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Monoclonal Antibodies as Therapeutic Agents

Development and optimization of monoclonal antibodies as therapeutic agents

Abstract: As medicinal treatments move to a more personalized approach, new developments must be made in order to assist in treating each patient on an individual basis. Within the past thirty years monoclonal antibodies (mAbs) have transformed from being scientific tools, to powerful therapeutics to help fill in the gaps in personalized medicine. Initially, mAbs emerged as cancer therapeutics, however these antibodies can be designed to have specificity for any cell surface target. This versatility lends itself to development of mAbs for ailments such as diabetes, arthritis, multiple sclerosis and Alzheimer’s disease. As a result, the industry is growing exponentially and is currently valued at billions of dollars. This review will focus on various methods of action for mAb-based treatments, as well as current limitations of these treatments, and development of new mAbs. It will also go into basic background, covering the history of mAbs as a therapeutic agents and the evolution they’ve undergone in the past 30 years.
1.0 Introduction:

Name
IgM
IgG
IgA
IgE
IgD
Size (kDa)
900
150
385
200
180
% found in Serum
10
75
14
0.002
1
Fixes complement
Yes
Yes
No
No
No
Function

– Expressed on surface of B cells
– In a pentameric form
– Strong avidity, allowing it to eliminate pathogens in B cells humoral response, until enough IgG is generated.
– Workhorse of the immune response system, main attack force against pathogens.
– Only antibody able to cross the placenta to give immunity to the fetus.
– Found in mucosal membranes.
– Resistant to digestion
– In a dimerized form
– Prevents colonization by pathogens
– Binds to allergens, and triggers a histamine response
– Protects against parasitic worms.
– Receptor for antigens on B cells not exposed to antigens yet.– Activates other parts of the immune system response

Table 1: Here is described the five major classes of antibodies. The vary greatly in size and function. One thing to note is the table line “fixes complement”, essentially if the antibody is able to fix it’s complement it is binding the serum complement to the product of the resulting interaction of the antibody and it’s antigen. This in turn can result in lysing of the microbes that have entered the host, can also be called cell lysing.
The body has many defense systems against antigens. One especially important molecule to antigen defense are antibodies. Due to their specificity as a result of being created specifically for individualized antigens, and their high affinity of binding they play an essential role in humoral affinity. However, their activity is not only related to binding of antigens, they also are receptor molecules and in turn promote a response that recruits immune cells for other effector functions of the immune system. There are a various classes of antibodies that break down into smaller subclasses; the five main classes with diverse functions are presented: immunoglobulin (Ig)A, IgD, IgE, IgG, and IgM (Table 1). Despite the various classes of antibodies a majority of them are IgG’s, they constitute approximately 75% of the serum immunoglobulin repertoire. Within this class there are another four subclasses of IgG’s, these molecules vary in their abundance and the respective effector effect each invoke.
IgG’s are generally conserved between its four various subclasses, which contain two light chains and two heavy chains. In IgG’s the light chain will have two domains a variable (VL)­ and constant domain (CL). There is also another chain called the heavy chain, this structure has one variable domain, and three different constant domains (CH1, CH2, CH3) These domains are abbreviated in such a way that it denotates the domain it is part of and the chain it is associated with in the subscript. The variance between the four subclasses of IgG’s are the locations of disulfide bonds and amount of disulfide bonds as shown. (Figure 1)

IgG1

IgG2

IgG3

IgG4
Figure 1: Shows the four classical structures of IgG’s. The main difference to notice is the variance in location of the disulfide bond between VL (purple) and CH (green). These disulfide bonds link the light and heavy chains together. However the disulfide between the two heavy chains (green) vary in the amount there, these specific region where these bonds form are called the hinge region.
The way the domains are broken up in IgG’s are not by chance, they are split by their bioactivity into various subdomains which are ordered in a logical manner. IgG’s are categorized in subdomains known as the crystallizable fragment (Fc), the complementarity determining regions (CDR), and the antigen-binding fragment (Fab). These domains are further explained in Figure 2.
Initially mAbs were recognized as biological tools and were essential for applications in pathological diagnosis and laboratory investigation. Due to specificity in binding, they were used to identify phenotype of blood cells, and other tissues as well as other diagnostic/imaging techniques, such as immunohistochemistry, flow cytometry and various other. (Weiner 2015) Early research showed that monoclonal antibodies (mAbs) could be easily and efficiently produced through hybridoma technology (a technology that won the Nobel peace prize), allowing them to be applied to the research mentioned earlier.
Only 30 years ago were mAb’s proposed as a possible therapeutic for cancer. Initially murine mAbs were trialed as a cancer treatment, and the results from the study were disappointing. Murine mAbs, are derived from mice, specifically laboratory mice. This led to troubles when administering murine mAbs to humans, due to the suboptimal compatibility of the mAb with the human immune system and low half life. Specifically the mAbs had a poor ability to recruit host cell effector functions, as well as poor penetration in tumor sites. Though when able to access those functions it was found that murine mAbs were poor at producing a cytotoxic effect on tumor cells, and generation of complexes that stimulated minimal allergic reactions to full out anaphylactic shock. This poor interaction, and allergic reaction is known as the human anti-murine antibody (HAMA) response. Despite the setbacks, information was elucidated that showed a mAb therapy is possible.

Figure 2:Here is described the structure of an IgG, with it’s domains and subdomains. The first section to talk about is the Fc region, also known as the crystallizable fragment region. It is the tail region of the antibody and the region that interacts with cell surface receptors. Most effector functions are produced after binding with this region. Important thing to note about the Fc is that it contains a highly conserved N-glycosylation site, which is important to F­C receptor mediated function. The Fab region is also known as the antigen-binding region, as in its name this is the location of where the antibody binds to antigens, specifically called the paratope. The paratope is located in the variable domain, which is the reason for the high specificity of the antibody to its corresponding antigen. In the Fab region there is another region called the CDR region, which stands for complimentary determining regions. The CDR is determined by B or T-cells interacting with the antigen and determines the epitope of the antigen. Once interacting with an antigen, the B-cells produce an antibody with a CDR region that has a specific interaction with the epitope, also known as the paratope (as mentioned earlier)
As science developed techniques were discovered that allowed genetic modification of murine mAbs to produce chimeric mAbs, that allowed the ushering in of successful mAb therapy. (Dowling, Chavaillaz et al. 2005) By chimeric, the mAb is now a hybrid of mouse/human mAb and behaves more like a natural human mAb. This change allows the host to less likely view the mAb as foreign antigen, as well as increasing the half life of the molecule. In turn the chimeric nature allows the mAb to induce normal effector functions, as well as induce proper interactions with malignant cells.
The next advent in mAb development were humanized mAbs. This may sound similar to chimeric structures when explained, however the key to understanding this difference is as opposed to replacing the Fc region in chimeric mAbs, there is a substitution where rodent sequences are exchanged for human sequences except in the Fab region, specifically the CDR where paratope binding is done.
This may seem odd to take these approaches of generating various mAbs degrees of humanization as it would be ideal to develop fully human mAbs. However this was hard to do initially due to challenges related to a lack of a stable human myeloma fusion partner. Though this challenge was eventually overcome due to phage-display platforms, and transgenic mouse platforms. These methods are both extremely versatile. For phage-display platforms it was discovered that foreign DNA sequences could be cloned into bacteriophages such that the cloned sequences would be expressed on the surface of the phage as fusion proteins, in turn they would then be enriched for specific sequences. This was combined with PCR amplification methods for cloning expressed Ig variable region cDNA in order to create a library of phage fusion proteins that could be used rapidly to access target-specific mAbs without hybridoma clones. (Lonberg 2008) Later it was shown that genetically engineered mice were able to express fully human antibodies that could be accessed by conventional hybridoma technology, allowing another technique to produce fully human mAbs. Despite the possibility of using fully human mAbs there are still issues of immunogenicity, which is why the development from murine to fully human mAbs was necessary for the furthering of mAbs as therapeutic agents.

Antibody Type
Murine
Chimeric
Humanized
Human
% Human
0 %
65 %
90 %
100 %
Generic Naming Suffix
-omab
-ximab
-zumab
-umab
Year and First Drug Released
Muromab
1986
Abcximab
1994
Daclizumab
1997
Adalimumab
2002
Function of Drug
Reduces acute immune response in patients undergoing organ transplants
Binds to glycoprotein IIb receptor of human platelets and in turn inhibits platelet aggregation
Binds to CD25, an alpha subunit of the IL-2 receptor of T cells. Helps treat patients with relapsing multiple sclerosis. However off market due to causing encephephalitis.
TNF inhibitor that is used to treat rheumatoid arthritis, ankylosing spondylitis, Crohn’s disease, ulcerative colitis, and psoriasis.
Table 2: In this model the mAb’s are colored with red and green. Red signifies human sequences while green signifies murine/mouse sequences to generate the structure. As the mAb is filled with more red, the chances of immunogenicity decreases, with green being extremely high while full red will become low. Unfortunately due to the nature of mAb’s it’s still possible for a fully human mAb to produce an immune response within a human host.
2.0 Optimizing and Designing Antibodies
Due to their unique structure and various forms mAbs show why they are great for therapeutic use but also why they are challenging molecules to develop. There are factors that are essential to antibody design and that should be kept in mind: conformational stability, binding affinity and specificity, colloidal stability, effector function, antibody design, cytotoxicity (antibody drug conjugates), and bi-specificity. Each of these properties can be easily changed sequentially however, changes in one property can lead to deficiencies in others. While optimizing these categories simultaneously is prohibitive due to the fact such a large library must be built to accommodate the modifications.
2.1 Antibody Binding Affinity and Specificity
The first thing to focus on with antibody design is to be able to have it recognize the antigen with high affinity and specificity. To modify this, changes must be made in the CDRs, there are a few approaches that allow the redesigning of mAbs such as de novo design, and motif-grafting.
De Novo design is essentially starting anew. To redesign the CDR’s of the mAb a computational approach is taken named OptCDR (Optimal Complementarity Determining Regions). This has been created to design
References:
Weiner, G. J. (2015). “Building better monoclonal antibody-based therapeutics.” Nature Reviews Cancer 15: 361.
Tiller, K. E. and P. M. Tessier (2015). “Advances in Antibody Design.” Annual Review of Biomedical Engineering 17(1): 191-216.
Lasch, S., et al. (2015). “Anti-CD3/Anti-CXCL10 Antibody Combination Therapy Induces a Persistent Remission of Type 1 Diabetes in Two Mouse Models.” Diabetes 64(12): 4198.
Raedler, L. A. (2016). “Darzalex (Daratumumab): First Anti-CD38 Monoclonal Antibody Approved for Patients with Relapsed Multiple Myeloma.” American health

Genetically Modified Foods: Health, Effects on Ecosystems and Economic Viability

Genetically Modified Foods
Walking into a supermarket, have you ever wondered what genetically modified foods (GM foods) actually are and the difference between GM foods and non-GM foods? GM foods are defined as foods produced from or using “organisms in which the genetic material has been altered in a way that does not occur naturally by mating and/or natural recombination” (World Health Organization 2014). Because of an increasing presence of GM foods in markets, I became interested in exploring the possible drawbacks of GM foods in the long-term. Therefore, I chose to analyze three different aspects of GM foods—health and safety, effects on ecosystems, and economic viability—in the long-term, to help myself and others make a decision on whether GM foods should be continued to be created and consumed.
A goal of GM foods is to help increase the nutritional value of foods. A GM food known as golden rice aims to create β-carotene (beta carotene) in rice plants to help fight vitamin A deficiency. However, in the most recently created strain of golden rice, GR2E, β-carotene levels in the rice were so low that it would require a person to consume 3.75 kg of golden rice per day to receive the necessary amount of β-carotene (Wilson and Latham 2018). Considering that the research on golden rice has been going on since 1982, there is no telling how much more time and money will be needed for the research to reach its goal.
The consumption of GM foods is linked to a variety of health complications. Research has shown that the consumption of GM foods is linked to organ toxicity, allergies, immune system disorders, accelerated aging, infertility, and gluten disorders. In fact, the American Academy of Environmental Medicine is urging doctors to put their patients on GMO-free diets as a result of these harmful impacts (Dean and Armstrong 2009). For example, the presence of Bt-toxin, which is derived from Bacillus thuringiensis, in genetically modified corn leads to production of Bt-toxin that pokes holes in cell walls, which is linked to diseases including cancer and leukemia (Smith n.d.). As a number of potentially deadly diseases have been identified to be caused by consumption of GM foods in the short term, there is a strong possibility of the existence of other diseases that have not yet been identified that may prove deadly in the future.
While people have claimed that extensive testing has been done to prove the safety of GM foods, the substantial equivalence with non-genetically modified products is not a proof of harmlessness. The chemical composition of food is an important indication—however, due to insertional mutagenesis or new metabolites, not all herbicide residues, unintended or unknown metabolites, and insecticide toxins are assessed (De Vendômois et al. 2010).
A bias can also be seen in the fact that the regulatory toxicological tests that were presented to authorities were done solely by the companies developing industrial products. Few studies have been conducted by independent groups, while many industry-funded studies have been conducted. Industries tend to only publish studies that help support their agenda—for instance, in the bisphenol A controversy, an analysis of all performed studies revealed that none of the industry-funded studies showed adverse effects of bisphenol A. However, 90% of government funded studies showed hazards at various levels and doses (Vom Saal and Hughes 2005). Therefore, as less biased studies have exposed serious risks in GM foods that cannot be tolerated in the status quo, GM foods therefore do not uphold long-term sustainability.
Another goal of GM foods is to increase yield of crops to help increase food production around the world. Since 1987, several thousand experimental GE-crop field trials have been conducted, and at least 3,022 applications were approved for traits often associated with yield, such as tolerance to abiotic stress or disease resistance, in which at least 652 trials named yield as the particular target trait (Gurian-Sherman 2009, 3-4). However, the only transgenic food crops that showed significantly improved yield were varieties of BT corn. The conclusion that can be drawn is that of several thousand field trials, many of which have been intended to raise operational and intrinsic yield, BT corn is the only crop that has succeeded. As a result, future potential yields of GM foods should be carefully considered.
In addition, Monsanto’s genetically-modified soybeans are both harmful for the planet’s ecosystem and not helping farmers produce greater yields. The crop is a leading cause of colony collapse disorder, which is also known as the bee death phenomenon, meaning that it is also a significant threat to the entire food supply. Without bees, 80 percent of all flowering plants and more than 75 percent of all the fruits, nuts, and vegetables grown for human consumption would cease to exist (Huff 2014). The U.S. Environmental Protection Agency (EPA) has also admitted that there was no difference in soybean yield when soybean seed was treated with insecticides and when soybean seed did not receive any insect control treatment.
The increased adoption of GM foods has increased the use of weed-killing herbicides, which is harmful to the planet’s ecosystem, as weeds have become more resistant. Research conducted by Federico Ciliberto of the University of Virginia spanning 14 years shown significant increase in the use of herbicides in plants such as soybean, which had the adopters of GM crops using 28% more herbicides than non-adopters (Newman 2016). Because weeds are developing resistances over time, more and more herbicides must be used to destroy them, leaving more traces of the harmful chemicals that herbicides contain.
Furthermore, commonly used chemicals have found to be harmful. For instance, polyethoxylated tallowamine (POEA), an ingredient used in Roundup, a top-selling weed killer, has been found to be deadlier to human placental, embryonic, and umbilical cord cells than the herbicide itself. In addition, the EPA considers glyphosate, which is the main chemical associated with herbicides, to have low toxicity when used in the recommended doses, but with the buildup of weed resistance, the greater doses of herbicides used could have higher unsafe levels of toxicity, leading to environmental and health issues (Gammon 2009). As a result, there is potential for increased production of GM foods to lead to ecological collapse in the long-term.
Another goal of GM foods is to help farmers gain more income through a more stable crop. A study following annual income of farmers globally from 1996 to 2012 found that many farmers, especially in developed countries, benefited from lower costs of production (Brookes and Barfoot 2014). However, small-scale farmers have trouble seeing economic gains because of the price of seed and lack of access to credit, because they often need institutional support, such as access to affordable markets, and may need assistance in “improving soil fertility, increasing nutrient availability, and optimizing plant density” (National Academies of Sciences, Engineering, and Medicine 2016).
Additionally, GM foods are currently monopolized by large corporations. Monsanto, DuPont/Pioneer, Syngenta and Dow AgroScience own 80 percent of the U.S. corn market and 70 percent of the soybean business for GM foods (Roseboro 2013). Specifically, new genetically engineered plant technologies and resulting GM plants and seeds have been patented, because patented seed costs are controlled by corporations focused on maximizing profits. Farmers contracted with Monsanto must pay numerous fees in addition to the higher cost of genetically modified seeds that farmers are required to buy fresh, annually.
Furthermore, GM seed prices have been steadily increasing. According to the U.S. Department of Agriculture’s Economic Research Service, the average per-acre cost of soybean and corn seed increased 325 percent and 259 percent between 1995 and 2011, respectively (Roseboro 2013). Non-GM seeds are also withheld from farmers, so they end up facing decreased options at significantly higher prices. As a result, the goal of helping farmers’ income has proven to be not viable both in the short-term and the long-term.
In addition, while people claim that eliminating GM foods would restrict valuable genetic research, preventing scientific progress, complex contracts and ambiguous patents by large agricultural corporations prevent this scientific progress in the first place (McIntyre et al. 2009, 6-7). Especially in developing countries, patents drive up costs and restrict experimentation by individual farmers while also undermining local practices for securing food and economic sustainability. Moreover, there is particular concern regarding present intellectual property rights instruments, because of concerns that they may inhibit access to assets of vital importance to the independent research community, specifically in view of the need for analyses and long-term experimentation on climate change impacts. Therefore, even if GM foods are not eliminated, the mere cost of trying to conduct research is already inhibiting potential research from occurring, preventing scientific progress.
There are still too many unknowns when it comes to genetically engineering our food, meaning that there is no way to properly predict long-term effects consuming GM foods may have. A study has revealed that 5% of a host’s genes had their levels of expression changed after just one foreign gene was inserted (Smith n.d). This change is in addition to any changes that may be present due to deletions and mutations in the host genes. 5% is a massive change, which may contribute to a wide variety of adverse health-related side effects that have yet to be discovered. Given that the study of genes is still ongoing, this 5% could affect genes that display pleiotropy or polygenic inheritance, thus inadvertently giving rise to other unpredictable impacts on health.
The safety of GM foods for consumption, ecologically, and economically are assessed differently by country and by local conditions (World Health Organization 2014). This makes it important for consumers to conduct to research on what national policies are in place to determine the safety of GM foods. Furthermore, unless some world standard is specified for regulating GM foods, there is no way to properly conduct studies to try to determine the long-term effects GM foods may have.
Works Cited
Brookes, Graham, and Peter Barfoot. 2014. “Economic Impact of GM Crops.” GM Crops

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