hits home: Evolving Medic, with more than 150 videos, has more than 36,000 subscribers. The channel has attracted partnerships with various healthcare-related companies, including a scrubs company and various education-based resources.

"Multiple prospective students to Mayo Clinic Alix School of Medicine have indicated that JR's videos were inspiring to them as they considered pursuing medicine as a career and as they navigated applying to medical school," says Megha Tollefson, M.D. , associate dean of admissions. "He's absolutely had a positive impact on students nationwide."

Smith will continue his training as a resident in orthopedic surgery at Mayo Clinic School of Graduate Medical Education. He intends to continue posting advice for future doctors. "As I progress throughout my career, I'm sure my audience will grow to include physicians as well," he says. "My desire is to share the guidance, resources and knowledge that may be limited for some with the hopes of creating a more equitable environment for all students to succeed."

Mentors make the difference

For Nihal Satyadev, M.D., a first-year neurology resident in Mayo Clinic School of Graduate Medical Education , a passion project focused on Alzheimer's disease made him realize the scope of the problem  — and also solidified his career goals to address it.

As a college student and an aspiring clinician, he learned about the ways in which Alzheimer's disease is becoming a public health crisis. Along with classmates, he began an undergraduate club for students interested the topic. "What began as a few friends meeting at my apartment grew into one of the largest student groups on campus and ultimately the leading national youth Alzheimer's nonprofit," he says.

The project led him to reach out to Alzheimer's experts at Mayo Clinic, including Ronald Petersen, M.D., Ph.D. , who became mentors. "Early in the journey," Dr. Satyadev says, "I met with members of Dr. Petersen's team, including [research operations program manager] Angela Lunde at Mayo Clinic in Rochester, who were instrumental in helping me understand the intricacies of population level research and community interventions for dementia."

The initiatives of Youth Movement Against Alzheimer's aim to involve young people in addressing the public health crisis, for instance, by providing companionship to patients and giving time off to family caregivers. Dr. Satyadev was involved for eight years as the group continued to grow, and stepped back as it took on fulltime staff under the management of Hilarity for Charity, the nonprofit started by Lauren and Seth Rogen.

For Dr. Satyadev, the passion project was just a beginning. The connections he has made at Mayo, in particular with neurologist and dementia specialist Gregg Day, M.D. , helped solidify his commitment to becoming a neurologist who specializes in neurodegenerative diseases. Now conducting research projects with Dr. Day during his spare hours, Dr. Satyadev is aiming to develop new strategies to diagnose and treat Alzheimer's disease and related dementias.    

"Working on the nonprofit helped me realize addressing this national and global challenge requires a lifetime commitment," he says.

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Functional Protein Engineering became the hallmark in biomolecule manipulation in the new millennium, building on and surpassing the underlying structural DNA manipulation and recombination techniques developed and employed in the last decades of 20th century. Because of their prominence in almost all biological processes, proteins represent extremely important targets for engineering enhanced or altered properties that can lead to improvements exploitable in healthcare, medicine, research, biotechnology, and industry. Synthetic protein structures and functions can now be designed on a computer and/or evolved using molecular display or directed evolution methods in the laboratory. This review will focus on the recent trends in protein engineering and the impact of this technology on recent progress in science, cancer- and immunotherapies, with the emphasis on the current achievements in basic protein research using synthetic antibody (sABs) produced by phage display pipeline in the Kossiakoff laboratory at the University of Chicago (KossLab). Finally, engineering of the highly specific binding modules, such as variants of Streptococcal protein G with ultra-high orthogonal affinity for natural and engineered antibody scaffolds, and their possible applications as a plug-and-play platform for research and immunotherapy will be described.

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Generating Conformation and Complex-Specific Synthetic Antibodies

Construction of Synthetic Antibody Phage Display Libraries

Construction of Synthetic Antibody Phage-Display Libraries

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INTRODUCTION

“The power of evolution is revealed through the diversity of life” – this is the introductory sentence of the announcement for the 2018 Nobel Prize in chemistry awarded to Frances H. Arnold for the directed evolution of enzymes, and to George P. Smith and Gregory P. Winter for the phage display of peptides and antibodies. Now, scientists have taken control of evolution and can use it for purposes that bring the greatest benefit to humankind. Enzymes produced through directed evolution are used to manufacture everything from biofuels to pharmaceuticals, and antibodies evolved using phage display combat autoimmune diseases and cure cancer [ 1 ]. Although, due to the new algorithms and advances in computer performance, novel and improved synthetic protein structures and functions can now be designed entirely in silico by rational molecular and de novo design [ 2 ], synergistic combination of the computational design and in vitro evolutionary approaches produces variants superior to those that could be generated by the design only [ 3 ]. Thus, Protein Engineering, a multidisciplinary field that organically integrates chemistry, physical and molecular biology, protein and computer sciences, has arrived to complement and succeed to Gene Engineering, one of the major achievements in Molecular Biology.

Arnold’s seminal invention of directed evolution in 1996 [ 4 - 7 ] resulted in the tremendous boost to enzyme-engineering possibilities well surpassing the limits of nature. Highly artificial selection environments, diverse catalytical reactions and non-natural substrates, incorporation of synthetic amino acids and chemical modifications into proteins, all these achievements have drastically broadened the boundaries of potential applications for the engineered enzymes: efficient and cost-effective, green, and sustainable biocatalysts were produced by the directed evolution for current industrial, pharmaceutical, and research applications [ 8 ].

Traditionally, the term “directed evolution” has been strictly referring to the Arnold’s method of enzyme improvement through iterative cycles of protein diversification at the gene level (using random mutagenesis) and high-throughput screening of the encoded variants at the protein level. Alternatively, in phage display, the DNA variability is introduced by the initial library of billions of protein or peptide variants, and then it undergoes a huge reduction in the sequential rounds of panning and amplification, resulting in a limited number (usually, dozens or less) of unique phage clones. In both cases, amplification of the selected molecular variants requires an unambiguous physical genotype–phenotype linkage, which is readily provided by a single cell or a phage particle. As mentioned above, principal distinction between the phage display and directed evolution is that DNA does not undergo further diversification between the rounds of phage display panning. Therefore, molecular evolution achieved in phage display by the sequential enrichment of the most-fitted variants is rather reminiscent of the genetic bottleneck effect in nature, unlike the gradual canonical directed evolution which, step by step, selects more and more evolutionary advanced molecules. By its very nature, based on molecular selection by the phage binding, the phage display technology cannot address modulations and tuning of enzyme catalysis and is usually directed toward enhancement of the structural stability of the protein and binding affinities to antigens, partner proteins, or ligands, demonstrating its tremendous power particularly for specific peptide and antibody generation and maturation.

Since Smith and Winter published their pivotal studies in 1980s-1990s [ 9 - 14 ], the scope and capabilities of phage display have been enormously expanded [ 15 - 20 ]. Construction of large numbers of phage display peptide and antibody libraries containing 10 11-12 unique members have been achieved [ 18 , 21 , 22 ] and are commercially available (New England BioLabs, MoBiTec GmbH, and Creative Biolabs, Shirley, NY, USA)

Synthetic antibodies, tailored to a specific antigen or antigen epitope hugely helped to overcome protein structure determination challenges as well as powered up many medical endeavors. Multiple modifications of the original technology of peptide phage display, including cyclic and artificially linked peptides [ 23 ], resulted in varieties of the cancer-specific ligands validated in cancer diagnostics and therapy [ 24 ].

Undoubtedly, the progress in protein engineering greatly affects the landscape of cancer- and immunotherapeutics beyond the production of new potent peptide-based ligands or synthetic and in vitro matured antibodies. Applications of the antibody-drug conjugates, bispecific antibody-based molecules, and other antibody derivatives are empowered by the highly discriminative antibody-driven targeted cell delivery and well-controlled effect on the cells of choice. The targeted cell type is defined by the antibody specificity to one of the cell–surface receptors, and its fate – by the built-in effector moiety. The antibody-drug conjugates are able to kill cancer cells by binding to the tumor-specific receptors and directionally dispatching their “payloads” – potent cytotoxic small molecules [ 25 ]. Cytolytic effect of the bispecific T-cell engagers (BiTEs) depends on their ability to induce synapse formation between the cancer and T cells by bridging together their receptors targeted by two specificities [ 26 , 27 ]. In addition of being a part of anti-cancer therapy, the antibody-based reagents could specifically inhibit inflammatory pathways and have been developed to treat diseases and conditions caused by excessive inflammation and autoimmunity [ 28 , 29 ]. To date, hundreds of different antibody-based formats have been engineered for therapeutic purposes, with many representing BiTEs [ 30 ], and new constructs are constantly emerging [ 31 ].

Here, we review the antibody phage-display technology in detail – from the library construction methods to synthetic antibody (sAB) production and characterization. Using the phage-display pipeline established in the Dr. Kossiakoff Laboratory (KossLab) at the University of Chicago, as an example, the state-of-the-art progress in sABs custom-tailoring for challenging applications, such as protein structure determination of membrane proteins is addressed. Then, the sABs high potency as energy and functional probes, conformational and crystallization chaperons, fiducial markers for single-particle cryo-electron microscopy (cryo-EM), and superb antigen-binding reagents is discussed. Next, reciprocal maturation of the uniquely specific affinity between the protein G (variant GA1) and the engineered sAB scaffold (Fab LRT ) aiming further higher-order construction efforts, such as multivalent and multi-specific antibody arrangements, is described. The resulting novel, easily manipulatable, multifunctional, and sturdy GA1-based plug-and-play platform utilizing Fab LRT molecules as the interchangeable elements, and its possible applications in basic science, biotechnology, and medicine are considered in the conclusion.

ANTIBODY PHAGE DISPLAY ANTIBODY FORMATS

Immunoglobulin G (IgG) is the most common type of human antibodies. IgGs, which are complex, bi-valent, multi-subunit proteins with many disulfide bonds and are unsuitable for bacterial expression. Therefore, smaller, mono-valent antigen-binding fragments such as scFv (single chain fragment variable) or Fab (fragment antigen-binding) are the most often used formats for phage display [ 32 ] ( Fig. 1a ). Fab molecule is a heterodimer containing a full-length light chain (Fab) and an Fd fragment of a heavy chain (VH and CH1 domains) usually linked together by a disulfide bond at their C-termini (this linkage is not essential for heterodimerization [ 33 ]. These chains, of approximately equal masses, comprise ~50 kDa Fab. The scFv molecule is half a size (~25 kDa) and consists of two variable domains (VH and VL) fused together by a small flexible peptide linker of ~15 aa ( Fig. 1a ) Notably, while Fabs generally maintain full antigen-binding affinity upon reformatting into the full-size IgG and back (taking into account the 2-fold difference in the format valency), the scFv affinity can be drastically changed upon reformatting and sometimes is influenced by the VH/VL domain mutual orientation and/or linker length, and, also, this format is prone to oligomerization [ 34 , 35 ].

Antibody formats used in phage-display technologies. a) Schematic structures of human IgG antibody and fragments: HC, heavy chain; LC, light chain; VH, variable domain of the heavy chain; VL, variable domain of the light chain; CH1-CH3, constant regions of the heavy chain; CL, constant domain of the light chain; Fc, fragment crystallizable; Fab, fragment antigen binding; Fd, heavy chain of the Fab; CDR, complementarity-determining regions of the heavy chain (H1-H3) and of the light chain (L1-L3); scFv, single chain fragment variable; b) camelid heavy-chain IgG antibody and VHH nanobody depictions. The schemes were generated using Biorender ( https://www.biorender.com/ ).

Both scFvs and Fabs have been displayed on the bacteriophage surface in the Winter’s pioneering works [ 24 , 36 ] and are still the most popular for construction of sAB phage display libraries. However, combinatorial arrays [ 37 ] and some other new formats, such as nanobodies representing a single VHH domain of an unusual homo-dimeric camelid IgG [ 38 ] ( Fig. 1b ), are successfully challenging the originals. A dozen of therapeutic antibodies have been commercialized that have been discovered by means of antibody-displaying phage libraries including the world best-selling rheumatoid arthritis treating antibody adalimumab [ 39 ], with many more in the pipeline [ 40 ].

In addition to phage display, a great variety of display platforms have been developed to better suit rationale and particularity of the selections. Large libraries of recombinant cell-surface proteins were constructed and successfully applied for display on the baculovirus and bacterial, yeast, insect, and mammalian cells [ 41 - 44 ]. Molecular displays on the surface of eukaryotic cells are highly permissive for multi-domain proteins and multi-molecular complexes, as well as for synthesis of functional mammalian proteins undergoing essential posttranslational modifications that are supported by the eukaryotic cytoplasm [ 45 ]. The alternative cell-free methods, like ribosome display [ 46 ] and mRNA display [ 47 ] offer their own advantages, such as speedy turnover, enormous library size, and potential incorporation of non-natural amino acids and chemical modifications into the proteins. However, despite of all the advantageous features of other display techniques mentioned above, phage display remains the most popular display platform today.

M13 BACTERIOPHAGE

This chapter is dedicated to M13 bacteriophage structure and physiology that have been studied for decades and can be skipped by the educated readers, however, it contains information important for better understanding of the M13 phage display design specifics, advantages, and limitations.

Bacteriophages M13, fd, and f1 belonging to Ff (F-pilus specific filamentous) phages and almost identical in every aspect (98% identity at the DNA level) were the first used in phage display [ 48 ]. Later, the tailed bacteriophages T4 and T7 have also been successfully tested as display platforms [ 49 ]. The most developed of those, T7 phage display, demonstrate several particular advantages over the classic M13 platform, that could be important in some special applications: (i) T7 phage contains double stranded DNA, which is more stable and less prone to mutation during replication as compared to the single-stranded M13 phage genomic DNA; (ii) foreign cDNA or bacterial genomic libraries could be directly inserted into the T7 phage ds DNA genome; (iii) T7 phage does not depend on a bacterial protein secretion pathway and has a lytic life cycle. However, the M13 phage has its own pluses, making it the most developed and popular antibody-display platform to date.

M13 phage has high capacity for replication and can accommodate large foreign DNA as a highly transformable M13-based dsDNA phagemid vector, mimicking phage replicative form (RF) and producing circular ssDNA phagemid molecules that can be packaged into infectious phage particles. M13 bacteriophage is non-lytic: the progeny phage filaments are secreted from the intact bacterial cell (although growth rate of the infected bacteria slows down). Consequently, M13-based phage display libraries can be stored as frozen E. coli stocks. The extraordinary stability of M13 bacteriophage particles in a wide range of temperature, pH, and solvent composition allows for prolonged incubations under various conditions of the phage-displayed antibody libraries during the experimental procedures, and the library storage in frozen or desiccated state for years [ 50 ].

The single-stranded circular phage M13 genomic DNA (6400 nt) encoding 11 phage-specific proteins in total is protected by the stable protein capsid composed of approximately 2,700 copies of the major coat protein gp8 forming the cylinder, and several copies of four other minor coat proteins at either end of the phage filament: gp3 (five copies) and p6 at one end; gp7 and gp9 at the other. The infectious M13 phage filament has a length of <1 µm and a diameter of ~7 nm [ 51 ].

The first step of M13 infection involves recognition of the F pilus on the surface of the E. coli by gp3, followed by gp3 binding to the tolA-based bacterial complex that bridges between the outer and inner membrane of the host [ 32 ]. After penetration through the periplasm the phage genomic ssDNA is got stripped of gp8 and released into the host cytoplasm, where it is rapidly converted to a RF dsDNA by E. coli enzymes. This initiates transcription from 5 constitutive promoters, producing mRNAs encoding all 11 phage proteins. Hundreds of the single-stranded copies of the M13 genome are made by the mechanism called rolling-circle replication using RF DNA as a template: some of them are converted into more RF molecules, while the majority are destined to be packaged into the phage progeny. As freshly synthesized phage proteins accumulate, ~750 gp5 dimers cooperatively cover the whole length of M13 circular ssDNA except for a short 77 base-long section, known as the packaging sequence. This gp5 shell packs genomic DNA into a flexible rod protected from nucleases, thus forming the intracellular precursor of the extracellular virion. Binding of the DNA packaging-signal at the forward end of the precursor by the assembly protein complex in the inner membrane initiates the phage extrusion. Next, the membrane-associated gp8 oligomers are built into a spiraling array around phage DNA, replacing gp5. This nascent filament passes through the periplasm-spanning channel and a porin-like structure composed of 14 subunits of gp4. Finally, the inner membrane inserted gp3 and gp6 proteins are attached to the proximal end of the extruding virion particle completing the phage assembly.

The details of M13 phage replication and assembly are reflected in a vector construction for displaying of foreign polypeptides as coat-protein fusions on the phage. As a rule, the gene of interest is fused to the gene of one of the coat proteins cloned into the plasmid, which carries both plasmid dsDNA and f1 ssDNA origins of replication, f1 DNA packaging signal, and an antibiotic resistance gene [ 52 ]. This vector, called a phagemid, can be transformed and amplified in E. coli as a regular plasmid. Since this phagemid mimics RF of the phage DNA, the phagemid-transformed F pilus + cells can efficiently produce ss phagemid DNA and encapsidate it into the infectious phage progeny with the assistance of the M13 helper phage infection that provides all the phage proteins required for phage biogenesis. A mutation, that makes f1 DNA packaging signal of helper phage defective is disadvantageous for packaging of the helper-phage ssDNA, and thus favors formation of the phagemid-containing virions. Since these virions display the phagemid-encoded fusion protein, it provides physical link between the phage genotype and phenotype, realizing the key principle for phage-display as a molecular evolution-directed system [ 53 ]. In order to eliminate the need for the helper phage and avoid the helper-phage contamination in the phage progeny, bacterial “packaging” cell lines containing M13-phage based helper plasmids lacking the ssDNA replication origin, have been constructed [ 54 ], however, the classic way featuring the helper-phage superinfection is still very popular.

By now, every M13 coat protein has been tried for molecular display as both N- and C-terminal fusions [ 55 ]. Nature of the coat protein to be used as the fusion partner as well as size and structural characteristics of the proteins chosen to be displayed should be considered for the display success. As a rule, large, structurally complex, and multi-subunit proteins, like antibodies, cannot be expressed in bacterial cells, and this automatically makes them inappropriate for bacteriophage display. Additionally, fusion proteins could be rejected after the synthesis at different stages of phage maturation. Such obstacles as steric hindrance or wrong surface texture could prevent some bulky, or highly charged, or overly hydrophobic fusion proteins from proper incorporation into the phage particles both prior to phage assembly – upon membrane association, or during recognition by the assembly complex, or after the capsid completion – at the phage extrusion step due to insufficiently wide gp4 channel opening for the particle decorated with a fully folded and assembled fusion proteins. While the major coat protein gp8 supports high multivalency of the display and is frequently used for the display of peptide libraries, even antibody fragments are too large for gp8 display. Historically, gp3 was the first display support that was successfully demonstrated to work in the antibody libraries displaying scFvs or Fabs [ 24 , 36 ] and is the most commonly used today. To be displayed, usually the antibody fragment is fused to the N-termini of gp3 or its C-terminal domain, which itself assembles into a capsid tip, while it is the N-terminal domain of gp3, which recognizes the E. coli F-pilus. The wild-type gp3, product of the copy of unmodified g3 in the helper-phage DNA, is incorporated into the pentameric gp3 arrays at a higher proportion over the fusion protein, thus ensuring infectivity of the progeny virions [ 56 , 57 ]. Moreover, the ratio of the fusion to wild type gp3 available for the phage assembly is such, that not every progeny particle displays the antibody. However, this is not usually problematic for the library performance, since the standard antibody phage library has a density of 10 13 cfu/ml, which is 100 times greater than the typical antibody libraries.

M13 PHAGE ANTIBODY LIBRARIES

Natural antibody libraries. Antibody-displaying phage libraries can be either natural or synthetic, or both, depending on the origin of the diversity component, incorporated into the antibody-fragment scaffold. Diversity of the natural libraries is provided by the antibody repertoire acquired in the live immune system by gene rearrangement [ 12 ]. Each of the three major human immunoglobulin gene loci (one for HC and two – lambda and kappa, for LC, light chain), contains multiples of variable (V), diversity (D, only for HC) and joining (J) genes. V(D)J recombination, different in each individual lymphocyte, causes one random copy of each type of the gene segment to be sequentially joined together, generating 1×10 11 unique heavy-chain sequences and an enormous potential number of naïve paired antibody perhaps in the range of 10 16 -10 18 unique antibody sequences [ 58 ]. Real diversity of the naïve human antibody repertoire is estimated to be at least 10 12 unique combinations. Since the number of peripheral blood B cells in a healthy adult human is on the order of 5×10 9 , the circulating B cell population samples only a microscopic fraction of this diversity.

Antibody diversity is, mostly, present in their six complementarity determining regions (CDRs), highly variable structural loops undergoing clonal selection in the immune system. Three of them: L1, L2, and L3 are located in VL, and three: H1, H2, H3 – in VH. In general, the CDR loops in HC are more frequently involved in antigen binding than those in the LC. Contribution of each of the six CDR loops to antigen recognition is individual and each residue position within a single CDR may play a completely different role depending on the antigen–antibody complex [ 59 ]. Five of the six CDRs of a restricted variety and length from 3 to 15 residues adopt a limited number of backbone conformations, while H3 is longer, more compositionally and structurally diverse [ 60 ] and is considered to be the most important for antigen recognition [ 61 ].

Standard natural combinatorial antibody libraries are constructed by random pairing of VH and VL repertoires from cDNA of the pool of mammalian B cells into a phagemid vector using PCR cloning. The antibody assortment can be enriched for the desired-target binders by prior immunization. The resulting immune library does not need to be as large as naïve libraries, since the immunized pool of lymphocytes would contain multiple proliferated B-cell clones targeting the antigen. Some naïve antibody libraries represent 10 11 individual clones or more [ 18 , 21 , 22 ], reaching practical limit of the phage antibody library size preset around 10 12 value, due to the technical limitation of bacterial transformation, culturing, and storage [ 62 ]. While theoretical diversity of the naïve combinatorial libraries is much higher than their achievable size, there is significantly less variety of the naturally paired VH-VLs in the mammalian immune system. A miniature phage library containing ~10 6 naturally paired scFvs derived from cDNA of the individual B cells was constructed using a single-cell microfluidics that allowed to mine for an extremely rear antibody variants as well as to display natural antibody paratopes from hundreds of donors in one phage library [ 63 ]. This technique was advanced to high-throughput single-cell activity-based screening and sequencing platform of thousands of antibodies from the mice immune cells and activated human memory cells [ 64 ]. At present, there are dozens of commercially available natural antibody libraries in multiple formats, including chicken scFvs and camelid VHHs (Creative Biolabs).

Natural antibody libraries have been proven to be excellent suppliers of high-quality antibodies suitable for therapeutic applications in medicine [ 65 ] and veterinary [ 66 ], and many such antibodies are in clinical use or at various stages of therapy development [ 40 ].

Synthetic antibody libraries. Most of the sAB libraries use the natural Ig framework, however, a number of modules representing protein-binding polypeptides from natural sources were engineered into the stable spatial arrangements of several diversified loops featuring compact antigen-binding structures [ 67 ]. When implemented in the phage display libraries, they yielded high-quality non-Ig binders that could be of great interest for basic and applied scientific research due to their robust folding, high solubility, and small size. A number of non-Ig binders for health-relevant targets are, presently, at different stages of clinical trials [ 68 ]. Nonetheless, despite the constant challenge from other emerging formats, the classic libraries based on the Ig-fragment scaffolds remained the most desirable for sAb production [ 69 ].

The CDR loops of a sAB library need to be designed so that the resulting library is enriched with diverse, yet nature-like sequences, different in length and composition [ 70 ]. As discussed above, the pre-existing variety of CDRs incorporated into the natural antibody libraries originates from the operational immune system in vivo [ 12 ], therefore, quality of the natural library depends, mostly, on the efficiency of the variety incorporation into the library, i.e., on the library size. On the contrary, theoretical number of the possible combinations in the case of synthetic CDR variants has almost no upper limit: e.g., complete randomization of just a single piece of 10 codons produces more than 10 13 (20 10 including stop codons) of clone variations – amount, practically unattainable in a phage library. Therefore, smart diversification strategy considering each CDR length variability and randomization method as well as their magnitude became the key issue in the design of a quality sAB library [ 69 ].

The early examples of sAB libraries incorporated random CDR sequences of different length by means of degenerate oligonucleotides synthesizes from different nucleotide mixtures at different codon positions [ 71 ]. Due to genetic code redundancy, all 20 amino acids are encoded by NNK or NNS degenerate codon (N is any of the four deoxyribonucleotides; K = G or T; S = G or C), removing two out of three stop codons (TGA and TAA, but not TAG) and reducing the number of possible codons from 64 to 32, thus, decreasing the number of unique DNA sequences by two-fold. Other combinations of nucleotide mixtures can further reduce the number of codons and encoded amino acids in the sets. Examples of the commonly used degenerate codons in CDR design include KMT (M = A or C) that encodes Ala, Asp, Ser or Tyr; WMC (W = A or T) for Asn, Ser, Thr, or Tyr; and RRT (R = A or G) for Asn, Asp, Gly, or Ser. Degenerate oligonucleotides are easy to design and are cost-efficient – highly functional antibody libraries of 10 8 -10 9 unique clones have been constructed using this method [ 72 ]. Although the smaller nucleotide sets are highly desirable due to the library size limitations, there are many mutually exclusive amino acids in these sets. This problem is resolved by the more expensive but fully codon-specific synthetic methods, such as trinucleotide phosphoramidite synthesis [ 73 ] or dsDNA triplet-based Slonomics [ 74 ], dictating incorporation of only the chosen codons at the desired ratio into each position. Consequently, CDRs can be designed to have a smaller number of variants, while featuring more nature-like distribution of amino acids, thus making the sABs to better resemble natural antibodies and improving the clone-representing potential of the library. Aiming for the library size ≤10 10 unique CDR combinations can significantly improve the clone coverage and sampling power of the phage display.

As was stated above, theoretical size of the library depends on its design: the number of CDRs touched, the length range of the CDR loops and quality, and quantity of the codon-randomization events in each CDR. Following the natural antibody tendency favoring the highest variability and paratope involvement in L3 and H3 CDRs, most sAB libraries that have only H3 and L3 diversified and just one of the canonical natural sequences incorporated in place of each of other four CDRs, proved to be very efficient [ 73 , 75 , 76 ]; however, some high-quality libraries have all six CDRs diversified [ 72 , 77 , 78 ]. Surprisingly, the minimalist libraries, containing sequences of only two residues, Tyr and Ser, in their CDRs, were proven highly effective in generating specific antibodies against a wide array of antigens providing a rationale for high abundance of these two residues in the CDRs of the natural immune repertoire [ 62 , 79 ]. Due to the simpler design, these libraries are relatively easy to construct, and, due to the smaller theoretical size, they possess higher sampling power. Involvement of more of CDRs and CDR positions into deeper diversification, while producing an exponentially increasing number of possible variants, allows to build more enhanced and sterically extended paratopes with higher conformational diversity, thus, facilitating efficient recognition of various epitopes of different types of antigens.

As was mentioned above, natural antibody libraries have a huge advantage over any sAB library since they contain only expressible and stable CDR variants that have passed multiple control points of the immune system. In addition, chemical synthesis of CDRs is inherently compromised by synthetic errors, introducing all sorts of mutations that generate unwanted amino-acid substitutions and stop codons resulting in nonfunctional antibody clones [ 72 , 77 ] To eliminate poorly expressed clones and non-productive frame shifts, a proofreading step, such as selection of ampicillin-resistant sAB – β-lactamase genetic fusions, has been added as the final step of library construction [ 77 ].

Another important aspect defining functionality of the sAB phage display is a balance between the theoretical number of possible variants set by the design, and the number of unique phage clones achieved at a construction step, i.e., the library sampling power. Clone underrepresentation in a library can also be caused by clone-specific impediments, like slower replication or assembly rates, reducing or even eliminating the pool of most troubled variants. The higher-order diversification design may account for such astronomical calculated number of the unique variants that the realistically limited of manageable phage library variety of 10 12 clones rationalized above would constitute only a microscopic fraction of the theoretical prospects. Due to this problem, more extensive and deeper diversification would not allow for selection of the ideal antibody variants, covered by the design but not even present during the phage panning, however, there would be a greater variety of the second- and third-best clones, suitable for the task. sAB phage libraries of this kind have produced a wide range of high-quality antibodies for a broad spectrum of antigens, although their theoretical variety is hugely under-sampled. The well-designed sAB libraries have several distinct advantages, which include optimal variability, size, clone representation and density of the display, high sAB expression, solubility and stability, and ease of further engineering and optimization.

THE KOSSLAB PIPELINE

Synthetic-antibody library construction. The KossLab pipeline have been constructed based on the scaffold from a humanized Fab 4D5 Herceptin framework (here, Fab S ) engineered for high stability and efficient phage display. Combination of the mixed nucleotide and trinucleotide phosphoramidite based methods were used for synthesis of randomized DNA oligonucleotides that were next cloned into the Fab framework using Kunkel mutagenesis method [ 80 ].

In the library, limited amino acid diversity is introduced into all three HC CDRs (H1, H2, and H3), as well as the third CDR of LC: L3, while L1 and L2 have fixed canonical loops: SVSSA and SASSLYS, respectively. L3 length varies from four to six residues. In H3, the loop lengths range from 6 and 20 residues (sub-library A), 7 and 15 residues (sub-library B) or 6 and 17 residues (sub-libraries C and D). H3 design in the most complex sub-library D (6 to 17 residues) was based on a tailored oligonucleotide synthesis strategy that biased the sequences in favor of Y, S and G but allowed for 19 of the 20 genetically encoded amino acids (only C was excluded) and potentiated high level of diversity amongst the H3 loops in terms of both length and sequence. The pooled library consists of 10 10 unique variants, huge under-sampling of the theoretical diversity (by more than 20 orders of magnitude) [ 62 ]. Extensive exploitation of the library over the years in hundreds of successful selections resulted in multitudes of high-affinity antibodies with dissociation constants in the sub-nanomolar to single-digit nanomolar range against a wide variety of protein antigens [ 81 - 100 ] confirming that this fully synthetic library with the limited diversification has essentially recapitulated capacity of the natural immune system.

Target immobilization and phage elution. Production and immobilization of the target-protein in an accessible and native functional state is the next critical step in the selection-project progress after obtaining high-quality phage-displayed sAB library. The original and still frequently used way of antigen immobilization for biopanning is passive protein adsorption on the activated plastic surfaces in random orientation through multiple non-specific non-covalent interactions [ 101 ]. However, adsorption is usually accompanied by some degree of structural change of the antigen, and there is a possibility that the antibodies isolated against the passively adsorbed antigen may not recognize the free native antigen in solution. Nevertheless, passive immobilization is appealing due to simplicity of the method and frequent success of selections that yield clones that recognize and bind functional protein.

At present, the most common immobilization method avoiding adsorption-induced conformational changes of the antigen is based on the extremely tight biotin–streptavidin interactions [ 102 ] that are widely used in various research and biotechnology applications requiring tight and specific intermolecular interactions (detection and isolation of proteins, nucleic acids and lipids, protein purification, new-generation DNA-sequencing, mass-spectrometry based proteomics and many others).

Protein antigen could be chemically conjugated with biotin [ 103 ] using commercially available biotinylation reagents targeting variety of specific functional groups or residues, including primary amines, sulfhydryls, carboxyls, and carbohydrates [ 104 ]. Chemical biotinylation can result in multiplicity of protein modification spots and masking or altering natural epitopes of the antigens by biotin moieties. Another, the least structure-disturbing way for biotin attachment, is through a genetic linkage of the antigen to Avi-tag, a peptide (GLNDIFEAQKIEWHE) specifically recognized and efficiently biotinylated by the biotin ligase BirA. BirA could modify Avi-tagged proteins in vitro , after purification [ 105 ], or in vivo [ 106 ], during expression in E. coli bearing BirA plasmid; in every case high-level of antigen biotinylation has been demonstrated (70-90%). This modification level is sufficient for efficient capture of the antigen on the streptavidin-coated microplates or streptavidin-coupled magnetic beads followed by the library panning.

There are other affinity tags options for tethering proteins in a relatively uniform orientation with minimal disruption of native conformation [ 107 ] including metal ion- [ 108 , 109 ], antibody- [ 110 ], and protein–ligand capture technologies [ 111 ] that have been used for the target immobilization. Depending on the antigen immobilization technique, elution methods also vary widely. The most-common eluants are diluted solutions of HCl or Glycine (adjusted to pH 2-3) that disrupt molecular interactions between the antibody displayed and the antigen immobilized (and the biotin–streptavidin complex too). Although there is almost no risk of eluting phage incompletely, the prolonged exposures to low pH could affect phage infectibility. If this becomes a problem, a gentle and highly specific alternative would be competitive elution with the antigen excess. This mostly releases the target-bound phage limiting selection of the non-specific clones [ 112 , 113 ], but requires additional amounts of the purified antigen. The most common affinity-independent elution method entails insertion of a unique proteolytic site next to the affinity tag [ 114 ]. Cutting-off the affinity tag would trigger release of the target–bound phage exclusively, enhancing specificity of the selected phage clones comparably to the competitive elution. In addition, phage elution of any kind can be skipped, if no phage tittering is needed, instead, E. coli cells could be directly infected with the antigen–bound phage in selection wells or by addition of the phage pulled out on magnetic beads.

Although there are many other types and varieties of protein-binding chemistry, the exceptionally-high femtomolar affinity of a biotin–streptavidin pair makes this technique generally superior to others, since dissociative loss of the target during multiple washing steps at lower immobilization affinities could ultimately impede quality of the selection. In addition, the biotin–streptavidin binding, as many other receptor–ligand interactions, immune complexes included, is easy to eliminate at low pH, which could be useful during the system set-up and testing.

Several modifiers and tags for target biotinylation have been applied in the immobilization protocols at KossLab pipeline at different periods and for specific antigens (i.e., natural non-recombinant proteins could be only biotinylated in a chemical reaction), including the SNAP-mediated biotinylation and immobilization that is discussed next.

SNAP-tagged targets: immobilization and specific proteolytic elution. Although the biotin–streptavidin binding is not covalent, it serves all intents and purposes in biopanning, since the binding is almost irreversible (K D ≈ 10 −15 M). However, there are several popular novel protein tags that could be attached covalently to the specific chemical moieties of the insoluble matrix among multiple other useful soluble carriers. Such enzyme derivatives, as, HaloTag (Promega, USA) [ 115 , 116 ], SNAP-tag [ 117 - 119 ], and CLIP-tag [ 119 , 120 ] (both – New England Biolabs) represent self-labelling tags that catalyze covalent attachment of the exogenously added specifically designed synthetic ligand. These systems have been exploited for years in vitro and in vivo for protein labeling, localization, and fluorescence, super-resolution, and electron microscopy imaging. HaloTag is a derivative of a bacterial enzyme haloalkane dehalogenase covalently linked through a reactive chloroalkane linker to a functional group of choice. Both SNAP-tag and CLIP-tag are engineered variants of the human DNA repair enzyme O(6)-alkylguanine-DNA alkylotransferase that catalyzes transfer and attachment of the alkyl group via a thioether bond to the reactive cysteine of the enzyme. SNAP-tag has been engineered to reacts with O(6)-benzylguanine, while CLIP-tag – with O(2)-benzylcytosine derivatives. The discriminatory substrate binding and choice of the fluorescently labeled substrates, makes these two tags highly applicable for orthogonal labeling of proteins in living cells [ 120 ].

Availability of various other SNAP substrates, besides fluorescent, like SNAP-biotin and SNAP-capture magnetic beads (NEB), allowed us to develop and successfully apply several variants of the antigen-immobilization technique based on the SNAP-tagging of target proteins. In addition to the classic biotin–streptavidin based immobilization of the SNAP-biotinylated target, the novel way of direct covalent attachment onto the SNAP-capture magnetic beads has been tested. Covalent nature of the direct capture and extreme substrate specificity of the engineered SNAP-tag allowed for efficient immobilization of not only highly or partially purified targets but even immobilization from the crude lysates of the cells expressing SNAP-tagged proteins. This direct covalent capture could be hugely beneficial for the proteins with IMAC-incompatible characteristics or complicated purification protocols, despite the fact, that, in our hands, the SNAP-capture beads demonstrated reproducibly somewhat lower antigen-binding capacity and specificity for phage binding, compared to the best-performing Streptavidin-coupled magnetic beads (Dyno beads, Invitrogen, USA). Also, further improvement and optimization of the surface chemistry and ligand density of the SNAP-capture beads should eliminate this problem in future.

Biotinylation of the SNAP-tagged protein is a bi-molecular reaction, simple, fast, completed within a short time, and irreversible that does not require any substantial excess of the SNAP-biotin reagent and, subsequently, is free of the purification step prior to the binding to streptavidin magnetic beads. We also have inserted a Thrombin-cleavage site between the tag and the antigen in the commercial SNAP-vector (New England Biolabs) to ensure fast and gentle elution of the enriched antigen-bound phage from the magnetic beads without acidic treatment. Thrombin-cleavage site was chosen because it had the highest cleavage rate, compared to some other highly specific proteases [ 121 ], like TEV, PreScission, or 3C protease; their cleavage sites were introduced into the experimental SNAP-fusion proteins as well and the cleavage rates were tested.

Theoretically, cleavage should release only the target-specific phage, while the SNAP-tag-bound phage should remain attached to the beads, however, natural spontaneous phage dissociation could significantly contaminate the eluted phage. Therefore, to avoid phage binding to the immobilized SNAP-tag, a soluble SNAP-tag competitor is added during the panning step. In addition, we found, that, like with other solubility-enhancing tag proteins (MBP, NusA, thioredoxin, GST, SUMO and Fh8 tag [ 107 , 122 ]), fusion to the SNAP-tag (182 amino acid-long, well-structured, highly soluble, and stable protein) can drastically improve stability, solubility, and production of the hard-to-express, poorly folded, and structurally unstable proteins.

The modified library-panning protocol developed in the KossLab for the SNAP-tagged targets ( Fig. 2 ) has been successfully used, as demonstrated below, for sAB generation against dozens of targets, including viral proteins [ 89 , 123 ] as well as for reciprocal maturation of the engineered-protein G and Fab-scaffold interface.

Panning cycles of a phage-display library. The scheme represents principal steps of the cycle flow common in multiple protocols with highlighted specific details for the SNAP-tagged target immobilization and thrombin-mediated elution developed in the KossLab. 1) Target immobilization on magnetic beads. Immobilization of the SNAP-tagged target may be achieved by means of either direct covalent attachment to SNAP-capture beads or by biotinylation of SNAP-tagged target by SNAP-biotin prior to the Streptavidin-coupled bead binding. The LVPRGS thrombin-cleavage site in the linker between the target and SNAP-tag is shown. 2) Phage binding. Incubation of the phage-display library (at the first cycle) or enriched amplified phage (other cycles) with the bead–bound SNAP-target. 3) Multiple washes of the beads pulled out with a magnet to remove the unbound and weakly-bound phage. 4) Elution of the target-specific phages by thrombin cleavage of the linker; note that acid would elute all the bound phage non-specifically. 5) Amplification of the enriched phage by propagation in Escherichia coli aided by the helper phage. The new panning cycle starts as the amplified phage is added to the bead-immobilized target for binding. Normally, as the cycles progress, the amount of the target immobilized on the beads is decreased to benefit binding of the growing share of sABs with higher affinities. If improvement of the sAB dissociation rate is at question, the possible trick would be prolongation of the wash-time. Panning procedure could be manual, using magnets and magnetic racks, semi-automatic, on a magnetic particle processor (KingFisher, Thermo Fisher Scientific, USA), or fully automated – with the help of several robotic systems.

SYNTHETIC ANTIBODIES. WHAT IS SPECIAL ABOUT THEM?

sABs are superior to the naturally produced antibody in many special features and applications. To start with, sAB production is performed in vitro , free of demanding and expensive dependence on laboratory animals and can allow for isolation of very highly-selective antibody reagents confirmed to bind the native protein, or their certain conformations, or post-translationally modified forms. Also, sABs variety is not affected by such inherent limitations of the mammalian immune system as intolerance, or presence of immunodominant antigens, or clonal drift. Moreover, while natural monoclonal antibody reproduction requires continuous maintenance and storage of the hybridoma cell line, sAB clones can be indefinitely preserved in a form of frozen or lyophilized DNA. Due to the same AB scaffold used for cloning of all the variants, sABs are easy to sequence and reformat into a full antibody or expression vectors for production in bacterial or mammalian cells. This has driven development and validation of the high-throughput sAB expression platforms [ 124 ].

sABs could compete with monoclonal antibodies almost in any standard laboratory applications, like ELISA and related homogeneous and heterogeneous immunoassays, flow cytometry, immunostaining, immunoprecipitation, chromatin immunoprecipitation, and many others. However, methods, requiring recognition of SDS-denatured proteins (such as Western blotting) that are highly feasible for monoclonal antibody, could be problematic for sAB. Unlike the most of natural-antibody CDRs that should recognize a short antigenic peptide (8-18 aa long) presented on the immune cell and thus are specific for linear epitopes readily accessible upon antigen denaturation, sABs are selected by binding to the native-state proteins. In the native form, many such linear epitopes are often hidden or interrupted, and outcompeted by the three-dimensional epitopes exposed and more potent for in vitro selection. Moreover, the well-structured proteins with pronounced surface architecture and limited number of the defined low-energy states comprise the best sAB targets, while flexible, poorly folded, and unstable proteins are not appropriate targets for sABs selection but are as good as well-folded proteins for natural antibody production. Advantageously, in vitro selection potentiates unparalleled antibody customization, unachievable by natural production of immunoglobulins. By the means of precise control over the antigen composition and state under the highly-manipulatable panning conditions sABs can be tailored to: (i) target specific regions of the surface of a protein, (ii) recognize distinct conformational states of the antigen, (iii) induce desired conformational changes, and (iv) capture and stabilize transient states and multi-protein complexes [ 81 ]. These unique sAB capabilities are opening new possibilities for investigation of macromolecular structures and processes inaccessible to the traditional affinity-reagent technology.

sAB customization strategies. Different epitope targeting sABs and translational medicine. Like the natural process of antibody production, in vitro selection usually generates sAB variants recognizing antigen epitopes of high “immunogenicity”. However, unlike natural immunogenicity, which is contingent also on the epitope-specific antibody progression through the controls of the immune system, the in vitro antigen epitope dominance depends, mostly, on the binding affinity of the sAB phage clone, and, to a lesser extend, on the share of the clone in the phage pool under the panning.

By binding to different epitopes of the protein, sABs can affect protein function as inhibitors or activators, promote or suppress ligand binding and homo- and hetero-protein interactions. sAB-based activators and inhibitors of definite immune checkpoints have proved their clinical efficacy as immunomodulatory therapeutics activating immune responds in tumorigenesis or reducing inflammation in autoimmunity. Besides the checkpoint blockade, similarly to the natural monoclonal antibodies, sABs could impact a variety of other areas, such as, allergy, transplantation, and T-cell immunotherapies.

The KossLab pipeline production also have confirmed an undeniable potential of sABs targeting different antigen determinants in translational medicine. For example, among the selected binders that modulate isocitrate dehydrogenase enzymatic activity, the explicit one has demonstrated ability to reactivate the enzyme mutant form associated with brain tumors [ 90 ]. “Activator drugs” of this kind could be the key remedy for many genetic disorders or malignant cell transformations caused by mutations disrupting protein function. Furthermore, a panel of sABs binding to different regions of paramyxovirus envelope glycoproteins and affecting different processes of the viral entry into the cell has been used to understand the steps in viral membrane fusion leading to acute respiratory infections [ 125 ].

Among the set of generated actin-filament pointed-end binders, three sABs have demonstrated unique properties toward the actin-dynamic probing: one binder caps the pointed end, the second one crosslinks actin filaments, and the third severs actin filaments and promotes disassembly. This study may provide insights into the details of actin-filament rearrangements occurring in healthy and diseased human tissues, in innate and adaptive immune cells, during metastatic cancer and viral and bacterial infection [ 94 ].

A rapid and efficient screening to tailor phage-display for the selection of neutralizing antibody was set up in the KossLab for the Anthrax model. The high-affinity neutralizing sABs selected against edema toxin, an adenylate cyclase and a major mediator of anthrax pathogenesis, has demonstrated efficacy of the synthetic alternatives to the traditional antibody therapeutics: its potency to inhibit edema-toxin catalyzed cAMP production in human cells is comparable with that reported for the best-performing monoclonal antibodies [ 126 ].

There are also sAB applications, where it is advantageous to deal with a variety of sAB specificities targeting non-redundant epitopes on the protein [ 127 ]. Although these are often obtained spontaneously and can be easily characterized by epitope-binning methods [ 128 ], sometimes, there is one major epitope prevailing in the selection. Then, the epitope-masking strategy directing the sAB binding on the protein away from the immunodominant hot spot could be implemented during the selection step. In this case, the sABs targeting the predominant protein epitope is pre-bound to the protein, thus, excluding the epitope from the competition for phage binding and allowing for selection of the sAB variants specific for the secondary epitopes.

In the KossLab, this trick has been efficiently applied in many instances, in particular, for generation of the sABs targeting non-redundant epitopes in two viral proteins, C-terminal domain of Ebola Zaire Nucleoprotein (EBOV NP CT ) and Zika methyltransferase (ZIKV MT), while developing a novel protein-complementation (PC)-based wash-free immunoassay [ 123 ], relevant for point-of-care (POC) applications and described in a separate chapter below. And recently, the second sAB partner for SARS-CoV-2 detecting assay has been identified after masking the major epitope of the SARS-CoV-2 Spike protein receptor-binding domain by the predominant sAB, that, in addition, has demonstrated an effective inhibition of the virus entry into the cell and can be used for the virus neutralization. Ability of this sAB pair to detect SARS-CoV-2 has been proven in the PC-based immunoassay, answering an urgent need for novel SARS-CoV-2 POC diagnostics during the COVID-19 pandemic [ 85 ].

Conformational sABs. It is common, that the protein used as a selection target is not conformationally uniform and can represent a dynamic or static mix of low-energy states, sometimes caused by contaminating ligand molecules or certain metal ions. Under such conditions, generation of a conformationally-selective antibody recognizing just one of the antigen states is accidental. Alternatively, a specific conformational state of an antigen could be targeted already at the design conception. As an illustration, adding or omitting maltose at the panning step in the maltose-binding protein (MBP) system resulted in the selection of a cohort of three classes of high-affinity and specificity MBP binders: endosteric (binding to the maltose-binding pocket), allosteric (opposite to the maltose-binding pocket), and peristeric (close to the maltose-binding pocket) [ 88 , 93 ]. These sABs stabilize MBP in different conformational states as has been revealed in the respective crystal structures. Respective modulations of the MBP ligand-binding affinity in competitive, allosteric, or peristeric manners, were used as probes to quantify energy contributions of the ligand binding to the conformational changes in proteins. Further development of this approach for more biological systems could in principle advance analysis of the energy landscapes of many systems including highly challenging regulatory proteins that control physiological responses to environmental changes.

sABs as tools for protein structure determination. As already mentioned, sABs represent indispensable tools for protein structure determination. They can be successfully used as fiducial markers for SP cryo-EM by adding mass (50 kDa for Fab) to the particle and assisting in its orientation [ 129 ]. Also, being easily crystallizable proteins with the known scaffold structure, sABs are widely used as crystallization chaperons that bind to a target of interest, enhance crystal packing, and provide high-quality phasing information. They have efficiently facilitated structure-determination of RNAs, RNA–protein complexes, protein complexes as well as individual proteins inherently recalcitrant to generate stable crystal lattices [ 83 , 84 , 130 ]. By driving a protein or its flexible parts to adopt and halt a uniform conformation, the conformation-specific sABs can enable crystallization of unstable, multi-conformational, multi-component or multi-subunit structures, membrane proteins and their complexes [ 81 , 82 , 98 , 131 ].

Membrane proteins and nanodisc-based phage display. As it has been brought up by many investigators, membrane proteins continue to be among the most challenging targets in structural biology. The naturally programmed conformational changes in the membrane proteins are induced by external stimuli and are in the core of the signal transduction mechanism. Sophisticated interrelation between the structural and functional transformations underlying the transduction process is one of the most complicated though most exciting areas of the today’s membrane-protein science. Membrane proteins are difficult to purify in a native form, and conditions matching their natural surroundings are hard to reproduce for crystallization purposes. Until recently, most sAB selection protocols targeting membrane protein have been performed in detergents, that are poor mimics of the native lipid environment. To produce sABs while resolving this problem, a novel approach, that combines the power of phage display with the benefits of embedding membrane protein targets in the lipid-filled nanodiscs has been developed in the KossLab [ 91 , 132 ]. Nanodiscs, small (5-50 nm in diameter) discoidal particles consisting of lipids enclosed by the membrane scaffold proteins [ 133 , 134 ] have been widely used in investigation of functional and structural properties of the membrane proteins as a sophisticated membrane mimetic system with precise control over their size and composition. For the purpose of application in phage display, the scaffold proteins of the nanodiscs are biotinylated enabling their pull-out by the magnetic beads. The nanodisc-based native-like lipid environment and the overall configuration of the system allow the partially imbedded target protein to adopt characteristic structural transitions, and the sABs to bind to these transient conformations. The novel approach developed in the KossLab yielded a rich pool of sAB, that could be used as crystallization chaperones, fiducial markers for SP cryo-EM or energy probes for different conformational states of a number of membrane proteins, laying foundation for elucidation of the high-resolution structures of the functionally relevant protein conformational states and understanding their dynamic interplay.

sABs as “universal” fiducial markers for SP cryoEM . SP cryo-EM has emerged over the past two decades as a powerful structural biology tool for challenging macromolecular systems: it does not entail crystallization or phase determination problem and requires only small amounts of the experimental sample [ 129 ]. In 2017 the Nobel Prize in Chemistry was awarded jointly to Jacques Dubochet, Joachim Frank, and Richard Henderson “for developing cryo-electron microscopy for the high-resolution structure determination of biomolecules in solution” thus highlighting this technology as one of the greatest benefits to humankind [ 135 ]. Since SP cryo-EM success relies on the accurate assignment of particle location and orientation and their sufficient mass, only symmetric and heavy objects, like viruses, were appropriate for the method at the times of method conception [ 136 , 137 ]. With the development of supportive new technologies, structures of many large protein complexes and oligomeric membrane proteins have been resolved to nearly atomic resolution by SP cryo-EM, the lower mass limit of the method has dropped to 50 kDa [ 138 , 139 ]. By increasing the particle mass and providing fiducial markers for particle alignments, sAbs significantly enable cryo-EM analysis and protein-structure determination [ 140 , 141 ].

The antibody-based fiducial markers have considerably aided the membrane-protein structural studies in the field of G-protein coupled receptor (GPCRs) [ 142 - 144 ]. There are hundreds of different GPCRs belonging to 3 major classes encoded in human genome. They play a central physiological role in the regulation of cellular responses to a wide variety of stimuli in both health and disease and thus represent one of the largest types of surface receptors targeted by drugs. In the KossLab, sABs have been generated to the two major G-protein subclasses binding GPCRs: trimeric G i and G s , as well as mini-G s with the goal to overcome specificity limitations of the fiducials tailored for a particular GCPRs. Epitope binning revealed that multiple distinct epitopes were targeted for each of trimeric G-protein, while some of the sABs cross-react between the trimeric G i and G s , suggesting their universal application across the subclasses. Similarly, sABs were generated to a representative of another class of GPCR signaling partner, G-protein receptor kinase 1, supporting the generalizability of the fiducial marker approach. EM data confirmed efficacy of the sABs as single and dual fiducials for multiple GPCR signaling complexes [ 87 ].

In addition, a universal sAB-targeted tag system that eliminates the need for a specific antibody for each individual membrane protein under structural study has been developed. To that end, an engineered variant of the apocytochrome b562 , 12 kDa protein BRIL has been chosen as such a tag since it contains terminal helical extensions easily adjustable for the distortion-free seamless connection to α-helices present either in the loops [ 145 ] or at the termini [ 146 , 147 ] of the membrane proteins. Several sABs generated and matured by the customized phage display selections against BRIL gained sub-nanomolar affinities and unhindered BRIL binding in multiple fusion systems. Negative-stain TEM and cryoEM structures of several examples of BRIL-membrane protein chimera demonstrated effectiveness of the sAB–BRILL systems as universal fiducial markers [ 86 ]. Thus, the technology is advancing towards making the “universal” sABs for the whole classes of proteins enabling their crystallographic and SP-cryoEM studies and potentially forming the “off the shelf” line of enhancement reagents. These would eliminate the need to incorporate the challenge of an antibody-engineering into the workflow of protein-structure determination projects.

Tools like sAB-based crystallization chaperones and fiducial markers have drastically improved the level and quality of structural analyses. To further improve effectiveness of the sABs as structure-determination chaperons, a special phage display engineering strategy, including a heat-stress step for the selected clones, has been used to generate Fab scaffold variants, that significantly reduces inherent flexibility of the “elbow” regions, which link the constant and variable domains of the Fab, disordering its structure. The strategy was validated using the previously recalcitrant Fab–antigen complexes: introduction of the engineered elbow region remarkably enhanced crystallization and diffraction resolution, while their high affinity and stability retained [ 148 ]. Also, sAB solubility, was addressed in the design of shotgun scanning libraries introducing aspartate as a negative design element at the antigen-binding site. The selected high-affinity variants featured a polar ring surrounding the paratope. This significantly enhanced sAB specificity and solubility, crucial for easily aggregating antibodies, while maintained its affinity to the antigen [ 149 ]. These universal improvements of the overall sAB rigidity and solubility have further shaped them for structural determination studies.

ENGINEERED PROTEIN G AND ANTIBODY-SCAFFOLD VARIANTS

Ig-binding proteins. Fortunately, nature has provided protein engineers not only with the IgG molecule as a framework for sABs but also with Ig-binding proteins enabling their isolation, detection, and assembly. Several species of infectious bacteria express multivalent Ig-binding proteins on their surface. Single domains from a number of almost identical entities comprising multidomain bacterial protein A ( Staphylococcus aureus ), protein G ( Streptococcus species C and G), and protein L ( Peptostreptococcus magnus ) have been characterized in detail both biochemically and structurally [ 150 ]. Each of these proteins has a different antibody binding profile in terms of the portion of the antibody that is recognized and the species and type of antibodies it binds. Protein A has greater affinity for the rabbit, pig, dog, and cat IgG whereas protein G has greater affinity for the mouse and human IgG. In addition to the predominant binding of both proteins A and G to the interface of CH2-CH3 heavy chains in the Fc (fragment crystallizable), they also bind to the Fab: Protein A – to VH, while protein G binds simultaneously, but very weakly, to CH1, a highly conserved domain across many isotypes and species, and CL kappa. Protein L only binds to VL kappa of the Fab part and exclusively [ 151 , 152 ].

Proteins A, G, and L have a scope of biochemical, biotechnological, and medical applications and are widely used in antibody purification and test methods like immunoprecipitation, ELISA, and Western blotting [ 153 ]. The “universal” recombinant fusion protein A/G comprised of four domains of protein A and two domains of protein G demonstrates additive properties of the two proteins and captures antibodies from the combined spectra of species and isotypes [ 154 , 155 ].

Recent engineering efforts in applied immunological and biochemical research have led to production of the synthetic ligands mimicking protein A and L (peptides, engineered protein domains, and designed artificial molecules). These mimetics can replace the originals for purification of antibodies, while surmounting some drawbacks such as high cost, low binding capacity, limited life cycles and so on [ 156 ].

An engineered GA1 variant. Protein G is used for IgG purification by the virtue of its affinity to the Fc portion of the molecule (K D ~ 10 nM), while its binding to the Fab CH and CL is pretty weak (K D ~ 3 µM). The 4D5 Herceptin Fab scaffold (Fab S ) of the KossLab pipeline containing E123S mutation in the Fab CL has been used for affinity maturation of protein G (a 65 aa-long C2 domain) [ 157 ]. The affinity maturation design involved soft randomization of the two points of contact with the Fab S scaffold: one, residues 15-24, involved in the formation of antiparallel β-strands with Fab CH and second, residues 37-43, interacting with the short non-conserved β-helical region connecting β-strands in CL. The matured variant GA1 demonstrated the highest improvement in the Fab S binding affinity, mostly due to substitutions of NDNG in the positions 40-43 for YVHE. In the crystal structure of GA1–Fab S complex, these residues form a structural helical cap in the GA1 interdigitating with the CL α-helical residues SQLKS improving the interface complementarity.

An engineered FAB LRT scaffold variant. Compared to the wild-type protein G, GA1 demonstrates a significant affinity boost in binding Fab S (K D ~ 50 nM) – adequate for applications that involve genetically linked GA1 strings to make multi-valent Fab assemblages [ 157 ]. However, binding is still characterized by fast dissociation kinetics that is not optimal for the desired non-equilibrium applications. To further improve affinity and dissociation kinetics of the GA1–Fab complex, we undertook a reciprocal phage display approach: Fab S scaffold was affinity matured against GA1 [ 123 ]. The library design was focusing on the residues 123-127 (SQLKS) of CL. Kunkle mutagenesis using NNK/NNT randomization yielded ~10 10 clones with theoretical diversity of ~1.7×10 7 unique codon variants ( Fig. 3 ). Several selected variants demonstrated significantly increased binding affinity and reduced dissociation rate. The best selected Lc variant with ~500-fold improved affinity to GA1 contained a serendipitous two amino acid deletion, which may have occurred during the synthesis of randomizing DNA oligonucleotides. Replacement of the original SQLKS sequence of Fab S with ΔΔLRT (Fab LRT ) resulted in the K d of ~100 pM, slow dissociation rate (~2.4×10 –4 s –1 ) and did not affect Fab stability or expression.

Construction of the phage-display library for Fab scaffold GA1-binding affinity maturation. Schematic representation of the degenerate-codon based randomization by Kunkel mutagenesis: a random Fab S -encoding phagemid containing dU substitutions for T was isolated from CJ236 E. coli strain (1), annealed with a synthetic DNA oligonucleotide comprised of a randomization sequence: LC 123 (NNK) 2 (NNT)(NNK) 2 LC 127 , flanked by 15 nt of the wild-type overhangs (2) used to prime the DNA synthesis by T4 DNA polymerase; the synthesized dU/T duplex phagemid molecule was transformed into the TG1 E. coli strain aiming for destruction of the wild-type dU-containing strand and efficient replication of the mutant T-containing strand (4). The following superinfection with M13 KO7 helper phage resulted in the mutant phage propagation (5).

The key structural basis underlying this ultra-high affinity of the Fab LRT was revealed in a crystal structure solved for the GA1–Fab LRT complex: a significant deletion-induced rearrangement and extension of the interface has packed the guanidinium group of Arg124 (replacing Lys126 in Fab S ) against the aromatic ring of Tyr40 of GA1 in a cation–π interaction, while providing H-bonding with the Tyr40 carbonyl via the secondary amine in ε position of Arg124 [ 123 ].

A NOVEL PLUG-AND-PLAY PLATFORM

The ultra-high GA1 binding affinity and extremely low dissociation rate of the Fab LRT encouraged us to develop a multivalent and multi-specific platform based on the GA1 module that could be easily modified just by exchange of the Fab component. Although the Fab LRT –GA1 interaction is not covalent, no exchange between the partners, which could compromise the performance of such a platform, has been detected within the timeframe of experiments.

There is a great variety of possible functionally relevant GA1 genetic fusions, allowing the researcher to design highly customized affinity reagents that can be assembled in a wide scope of different formats in a straightforward way ( Fig. 4a ). The simplest example is being a string of genetically linked GA1 modules ( Fig. 4b ) for binding multiple, same or different-specificity, Fab LRT molecules for the avidity boost or “randomized” multi-specificity, respectively.

GA1-based plug-and-play platforms. Schematic representation of the target-directed Fab LRT delivering GA1-fused effector, and some effector variant (a): GA1 modules to build multimeric strings for avidity enhancement (b); enzyme- or fluorescent tags for protein detection (c), split enzymes for protein complementation detection assays (d); GA1 fusions to Fc, mimicking IgG (e); GA1 fusions with Fab H (bi-Fab) or scFv for BiTE mimetics and some possible tri-specific binders (f). Change of only the Fab LRT component in the system will redirect its specificity.

GA1 could be genetically coupled with the enzymes and protein tags for antigen detection by means of Fab LRT ( Fig. 4c ): we have successfully applied GA1 fusions to β-lactamase (with a fluorogenic substrate) or SNAP-tag (modified with a fluorescent label) to be paired with the interchangeable Fab LRT of a variety of specificities for routine antigen detection (ELISA, CFC) and imaging purposes. A novel wash-free β-lactamase complementation-based detection assay ( Fig. 4d ) has been developed by fusing GA1 to two split β-lactamase complementation fragments [ 158 ] (details of this work are discussed in the next chapter). Also, linking of GA1 to the IgG Fc fragment replacing Fab, resulted in the IgG mimetic ( Fig. 4e ) with the interchangeable Fab LRT specificity that could be utilized in place of multiple IgGs in the Fc-mediated processes. In addition, the GA1 fusions to the trans-membrane domains (TMDs) of cell receptors made in the endoplasmic reticulum, anchors the exchangeable Fab component to the eukaryotic cell surface, thus, enabling investigation of the cell–cell signaling and interaction, and many other applications.

It turned out that the Fab-binding affinity maturation has been beneficial for GA1 in other aspects as well: it significantly reduced Fc binding and practically abolished GA1 affinity to all natural Fabs tested, including human kappa (Fab H ) – the parental to Fab S [ 123 ]. This made the GA1–Fab LRT interactions insensitive to the presence of endogenous IgGs, which are very likely in many samples as a background – an essential detail for an experimental platform. High specificity and orthogonal nature of the GA1–Fab LRT binding also allowed us to genetically connect a second specificity to the GA1 module in a form of Fab H , or scFv, without being concerned of their possible interaction and interference with the Fab LRT binding. The resulting bi-Fabs, functionally mimicking the bi-specific T-cell engagers (BiTEs), very efficient immunotherapeutics, have the advantage of one easily interchangeable specificity and are discussed in more details below. Fab LRT , Fab H , and scFvs containing and proximity-sensitive Fab LRT and split-scFv GA1-complexes represent two of many possible tri-specific formats ( Fig. 4f ). In addition, it is important to note, that the control over the linker length in the GA1 fusions could be an essential variable for applications in the systems with specific dimensional or steric demands.

A novel β-lactamase complementation-based assay. As mentioned above, we have developed a wash-free antigen-detection system based on the GA1 module fusions in a protein-fragment complementation format, widely used for identification and studying of protein–protein interactions. It is comprised of two fusions of GA1 to two complementary β-lactamase fragments (BLFs) individually pre-bound to the two Fab LRT component targeting non-redundant epitopes of the antigen of choice. When the fusions bind independently to the non-overlapping epitopes on the antigen by means of Fab LRT component, proximity of the BLFs attained within the immune complex should allow them to reassociate into the active enzyme, detectable with a fluorogenic substrate ( Fig. 4d ). Success of complementation and enzyme reactivation depend to a great extent on the appropriateness between the length of fusion linkers and the distance between the antigen epitopes targeted by Fabs.

The recent world threats caused by severe viral infections motivated us to choose Ebola and Zika virus proteins (EBOV NT CT and MT ZIKV, respectively) as antigens to be detected in the system and used initially for generation of the high-affinity sABs by phage-display selections reformatted next into the Fab LRT scaffold. The independent mode of antigen binding by the Fab pairs was confirmed by SPR. The 30 aa-long linker (~100 Å stretched) used in all GA1 fusions turned out to be sufficient for successful complementation; β-lactamase activity was readily detected by a fluorescent signal in the reactions containing viral antigens with antigen-appropriate Fab LRT pairs and complementary GA1-BLF [ 123 ].

This wash-free sandwich antigen-detection system has been also applied for SARS-CoV-2 detection using a pair of Fab LRT targeting two non-overlapping epitopes of the Spike RBD. The detection limit of the systems (~10 nM) falls within the standard range for a sandwich antigen-detection immunoassay, like ELISA. Together with the simple and fast non-wash procedure, reproducible quantitative results, and detected-antigen adjustability, easily achievable by Fab LRT pair exchange, this makes the protein-complementation GA1-based assay a suitable candidate for POC applications; in addition, it was shown, that its components can be freeze-dried and fully reactivated after rehydration [ 85 ].

The strong assay performance confirmed high potential of GA1 as an effective non-covalent bond between the BLFs and Fab LRT of any specificity, thus, laying the basis for numerous plug-and-play opportunities.

A novel plug-and-play BI-Fab format. Our next endeavor undertaken was to apply the ultra-high affinity GA1-Fab LRT platform for building bi-Fabs complexes mimicking structurally and, hopefully, functionally the bi-specific T-cell engagers (BiTEs), with an advantage of one varying specificity providing by the interchangeable Fab component in a plug-and-play manner.

Classical BiTEs represent engineered fusion molecules, consisting of two different scFvs of particular specificities – one, binding to T-cells via the CD3 component of the T-cell receptor complex, and the other – to a cancer cell via a tumor-specific molecule [ 27 ]. This engagement leads to the T-cell dependent death of the cancer cell.

The principle of two-molecule design of bi-Fabs exploits the GA1 module as a reliable non-covalent fixative for Fab LRT already experimentally approved in this capacity in the plug-and-play detection assay discussed above. Since GA1 orthogonal binding demonstrates no measurable binding to Fab H scaffold, it allows to genetically fuse Fab H to GA1 through a flexible linker without affecting the GA1-binding when Fab LRT is added to the fusion, forming a bi-Fab – a stable association of two distinct antigen-binding specificities [ 123 ].

To mimic the BiTE design in the bi-Fab format, we choose two antigen targets: (i) T-cell specific CD3 receptor commonly used in the BiTE therapy and as a specific cell surface marker, and (ii) HER2 receptor, highly over-expressed on the cell surface in many cancer cells such as breast-cancer cell lines, including SKBR3. To that end, three well-characterized antibodies were chosen: the already tested in the BiTE format humanized α-CD3 antibodies, OKT3 or UCHT1; and α-HER2 trastuzumab, highly therapeutic anti-cancer antibody. To construct bi-Fabs in either “polarities”, each antibody was reformatted in both Fab H and Fab LRT scaffolds and all three resultant Fab H variants were fused to GA1 at their Hc C-termini via a 13 aa-long GS linker. The bi-Fabs were assembled combining the CD3 or HER2 specificities reciprocally as either Fab H –GA1 fusion or Fab LRT ( Fig. 5 ).

Schematic representation of T-cell engagement in cytolytic synapse with cancer cell expressing HER2 receptor by means of bi-Fab inter-cellular bridging: it binds HER2 receptor with Fab H , genetically fused to GA1, while Fab LRT , non-covalently attached to GA1, binds to CD3δε co-receptor of T-cell specific receptor (TCR). Activation of the CD8 cytotoxic T-cell involves cytolytic granule fusion and release directed toward the cancer cell, while the activated CD4 helper T-cells increase production and secretion of cytokines interferon γ and IL2, activating cytokine production, cytolytic effect, and proliferation of peripheral blood immune cells. Lysis of the attached cancer cell is caused by the membrane perforation by perforin, and subsequent programmed cell death induced by granzymes, entering through the perforin pores into the cancer cell cytoplasm. Compilation of the T-cell activation effects is shown. Bi-Fab is colored as following: GA1 is shown in red; fused to it, Fab H (specific for HER2 receptor of cancer cell) – in green, and Fab LRT , specific to CD3 – in blue. Two different CD3-specific Fab LRT , derivatives of OKT3 and UCHT1 antibodies, were interchangeably used with trastuzumab-derived Fab H fused to GA1.

All the bi-Fab combinations showed efficient T-cell engagement activity, similar to the activity of the covalently linked BiTE, as measured in the PBMC-SKBR3 co-cultures by three readouts: (i) activity of the cytoplasmic enzyme, lactate dehydrogenase, released into the medium upon cell lysis caused by activation of cytotoxic T-cells; (ii) interleukin IL2, and (iii) interferon γ produced by T-helper cells. As expected, functionality of the bi-Fabs was absolutely dependent upon the genetic fusion of GA1 to Fab H , as no detectable activity was observed when all the individual components (Fab H , Fab LRT , GA1) were added as three separate unlinked entities [ 123 ].

Given the ease of interchanging Fab LRT in the bi-Fabs, we envision the following possible advances of the GA1-based plug-and-play BiTE mimetics. A high-throughput platform can be developed to profile bi-Fabs targeting many different cancer-specific cell-surface markers through the Fab LRT assortment. Then, the Fab LRT variety could target any number of surface antigens on a certain type of cancer cells, amplifying therapeutic effect. In addition, as in other GA1-fusion applications, the linker length could be easily adjusted tailoring the bi-Fab for the particular antigen or even antigen epitope. Multiple GA1 modalities could be also included into the design to exploit possible avidity effects in the resulting multi-valent bi-specific formats.

PERSPECTIVES

Protein engineering is now a mature field of protein science. While the ability to clone, express, and purify proteins is well established and is incorporated into the most standard operating procedures in the research laboratories, the field will continue to evolve as new technologies that build on those described here are brought into practice. One might argue that protein engineering has been practiced for hundreds of years through genetic selections. However, attempts to understand molecular basis of the phenotypic alterations and to rationally design them have only become possible through the relatively recent process involving integration of the protein structure information. An important aspect of protein engineering is that it has both basic and applied research applications. Thus, we can work on projects that build our basic understanding of the molecular basis of biological systems, while at the same time, produce molecules that are beneficial to health care and to society in general.

Abbreviations

bi-specific T-cell engagers

complementarity-determining region

constant regions of the heavy chain

constant domain of the light chain

fragment antigen binding

fragment crystallizable

heavy chain

Dr. A. A. Kossiakoff laboratory at the University of Chicago

light chain

• synthetic antibody

single chain fragment variable

engineered variant of protein G domain C2

variable domain of the heavy chain

variable domain of the light chain

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Acknowledgments

The author is deeply grateful to Tony Kossiakoff for critical reading and fruitful discussions of the review and to Erna Davydova for technical help.

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Translated from Uspekhi Biologicheskoi Khimii , 2022, Vol. 62, pp. 319-368.

In loving memory of Lev P. Ovchinnikov,

great scientist, wise mentor, and dear friend

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Davydova, E.K. Protein Engineering: Advances in Phage Display for Basic Science and Medical Research. Biochemistry Moscow 87 (Suppl 1), S146–S167 (2022). https://doi.org/10.1134/S0006297922140127

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Making medical discoveries: Temple senior’s journey in medical research

Reza Abdavies looks ahead to working as a clinical research assistant at the Children’s Hospital of Philadelphia.

Big Future: Clinical research assistant at the Children’s Hospital of Philadelphia (CHOP).

Why Temple: Growing up in rural Massachusetts, Reza Abdavies felt eager to explore a lifestyle filled with diverse cultures and vibrant urban life, ultimately leading him to Philadelphia. Drawn in by the vast array of extracurricular activities and the dynamic academic environment, Abdavies knew that Temple’s College of Science and Technology (CST) was where he could thrive. His decision was solidified after touring the campus and feeling an immediate connection to the community and its offerings. At Temple, Abdavies pursued his passion for biology, setting out for a career focused on helping others and making a difference. This journey has shaped the foundation of Abdavies’ eventual transition toward pre-med studies. 

Academic highlights: A pivotal moment in Abdavies’ academic journey at Temple was his immersion in Parasitology (BIOL 3243). The course emphasizes the identification, pathology and control of significant medical parasites, complemented by hands-on laboratory experience. Through his journey in the course, Abdavies found his perspective on medical research especially broadened. Supporting his development, Professor William Bowie, who taught Organic Chem Lab, provided mentorship to Abdavies. Throughout the course, Bowie clarified complex concepts, enhancing Abdavies’ confidence and proficiency in lab work.

Standout internship: Abdavies’ ambition and curiosity led him to the Lankenau Institute for Medical Research, where he delved into the effects of essential micronutrients and their effects on the epithelial barrier function. This significant internship experience not only resulted in a co-authored publication, but also solidified his passion for research, setting a firm foundation for his future career in medical research at CHOP. 

Philly life: Living in Philly, Abdavies has especially enjoyed the variety of culinary experiences within the city. One of his favorite restaurant discoveries has been Chubby Cattle, located in Philadelphia’s Chinatown. Closer to Temple, the campus’ food trucks have also remained some of Abdavies’ notable favorites.

Temple Made moment: One of Abdavies’ most fulfilling experiences was serving as a peer leader in CST. In this role, he helped incoming students navigate the transition to college life, empowering them in their personal growth and development. This position highlighted his own growth as an undergraduate and emphasized the impact of Temple’s community-oriented education.

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$1.6 million announced by Manchin to go towards Rainelle Medical Center and two other projects

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The two other medical research projects are focusing on vision and aging.

“I’m pleased HHS is investing more than $1.6 million to strengthen health services in Greenbrier County and advance important aging and vision research at WVU. I look forward to seeing the positive impacts of these important initiatives for years to come. As a member of the Senate Appropriations Committee, I will continue advocating for resources that support academic research and ensure all West Virginians have access to quality, affordable healthcare,” said Senator Manchin.

Each award are as followed:

• $1,184,522: Rainelle Medical Center, Greenbrier County
• $228,000: West Virginia University: Aging Research
• $228,000: West Virginia University: Vision Research

The total funding for these projects is $1,640,522.

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Twenty two students (twelve medical students, six students in Dental Medicine and four students in Pharmacy) have undergone their summer internship at the training and clinical facilities of Medical University-Varna. For two weeks interns trained at the academic and clinical facilities, under the direct supervision and guidance of leading professors in their respective fields. Summer…

Institute for Applied Medical Research

A pilot plant "Pul-Sar" in Dubna (Special Economic Zone "Dubna"

"Russian Medical School" is an educational institution for postgraduate medical education

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Developments

We cooperate with:

• N.N. Priorov Central Institute of Trauma Surgery (development of the newest methods of hemostasis during spine surgeries)
• M.F. Vladimirsky Moscow Regional Clinical Research Institute (clinical studies of endoscopic methods of hemostasis)
• N.V. Sklifosovsky Research Institute of Emergency Medicine (development of methods of emergency gastroenterological care)
• A.N. Rychikh Scientific Research Center for Coloproctology (colorectal surgery, endoscopy)
• Sechenov Moscow State Medical University
• Pirogov Russian National Research Medical University
• Pavlov Russian State Medical University
• Russian Society of Surgeons
• Children’s City Clinical Hospital named after Z.A. Bashlyaeva

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Dubna, Ulitsa Programmistov 4, building 6 Special Economic Zone “Dubna”

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Manchin announces $1.6 million for rainelle medical center, two medical research projects at west virginia university.

Washington, DC – Today, U.S. Senator Joe Manchin (D-WV), member of the Senate Appropriations Committee, announced $1,640,522 from the U.S. Department of Health and Human Services (HHS) for the Rainelle Medical Center and two West Virginia University (WVU) medical research projects focusing on aging and vision. 

“I’m pleased HHS is investing more than $1.6 million to strengthen health services in Greenbrier County and advance important aging and vision research at WVU,"  said Senator Manchin. “I look forward to seeing the positive impacts of these important initiatives for years to come. As a member of the Senate Appropriations Committee, I will continue advocating for resources that support academic research and ensure all West Virginians have access to quality, affordable healthcare.”

Individual awards listed below:

• $1,184,522 – Rainelle Medical Center, Greenbrier County
• $228,000 – West Virginia University: Aging Research
• $228,000 – West Virginia University: Vision Research

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Pullman-Moscow Regional Airport terminal project nears completion

The Palouse’s new air travel hub is nearing completion and set to open to the public later this month.

The Pullman-Moscow Regional Airport’s new 47,000 square foot terminal is opening May 22 after two years of construction . The $92 million project includes a brand new terminal nearly six times larger than the existing structure, as well as an additional 4,500 square foot expansion debuting later this summer.

Airport officials hosted a pre-opening tour of the new facility for Palouse-area media on Tuesday.

The new terminal includes capacity for three ticket counters, two TSA screening lanes and three rental car counters. Private charter security screenings will also be available in August with the completion of the western expansion. Passengers arriving into Pullman will no longer have to brave the weather conditions outside, and will instead pass through a modern security gate inside the terminal building.

In spite of the changes, Pullman-Moscow Regional Airport Board Chair and Pullman Mayor Francis Benjamin said the security experience is still likely to be the fastest passengers have experienced with air travel.

References to the Palouse and its institutions of higher education will be evident throughout the new airport. Artistic depictions of the Palouse punctuate each ticket counter, while Washington State University and University of Idaho iconography will be visible throughout the site. Glenn Johnson, longtime Pullman mayor and voice of the Cougs , welcomes visitors to the terminal and provides pre-boarding instructions.

WSU and UI, both heavy users of the airport, provided a joint $1 million in support for the project in 2021, with the cities of Pullman and Moscow contributing a further $2 million each.

Art Bettge, vice-chair of the airport board, noted during a tour of the facility that between the new terminal and the runway project completed in 2022, the Palouse has seen $250 million in investments for a local cost of about $18 million. The lion’s share of the two project’s funding has come from federal sources.

Unlike most airports, summer represents a slowdown for the airport, giving staff time to adjust to the new facilities before the number of flights is expected to pick up in the fall. The airport currently offers two flights to Seattle daily through Aug. 15, with that number rising to five or six thereafter with the addition of a daily flight to Boise. Additional destinations are also being considered.

The new terminal includes seating for upwards of 160 people, an outdoor seating area, a service animal relief area, and space for concessions both pre- and post-security screening. Approximately 70 people work at the airport, with additional staffing needs likely once the new terminal is fully online.

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Undergraduate students receive $61,000 in 2024–25 research fellowship funding.