How mRNA Therapeutics are Entering and Revolutionizing the Monoclonal Antibody Field—Part 1-of-2

Posted in: Therapeutics

Introduction: Advances in the antibody (Ab) field have always been deemed revolutionary. The first ever Nobel Prize was awarded for the discovery of immunization in 1901, and the honor was later extended for the respective discoveries of the principles for monoclonal antibody (mAb) production and Ab diversity in the late 80s. Today, mRNA therapeutics are stepping on the scene, providing a new modality for vaccination or treatment of various diseases. 

Although Zone in with Zon focuses on “what’s trending in nucleic acids research,” this blog on modified mRNA (mod RNA)-encoded Abs highlights both the nucleic acid and protein aspects of Abs. “Antibody OR antibodies” appear in ~1.3 million PubMed articles, ~4.1 million Google Scholar items, and ~121 million Google links. For this reason, this blog will be the first part of a two-part commentary, as I believe that the extensive science behind a subject as vast and important as Abs is worthy of a more in-depth discussion.

Frankly, I know relatively little about Abs or immunology, and what I do know has been acquired stochastically over my years as a researcher. Because of this, I did much more “homework” than usual in preparation of this two-part series. Part 1 will provide a historical perspective on the evolution of mAbs, a field that has been awarded three Nobel Prizes: in 1901 (the first ever Nobel), 1983, and 1987. Part 2 will then deal with mod mRNA-encoded equivalents of mAbs, and will be posted next time. 

Emil von Behring

History of Immunization: In an educational series on mAbs in medicine by Llewelyn et al., the discovery of Abs began with the use of passive immunization, which is thought to have been first used on Christmas night in 1891. The recipient was reported as a young boy in Berlin with diphtheria, who was cured by injection of diphtheria antitoxin. Passive immunization is based on work carried out by Emil von Behring (pictured here) and Kitasato Shibasaburō, who published a landmark article in 1890, in which they showed that serum from an animal actively immunized against diphtheria toxin could be used to neutralize even a fatal dose of the toxin in another animal. 

The potential for treatment in humans was immediately apparent, according to Llewelyn et al., and Behring set to work in trying to produce large amounts of antitoxin that could be effective against diphtheria in humans. In light of this, the pharmaceutical company Farberwerke Hoechst scaled up production by immunizing sheep, and the first large trials started in 1893, with good results. In the summer of 1894, the first British patients were treated, also with favorable results. These results were obtained with tetanus toxin as well. Behring’s pioneering work led to him being awarded the first Nobel Prize in Physiology or Medicine in 1901 for the development of serum therapies against diphtheria. To this day, diphtheria and tetanus, as well as pertussis, are routinely prevented by the “DTP” vaccine.

Antibody Structure: Despite the benefits of the original serum therapies, a major problem with passive serotherapy was toxicity. Anaphylactic reactions often occurred after giving horse serum, and “serum sickness” (fever, rashes, and joint pains) was common. Llewelyn et al. state that Behring believed that the antitoxin effect resided in the protein fraction of the serum, and that Behring went on to show that ammonium sulfate precipitated a fraction (now known as gamma globulin) that retained antitoxin activity.

The active components of gamma globulin, now known as immunoglobulins (Igs) or Abs, are what protect us against the effects of diphtheria and tetanus toxins. IgG, the prototype Ab, is a glycoprotein with a molecular weight of 150,000 Dalton (D). As depicted here, the molecule consists of two identical heavy chain-light chain heterodimers linked by a disulfide bridge to form a Y-shaped structure. Each heavy chain comprises one variable (V) and three constant (C) Ig domains, whereas each light chain consists of a single variable and a single constant Ig domain. Each domain comprises about 110 amino acids.

Antibody Function: The important features are the antigen binding site (Fab) and the Fc region (fragment crystallizable), which is responsible for initiating host defense mechanisms. The Fc region is the stem of the Y structure, and the two antigen binding sites are at the tip of the outstretched arms. The hinge region of the Ab is flexible so the antigen binding sites are widely and variably separated, which allows for the linking of two identical antigens by the same antibody. Many invading microorganisms such as bacteria and viruses have repeating subunits, which allow for much more effective binding (the avidity effect). The multiple binding sites also allow for aggregation and rapid clearance of antigens.

Although an Ab may have a direct neutralizing effect on a virus or toxin, Fc-mediated defense systems are needed to eliminate the source of the antigen. Abs can sensitize a target cell for attack by killer (K) lymphocytes (a white blood cell subtype) by coating (opsonizing) the cell with IgG. K lymphocytes recognize coated target cells because they have receptors for the Fc region of IgG on their surface. This mode of killing is known as Ab-dependent cellular cytotoxicity. Similarly, phagocytes (including neutrophils and macrophages) target an opsonized antigen through Fc receptors, as illustrated here. The combination of Ab and antigen can activate the classical complement pathway, and deposition of complement on the target cell can produce lysis or further opsonization.

3D rendering of antibody opsonization of influenza viruses. Viruses coated with Abs cannot penetrate into the target cell (left foreground) and are engulfed and destroyed by a macrophage (background cells). 

Genetic Basis of Antibody Diversity: The discovery that each Ab has its own amino acid sequence in the variable region seems to suggest that a separate gene is produced for each Ab. However, if this were truly the case, most of the human genome would be needed to code for the enormous number of Ab molecules that could exist. In 1965, Dreyer and Bennett hypothesized that each class of constant region is encoded by one gene, but that large numbers of genes exist for the variable domains (VH and V1). They suggested that Ab genes would be formed by recombination of these genes during B-cell development, and they were correct. 

VDJ recombination. Taken from commons.wikimedia.org and free to use.

According to Llewelyn et al., and as depicted here, the assembly of genes for variable domains occurs by random joining of a number of gene segments (V, D, and J for heavy chains or V and J for light chains). Additional diversity then results from loss or addition of nucleotides at the junctions between these gene segments. The VDJ segment is then apposed to the constant region at the mRNA stage, after the large nuclear RNA transcripts have been edited. The number of unique Ig variable regions due to random recombination of V, D, and J gene segments is very large. Moreover, the joining positions of VH, DH, and JH segments are not always the same, and the DH segment may end up in any of its three possible amino acid reading frames. Readers interested in a more detailed description of VDJ mRNA formation and antibody production can consult this helpful video lecture.

Susuma Tonegawa. Taken from commons.wikimedia.org and free to use.

This, along with random loss or addition of nucleotides at the boundaries between the segments, further increases diversity. Since any light chain is thought to be able to combine with any heavy chain, it is estimated that >1010 Abs can be produced from a relatively small number of DNA segments. Further diversity arises later through the introduction of scattered point mutations into the DNA of the assembled heavy and the light chains. This is known as somatic hypermutation, and it usually occurs during the immune response to foreign antigen. In 1987, Susumu Tonegawa was awarded the Nobel Prize in Physiology or Medicine “for his discovery of the genetic principle for generation of antibody diversity.

Hybridoma technology for mAb production. Taken from commons.wikimedia.org and free to use.

Monoclonal Antibodies: In 1975, George Köhler and César Milstein described an elegant system that could be used to obtain pure Abs of known specificities in large amounts, as depicted here in a slide provided by the U.S. National Cancer Institute. In their method, a mouse is immunized repeatedly with the desired antigen, and the spleen, which contains proliferating B cells, is removed. B cells normally die in culture, but can be immortalized by fusion with a non-secretory myeloma cell. The resulting hybridoma can then secrete large amounts of the Ab encoded by its B cell fusion partner. Supernatants from the hybrids that survive the selection procedure are tested for binding to the original antigen.

The 1984 Nobel Prize in Physiology or Medicine was awarded jointly to Niels K. Jerne, Georges J.F. Köhler and César Milstein "for theories concerning the specificity in development and control of the immune system and the discovery of the principle for production of monoclonal antibodies." The seminal work of Köhler and Milstein was already mentioned. Jerne, on the other hand, is known for three significant ideas, discussed in detail elsewhere. Briefly, (1) instead of the body producing Abs in response to an antigen, Jerne postulated that the immune system already has the specific Abs it needs to fight antigens. (2) It was known that the immune system learns to be tolerant to the individual's own self. Jerne postulated that this learning takes place in the thymus. (3) It was known that T cells and B cells communicate with each other. Jerne's network theory proposed that the active sites of Abs are attracted to both specific antigens (idiotypes), as well as to other Abs that bind to the same site. The Abs are in balance, until an antigen disturbs the balance, stimulating an immune reaction.

Left to right: Niels K. Jerne, Georges J.F. Köhler and César Milstein. Taken from linked wiki cites and free to use.

Human Monoclonal Antibodies (hu-mAbs)

Although mAbs were discovered to enable useful diagnostic methods, rodent mAbs have proven unsuitable and ineffective for treating humans, due to the fact that they not only initiated human defense systems poorly, but are themselves the target of an immune response that can greatly shorten their circulating half-life, according to Llewelyn et al. Fortunately, in the early 1990s, molecular biology and recombinant Ab production technology, in combination with detailed descriptions of Ab gene coding, led to a revolution in the mAbs industry. A 2019 review by Van Hoecke and Roose states that “these new technologies indeed paved the way for the generation of improved recombinant mAb formats (shown here), that gradually contained less murine sequences and ultimately culminated in the design of fully human antibodies.”

Overview of mAb variants used in therapy. Fully murine (left) or fully hu-mAbs (right), and recombinant species are used in therapy (middle). These include chimeric mAbs, composed of human constant regions and murine variable regions, and humanized mAbs, where the hypervariable CDR-domains of the murine Ab are grafted on a human Ab. Clinically applied examples of each are given, including their targets between brackets. Taken from Van Hoecke and Roose J. Transl. Med. (2019) 17:54. © The Authors 2019 (Open Access: This article is distributed under the terms of the Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium. 

Readers interested in hu-mAbs can peruse the >2,600 items in my search results for titles with the phrase “human monoclonal antibodies” in Google Scholar. Not surprisingly, according to Van Hoecke and Roose, the clinical use of mAbs today represents a rapidly growing market for the biopharmaceutical industry, with projected combined worldwide sales of nearly $125 billion in 2020. mAb therapies are now available to treat disorders ranging from rheumatoid arthritis, a condition that affects millions of patients, to rare diseases that affect just a few thousand patients, such as mantle cell lymphoma.  

According to Van Hoecke and Roose, 76 mAbs have been approved by the European Medicines Agency (EMA) and/or the U.S. FDA for therapeutic use, and over 50 mAbs are being investigated in late-stage clinical studies. Approximately 6 new mAb products are licensed every year. So-called check-point inhibitor mAbs represent a striking example of how therapeutic mAbs have revolutionized treatment options for patients. These types of mAbs have boosted the cancer immunotherapy field, and check-point inhibitors are now one of the most successful and important strategies for treating cancer patients.

Although the mAb industry is one of the fastest growing pharmaceutical sectors, the technical, regulatory, and strategic Chemistry, Manufacturing, and Controls (CMC) activities necessary to successfully advance new mAb products to clinical trials and to market approval are vast and challenging. Van Hoecke and Roose add that these challenges are inherent to the current manufacturing process of mAbs. mAbs are produced using mammalian cell lines and then purified from complex media, implying that an extensive purification process is needed in order to obtain a safe formula for the Ab from the cell culture supernatant, which must be free from viruses and other contaminants.

In addition to these hurdles, mAbs are prone to a wide variety of post-translational modifications, including glycosylation, deamidation, oxidation, incomplete disulfide bond formation, N-terminal glutamine cyclization, and C-terminal lysine processing. As these modifications can strongly impact the biological activity and therapeutic properties of the mAbs, they need to be characterized and controlled, which means they require the costly development and implementation of numerous analytical tools to assess their Quality Attributes. All these aspects represent further challenges in the production process. Furthermore, regulatory agencies are constantly seeking enhanced quality, while health care systems demand lower processing costs. 

In the second and final part of this two-part blog, which will be posted on July 23, 2019, I will explore how recent advances in biosynthetic, chemically modified mRNAs have enabled the use of the human body as its own bioreactor to produce mAbs.

Mark your calendar for that posting date. In the meantime, your comments are welcomed as always.

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