Monoclonal Antibodies as Therapeutic Agents

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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:

  1. Weiner, G. J. (2015). “Building better monoclonal antibody-based therapeutics.” Nature Reviews Cancer 15: 361.
  2. Tiller, K. E. and P. M. Tessier (2015). “Advances in Antibody Design.” Annual Review of Biomedical Engineering 17(1): 191-216.
  3. 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.
  4. Raedler, L. A. (2016). “Darzalex (Daratumumab): First Anti-CD38 Monoclonal Antibody Approved for Patients with Relapsed Multiple Myeloma.” American health & drug benefits 9(Spec Feature): 70-73.

 

 

 

 

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