Oct 2, 2014 - Dr. I.O. Sule. SUBMITTED .... toward creating a âmagic bulletâ. As early as 1900, Paul Ehrlich theorized that it should be possible to ... antibodies. Dr. Pieczenik suggested to Cotton what became the Cotton-Milstein experiment.
A SEMINAR REPORT
ON
MONOCLONAL ANTIBODIES
BY
AGBABIAKA, TARIQ OLUWAKUNMI MATRIC NUMBER: 11/55EJ042 Supervised by Dr. I.O. Sule SUBMITTED TO THE DEPARTMENT OF MICROBIOLOGY, UNIVERSITY OF ILORIN, ILORIN, NIGERIA IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF BACHELOR OF SCIENCE (HONS) DEGREE IN MICROBIOLOGY.
1
October, 2014
OUTLINE TITLE PAGE
i
TABLE OF CONTENT
ii
INTRODUCTION
1
HISTORY/DEVELOPMENT
6
IMPORTANCE
7
PRODUCTION AND PURIFICATION
11
FORMS OF MONOCLONAL ANTIBODIES
17
APPLICATIONS
19
LIMITATIONS
25
CONCLUSION
26
REFERENCES
27
LIST OF FIGURES Antibody and antigens
1
Antigen processing by T-cell
2
Structure of an antibody
3
Antibody isotypes of mammals
5
Steps involved in production and purification of monoclonal antibodies
12
Monoclonal antibody production
13
Monoclonal antibodies’ purification processes
16
Recombinant monoclonal antibody
17
Chimeric and humanized monoclonal antibodies
19 2
Different forms of monoclonal antibodies
21
Monoclonal antibodies for cancer
23
LIST OF TABLES Antibody isotypes of mammals
4
Clinically important monoclonal antibodies
9
3
1.0
INTRODUCTION
1.1
Antibody
An antibody (Ab), also known as Immunoglobulin (Ig), is a large Y-shaped protein produced by plasma cells that is used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. The antibody recognizes a unique part of the foreign target, called an antigen (Charles, 2001). Each tip of the "Y" of an antibody contains an active binding site called paratope (a structure analogous to a lock) that is specific for one particular active binding site called epitope (similarly analogous to a key) on an antigen, allowing these two structures to bind together with precision. Using this binding mechanism, an antibody can tag a microbe or an infected cell for attack by other parts of the immune system, or can neutralize its target directly (for example, by blocking a part of a microbe that is essential for its invasion and survival). The production of antibodies is the main function of the humoral immune system (Pier et al., 2004).
Figure 1: Antibody and antigens (Leja, 2014).
Antibodies are secreted by a type of white blood cell called a plasma cell. Antibodies can occur in two physical forms: a soluble form that is secreted from the cell and a membranebound form that is attached to the surface of a B-cell and is referred to as the B-cell receptor (BCR). The BCR is found only on the surface of B-cells and facilitates the activation of these 4
cells (i.e. B-cells) and their subsequent differentiation into either antibody factories called plasma cells or memory B-cells that will survive in the body and remember that same antigen so the B-cells can respond faster upon future exposure (Borghesi and Milcarek, 2006). In most cases, interaction of the B-cell with a type of white blood cell lymphocyte called T helper cell is necessary to produce full activation of the B-cell and, therefore, antibody generation following antigen binding (Parker, 1993).
Figure 2: Antigen processing by T cell (Sullivan, 2014).
Antibodies are typically made of basic structural units. Each of these units has two large heavy chains and two small light chains. There are several different types of antibody heavy chains, and several different kinds of antibodies, which are grouped into different isotypes based on which heavy chain they possess.
5
Figure 3: Structure of an antibody showing its various basic structural units (Webster, 2006).
Five different antibody isotypes are known in mammals, which perform different roles, and helps direct the appropriate immune response for each different type of foreign object they encounter (Market and Papavasiliou, 2003). The five isotypes are each named with an "Ig" prefix that stands for immunoglobulin, another name for antibody viz.: IgA, IgD, IgE, IgG, and IgM, and differ in their biological properties, functional locations and ability to deal with different antigens. These differences are depicted in the Table below.
6
Table 1: Antibody isotypes of mammals Name Types IgA 2
i.
Description Found in mucosal areas, such as the gut,
i.
respiratory tract and urogenital tract, and
Reference(s) Underdown and Schiff, 1986.
prevents colonization by pathogens. IgD
1
i.
Functions mainly as an antigen receptor
i.
on B cells that have not been exposed to
ii.
Geisberger et al., 2006. Chen, 2009.
antigens. ii.
It has been shown to activate basophils and mast cells to produce antimicrobial factors.
IgE
1
i.
Binds to allergens and triggers histamine
i.
Pier et al., 2004.
In its four forms, provides the majority of
i.
antibody-based immunity against invading
ii.
Pier et al., 2004. Udenze, 2014.
release from mast cells and basophils, and is involved in allergy. Also protects against parasitic worms. IgG
4
i.
pathogens. ii.
The only antibody capable of crossing the placenta to give passive immunity to the foetus.
IgM
1
i.
Expressed on the surface of B cells (monomer) and in a secreted form (pentamer)
with
very
high
i.
Pier et al., 2004; Geisberger et al., 2006.
avidity.
Eliminates pathogens in the early stages of
7
B
cell-mediated
(humoral)
immunity
before there is sufficient IgG.
IgD, IgE, IgG exists as monomers while IgA exists as a dimer in secretions and IgM exists as a pentamer.
Figure 4: IgD, IgE, IgG (Monomers); IgA (Dimer); IgM (Pentamer) (Mirello, 2014).
1.2
Monoclonal Antibody
Monoclonal antibodies (mAb or moAb) are monospecific antibodies that are the same because they are made by identical immune cells that are all clones of a unique parent cell. Monoclonal antibodies have monovalent affinity, in that they bind to the same epitope. Given almost any substance, it is possible to produce monoclonal antibodies that specifically bind to that substance; they can then serve to detect or purify that substance.
Put most simply, monoclonal antibodies are antibodies that are identical, each derived from one type of immune cell and each a clone of a single parent cell (Jones, 2014).
8
They play a major role in treating a wide variety of diseases including cancer, infectious diseases, allergy, autoimmune disease and inflammation. Monoclonal antibodies now belong to a well-established drug class that has a high success rate from first in human studies to regulatory approval: Typically 25%, which compares favourably with the 11% success rate for small molecule drugs (Shire et al., 2010).
2.0
HISTORY/DISCOVERY OF MONOCLONAL ANTIBODIES
In the 1970s, the B-cell cancer multiple myeloma was known, and it was understood that these cancerous B-cells all produce a single type of antibody (a paraprotein). This was used to study the structure of antibodies, but it was not yet possible to produce identical antibodies specific to a given antigen (Shailendra, 2012).
Like much in medical progress, the development of monoclonal antibodies has not been a straight line to good results. The technique brought a great deal of excitement at the onset, followed by disappointment. In recent years, however, the medical world has looked on mAb with renewed enthusiasm as many of the problems encountered early on were resolved.
When Georges Kohler and Cesar Milstein invented monoclonal antibodies in 1975, they were unlocking a door science had been knocking on for the entire twentieth century. What the scientists (who received the Nobel Prize for their discovery) had done was to take a giant step toward creating a “magic bullet”. As early as 1900, Paul Ehrlich theorized that it should be possible to develop a compound that would target and kill specific disease cells (i.e. a magic bullet), while leaving a patient’s normal cells unharmed (Jones, 2014).
9
Production of monoclonal antibodies involving human–mouse hybrid cells was described by Jerrold Schwaber in 1973 (Schwaber and Cohen, 1973) and remains widely cited among those using human-derived hybridomas, but claims of priority have been controversial (Hinali et al., 2013). Schwaber's work was only used for fusing human and mouse cells with the goal of eliminating chromosomes to identify genetic markers and not to create monoclonal antibodies. Dr. Pieczenik suggested to Cotton what became the Cotton-Milstein experiment where the Kohler-Milstein experiment was the control. This is all documented in the MRC, Cambridge, Archives and with letters from witnesses who later won the Nobel Prize i.e. John Sedat and his wife Elizabeth Blackburn. The invention was reduced to practice by Cotton and Milstein, and then by Kohler and Milstein (Kohler and Milstein, 1975). The key idea was to use a line of myeloma (tumour of bone marrow) cells that had lost their ability to secrete antibodies, come up with a technique to fuse these cells with healthy antibody-producing Bcells (hybridoma technology), and be able to select for the successfully fused cells. This was put into practice by Milstein and Köhler in their search for a laboratory tool to investigate antibody diversity (Marks, 2014).
3.0
IMPORTANCE
Since the first publication by Kohler and Milstein on the production of mouse monoclonal antibodies (mAbs) by hybridoma technology, mAbs have had a profound impact on medicine by providing an almost limitless source of therapeutic and diagnostic reagents. Therapeutic use of mAbs has become a major part of treatments in various diseases including transplantation, oncology, autoimmune, cardiovascular, and infectious diseases.
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The limitation of murine monoclonal antibodies due to immunogenicity was overcome by replacement of the murine sequences with their human counterpart leading to the development of chimeric, humanized, and human therapeutic antibodies.
Remarkable progress has also been made following the development of the display technologies, enabling of engineering antibodies with modified properties such as molecular size, affinity, specificity, and valency. Moreover, antibody engineering technologies are constantly advancing to enable further tuning of the effector function and serum half life (Nissim and Chernajovsky, 2008).
Monoclonal antibodies offer what many medical authorities view as some of the most promising—if persistently elusive—pathways for the treatment of cancer and other deadly diseases.
The extraordinarily specific nature of antibodies becomes a tool with wide and potentially revolutionary applications. In essence, they can be deployed to find a single targeted substance, such as an antigen found only on a cancer cell, and make it possible to pinpoint the cell and destroy it. In addition to cancer therapies, mAb also is used in diagnostic tests for everything from pregnancy, to AIDS, to drug screening. Furthermore, the antibodies can be used to lessen the problem of organ rejection in transplant patients and to treat viral diseases that are traditionally considered “untreatable” (Jones, 2014).
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Table 2: Some Clinically Important Monoclonal Antibodies Main
Types
Application
Mechanism/Target
Form
Rheumatoid
Inhibits tumour
Chimeric
arthritis
necrosis factor-
Crohn's disease
alpha (TNF-α)
Ulcerative
category Infliximab
Colitis Adalimumab
Rheumatoid
Inhibits tumour
arthritis
necrosis factor-
Crohn’s disease
alpha (TNF-α)
Ulcerative
Antiinflammatory
Human
Colitis Basiliximab
Acute rejection of
Inhibits interleukin
kidney transplants
2 (IL-2) on
Chimeric
activated T cells
Daclizumab
Acute rejection of
Inhibits interleukin
kidney transplants
2 (IL-2) on
Humanized
activated T cells Omalizumab
Moderate-to-severe
Inhibits human
allergic asthma
immunoglobulin E
Humanized
(IgE) Anti-Cancer
Gemtuzumab
Relapsed acute
Targets myeloid cell Humanized
12
myeloid leukemia
surface antigen CD33 on leukemia cells
Alemtuzumab
B-cell leukaemia
Targets an antigen Humanized CD52 on T- and Blymphocytes
Rituximab
Non-Hodgkin's
Targets
lymphoma
phosphoprotein
Chimeric
CD20 on B lymphocytes Trastuzumab
Breast cancer with
Targets the
HER2/neu
HER2/neu (erbB2)
overexpression
receptor
Humanized
Immunotherapy, targets
Anti-cancer and anti-viral
Bavituximab
Cancer, viral infections
phosphatidylserine
Others
Palivizumab
RSV infections in
Inhibits an RSV
children
fusion (F) protein
Chimeric
Humanized
Source: (Rang, 2003).
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4.0
PRODUCTION
AND
PURIFICATION
OF
MONOCLONAL
ANTIBODIES There are a large number of steps involved in the production of monoclonal antibodies and each of these may be carried out in many different ways. The diversity of published approaches reflects both individual biological problems and previous experience. The methods also vary in convenience, speed, reliability, and expense. There is no one right approach and ultimately each investigator must choose and adapt the published strategies to individual needs (Goding, 1996). Monoclonal antibodies are typically made by fusing myeloma cells with the spleen cells from a mouse that has been immunized with the desired antigen. However, recent advances have allowed the use of rabbit B-cells to form a rabbit hybridoma. Polyethylene glycol is used to fuse adjacent plasma membranes of both the myeloma and spleen cells (Yang and Shen, 2006), but the success rate is low so a selective medium in which only fused cells can grow is used. This is possible because myeloma cells have lost the ability to synthesize hypoxanthineguanine-phosphoribosyl transferase (HGPRT), an enzyme necessary for the salvage synthesis of nucleic acids. The absence of HGPRT is not a problem for these cells unless the de novo purine synthesis pathway is also disrupted. By exposing cells to aminopterin (a folic acid analogue, which inhibits dihydrofolate reductase, DHFR), they are unable to use the de novo pathway and become fully auxotrophic for nucleic acids requiring supplementation to survive. The selective culture medium is called HAT medium because it contains hypoxanthine, aminopterin, and thymidine. This medium is selective for fused (hybridoma) cells. Unfused myeloma cells cannot grow because they lack HGPRT, and thus cannot replicate their DNA. Unfused spleen cells cannot grow indefinitely because of their limited life span. Only fused hybrid cells, referred to as hybridomas, are able to grow indefinitely in the media because the
14
spleen cell partner supplies HGPRT and the myeloma partner has traits that make it immortal (similar to a cancer cell).
This mixture of cells is then diluted and clones are grown from single parent cells on microtitre wells. The antibodies secreted by the different clones are then assayed for their ability to bind to the antigen [with a test such as Enzyme-Linked Immunosorbent Assay (ELISA)
or
Antigen
Microarray
Assay
or
Radioimmunoassay
(RIA)
or
immunofluorescence]. The most productive and stable clone is then selected for future use.
The hybridomas can be grown indefinitely in a suitable cell culture medium. They can also be injected into mice (in the peritoneal cavity, surrounding the gut). There, they produce tumours secreting an antibody-rich fluid called ascites fluid.
The medium must be enriched during in-vitro selection to further favour hybridoma growth. This can be achieved by the use of a layer of feeder fibrocyte cells or supplement medium such as briclone. Culture-medium conditioned by macrophages can also be used. Production in cell culture is usually preferred as the ascites technique is painful to the animal. Where alternate techniques exist, this method (ascites) is considered unethical (Debtanu et al., 2012).
Figure 5: Schematic illustration of steps involved in production and purification of monoclonal antibodies (Kyowa, 2014).
15
Figure 6: Monoclonal antibody production (Anonymous, 2013).
Production of monoclonal antibodies does not yield pure product. Hence, the need for purification of the product yielded arises. After obtaining either a media sample of cultured hybridomas or a sample of ascites fluid, the desired antibodies must be extracted. The contaminants in the cell culture sample would consist primarily of media components such as growth factors, hormones, and transferrins. In contrast, the in vivo sample is likely to have host antibodies, proteases, nucleases, nucleic
16
acids, and viruses. In both cases, other secretions by the hybridomas such as cytokines may be present. There may also be bacterial contamination and, as a result, endotoxins that are secreted by the bacteria. Depending on the complexity of the media required in cell culture, and thus the contaminants in question, one method (in-vivo or in-vitro) may be preferable to the other. The sample is first conditioned, or prepared for purification. Cells, cell debris, lipids, and clotted material are first removed, typically by centrifugation followed by filtration with a 0.45 µm filter. These large particles can cause a phenomenon called “membrane fouling” in later purification steps. In addition, the concentration of product in the sample may not be sufficient, especially in cases where the desired antibody is one produced by a low-secreting cell line. The sample is therefore condensed by ultra-filtration or dialysis.
Most of the charged impurities are usually anions such as nucleic acids and endotoxins. These are often separated by ion exchange chromatography. Either cation exchange chromatography is used at a low enough pH that the desired antibody binds to the column while anions flow through, or anion exchange chromatography is used at a high enough pH that the desired antibody flows through the column while anions bind to it. Various proteins can also be separated out along with the anions based on their isoelectric point (pI). For example, albumin has a pI of 4.8, which is significantly lower than that of most monoclonal antibodies, which have a pI of 6.1. In other words, at a given pH, the average charge of albumin molecules is likely to be more negative. Transferrin, on the other hand, has a pI of 5.9, so it cannot easily be separated out by this method. A difference in pI of at least 1 is necessary for a good separation.
Transferrin can instead be removed by size exclusion chromatography. The advantage of this purification method is that it is one of the more reliable chromatography techniques. Since we
17
are dealing with proteins, properties such as charge and affinity are not consistent and vary with pH as molecules are protonated and deprotonated, while size stays relatively constant. Nonetheless, it has drawbacks such as low resolution, low capacity and low elution times. A much quicker, single-step method of separation is Protein A/G affinity chromatography. The antibody selectively binds to Protein A/G, so a high level of purity (generally >80%) is obtained. However, this method may be problematic for antibodies that are easily damaged, as harsh conditions are generally used. A low pH can break the bonds to remove the antibody from the column. In addition to possibly affecting the product, low pH can cause Protein A/G itself to leak off the column and appear in the eluted sample. Gentle elution buffer systems that employ high salt concentrations are also available to avoid exposing sensitive antibodies to low pH. Cost is also an important consideration with this method because immobilized Protein A/G is a more expensive resin.
To achieve maximum purity in a single step, affinity purification can be performed, using the antigen to provide exquisite specificity for the antibody. In this method, the antigen used to generate the antibody is covalently attached to an agarose support. If the antigen is a peptide, it is commonly synthesized with a terminal cysteine, which allows selective attachment to a carrier protein, such as Keyhole limpet hemocyanin (KLH) during development and to the support for purification. The antibody-containing media is then incubated with the immobilized antigen, either in batch or as the antibody is passed through a column, where it selectively binds and can be retained while impurities are washed away. An elution with a low pH buffer or a more gentle, high salt elution buffer is then used to recover purified antibody from the support.
To further select for antibodies, the antibodies can be precipitated out using sodium sulfate or ammonium sulfate. Antibodies precipitate at low concentrations of the salt, while most other
18
proteins precipitate at higher concentrations. The appropriate level of salt is added in order to achieve the best separation. Excess salt must then be removed by a desalting method such as dialysis.
The final purity can be analyzed using a chromatogram. Any impurities will produce peaks, and the volume under the peak indicates the amount of the impurity. Alternatively, gel electrophoresis and capillary electrophoresis can be carried out. Impurities will produce bands of varying intensity, depending on how much of the impurity is present (Chandel and Harikumar, 2013).
Figure 7: Monoclonal antibody purification processes (Kyowa, 2014).
19
5.0
FORMS OF MONOCLONAL ANTIBODIES
5.1
Recombinant
The production of recombinant monoclonal antibodies involves technologies, referred to as “repertoire cloning” or “phage display/yeast display”. Recombinant antibody engineering involves the use of viruses or yeast to create antibodies, rather than mice. These techniques rely on rapid cloning of immunoglobulin gene segments to create libraries of antibodies with slightly different amino acid sequences from which antibodies with desired specificities can be selected (Siegel, 2002). These techniques can be used to enhance the specificity with which antibodies recognize antigens, their stability in various environmental conditions, their therapeutic efficacy, and their detectability in diagnostic applications (Schmitz et al., 2000).
Figure 8: Recombinant monoclonal antibody production (Nabel, 2004).
20
5.2
Chimeric Antibodies
Early on, a major problem for the therapeutic use of monoclonal antibodies in medicine was that initial methods used to produce them yielded mouse, not human antibodies. While structurally similar, differences between the two were sufficient to invoke an immune response when murine monoclonal antibodies were injected into humans, resulting in their rapid removal from the blood, as well as systemic inflammatory effects, and the production of human anti-mouse antibodies (HAMA) (Khan, 2014).
In an effort to overcome this obstacle, approaches using recombinant DNA have been explored since the late 1980s. In one approach, mouse DNA encoding the binding portion of a monoclonal antibody was merged with human antibody-producing DNA in living cells. The expression of this chimeric DNA through cell culture yielded partially mouse, partially human monoclonal antibodies. For this product, the descriptive terms "chimeric" and "humanised" monoclonal antibody have been used to reflect the combination of mouse and human DNA sources used in the recombinant process (Chadd and Chamow, 2001).
21
Figure 9: Chimeric and humanized monoclonal antibodies (Saldanha, 2000).
5.3
‘Fully’ Human Monoclonal Antibodies
Ever since the discovery that monoclonal antibodies could be generated, scientists have targeted the creation of 'fully' human antibodies to avoid some of the side effects of humanised or chimeric antibodies (Lonberg and Huszar, 1995).
22
Laboratory mice provide a ready source of diverse, high-affinity and high-specificity monoclonal antibodies. However, development of rodent antibodies as therapeutic agents has been impaired by the inherent immunogenicity of these molecules. One technology that has been explored to generate low immunogenicity monoclonal antibodies for in vivo therapy involves the use of transgenic mice expressing repertoires of human antibody gene sequences. This technology has now been exploited by over a dozen different pharmaceutical and biotechnology companies toward developing new therapeutic monoclonal antibodies and currently at least 33 different drugs in clinical testing – including several in pivotal trials – contain variable regions encoded by human sequences from transgenic mice. The emerging data from these trials provide an early glimpse of the safety and efficacy issues for these molecules (Lonberg, 2005). XenoMouse, another transgenic system, has succeeded in recapitulating the human antibody response in mice by introducing nearly the entire human immunoglobulin loci into the germ line of mice with inactivated mouse antibody machinery. XenoMouse strains have been used to generate numerous high-affinity, fully human antibodies to targets in multiple disease indications, many of which are processing in clinical development (Jakobovits et al., 2007).
Transgenic mice have been exploited by a number of commercial organisations:
Medarex — who marketed their UltiMab platform. Medarex were acquired in July 2009 by Bristol Myers Squibb (Bloomberg, 2009).
Abgenix — who marketed their Xenomouse technology. Abgenix were acquired in April 2006 by Amgen (Amgen, 2006).
23
Figure 10: Different forms of monoclonal antibodies (Kyowa, 2014).
6.0
APPLICATIONS
6.1
Diagnosis tests
Monoclonal antibodies are used in diagnostic tests for everything from pregnancy, to AIDS, to drug screening (Jones, 2014). Detection of mycoplasmal infection using monoclonal antibodies has been successful in a number of formats. Detection of specific antibodies in serum using monoclonal-mediated ELISA and metabolic inhibition can determine the specificity of responses with pinpoint (epitope) accuracy.
From 1985 on, the application of hybridoma technology to the production of Mycoplasmalike organisms (MLO)-specific monoclonal antibodies has greatly benefited both detection and characterization of this group of mollicutes by overcoming the problems encountered 24
with polyclonal antisera. Indeed, for the first time, highly specific reagents were available for MLOs. The first MAbs were produced against a New Jersey strain of aster yellow MLO, using partially purified salivary gland preparations from infected leafhopper vectors as the immunogen. Since then, MAbs against a large number of MLOs have been obtained and serological techniques similar to those described next have been applied to the diagnosis of MLO infections and to the identification of insect vectors (Chester et al., 1996).
6.2
Therapeutic Treatment
Therapeutic use of mAbs has become a major part of treatments in various diseases including transplantation, oncology, autoimmune, cardiovascular, and infectious diseases (Nissim and Chernajovsky, 2008).
6.2.1 Cancer Treatment There are a number of ways that mAbs can be used for therapy. For example: mAb therapy can be used to destroy malignant tumour cells and prevent tumour growth by blocking specific cell receptors. Variations also exist within this treatment, e.g. radioimmunotherapy, where a radioactive dose localizes on target cell line, delivering lethal chemical doses to the target (Waldmann, 2003). One possible treatment for cancer involves monoclonal antibodies that bind only to cancer cell-specific antigens and induce an immunological response against the target cancer cell. Such mAb could also be modified for delivery of a toxin, radioisotope, cytokine or other active conjugate; it is also possible to design bi-specific antibodies that can bind with their Fab regions both to target antigen and to a conjugate or effector cell. In fact, every intact antibody can bind to cell receptors or other proteins with its Fc region (Carter, 2001)
25
Figure 11: Monoclonal antibodies for cancer. ADEPT, antibody directed enzyme prodrug therapy; ADCC, antibody dependent cell-mediated cytotoxicity; CDC, complement dependent cytotoxicity; MAb, monoclonal antibody; scFv, single-chain Fv fragment (Carter, 2001).
6.2.2 Autoimmune Diseases Monoclonal antibodies used for autoimmune diseases include infliximab and adalimumab, which are effective in rheumatoid arthritis, Crohn's disease and ulcerative Colitis by their ability to bind to and inhibit tumour-necrosis factor alpha (TNF-α). Basiliximab and daclizumab inhibit interleukin 2 (IL-2) on activated T-cells and thereby help prevent acute rejection of kidney transplants (Rang, 2003). 6.2.3 Human immunodeficiency virus type 1 (HIV-1) Treatment In humanized mice, combinations of monoclonal antibodies have been shown to suppress viremia. Administration of a cocktail of HIV-1-specific monoclonal antibodies, as well as the single glycan-dependent monoclonal antibody PGT121, resulted in a rapid and precipitous decline of plasma viremia to undetectable levels in rhesus monkeys chronically infected with the pathogenic simian–human immunodeficiency virus SHIV-SF162P3. A single monoclonal antibody infusion afforded up to a 3.1 log decline of plasma viral RNA in 7 days and also
26
reduced proviral DNA in peripheral blood, gastrointestinal mucosa and lymph nodes without the development of viral resistance. Moreover, after monoclonal antibody administration, host Gag-specific T-lymphocyte responses showed improved functionality. Virus rebounded in most animals after a median of 56 days when serum monoclonal antibody titres had declined to undetectable levels, although, notably, a subset of animals maintained long-term virological control in the absence of further monoclonal antibody infusions. These data demonstrate a profound therapeutic effect of potent neutralizing HIV-1-specific monoclonal antibodies in SHIV-infected rhesus monkeys as well as an impact on host immune responses (Barouch et al., 2013).
6.2.4 Ebola Haemorrhagic Fever Treatment Three anti-Ebola virus mouse/human chimeric mAbs (c13C6, h-13F6, and c6D8) were produced in Chinese hamster ovary and in whole plant (Nicotiana benthamiana) cells. In pilot experiments testing a mixture of the three mAbs (MB-003), it was found that MB-003 produced in both manufacturing systems protected rhesus macaques from lethal challenge when administered 1 h post-infection. In a pivotal follow-up experiment, it was found that significant protection (P < 0.05) when MB-003 treatment began 24 or 48 h post-infection (four of six survived vs. zero of two controls). In all experiments, surviving animals that received MB-003 experienced little to no viremia and had few, if any, of the clinical symptoms observed in the controls. The results represent successful post-exposure in vivo efficacy by a mAb mixture and suggest that this immuno-protectant should be further pursued as a post-exposure and potential therapeutic for Ebola virus exposure (Olinger Jr. et al., 2012).
27
7. 0
LIMITATIONS
All is not clear sailing from now on for antibody therapy - a number of challenges remain. For example, it has become clear that individual monoclonal antibodies for the treatment of rheumatoid arthritis have different kinetics and side effects that may affect their efficacy in vivo. As with conventional drugs, side effects of monoclonal antibodies must be dealt with, although they are more likely to be related to the mechanism of action than to unknown side effects often seen with pharmacotherapy. Such complications have already been observed with anti-tumour necrosis factor (anti-TNF) therapy, in which a recognizable increase in the incidence of infectious complications, particularly reactivation of tuberculosis, has occurred in patients undergoing this treatment (Nabel, 2004). Economic considerations offer one of the biggest challenges to further development of the technology. Developing the plants and “bioreactors” that can produce mAbs is costly and requires huge start-up costs and lengthy timeframes for construction and regulatory approvals. An industry estimate several years ago suggested that the need by 2010 would be for 25 new plants worldwide (in addition to the ten then operating) at a cost of $5 billion, if as expected some 100 new mAbs are ready for market by then. That picture is further complicated by what biotech companies say is a reluctance on the part of lenders to fund such projects, because the first generation mAbs of the 1980s attracted large investment and then failed to deliver—and that has meant a lingering bad taste in the mouths of many bankers (Jones, 2014).
28
8.0
CONCLUSION
Monoclonal antibodies have progressed from science fiction to reality. Kohler and Milstein’s reduction of the principle of Paul Ehrlich’s proposal of a “magic gun” into monoclonal antibodies solved a lot of the challenges of medicine in the 21st century and opened a number of doors of advancement of healthcare. However, as with much of the progress experienced in science, this development was not a merry-go-round ride. But, the opportunities embedded therein proved to be of greater motivation; coupled with advancement in molecular biology, all paved the way for the actualization of the Monoclonal Dream which is the production of a complimentary antibody for any antigen/disease (perhaps with greater emphasis on viral diseases and tumours which are traditionally deemed as ‘incurable’) without endangering the patient’s immune system. The absolute dream is not a reality yet. But, the present attitude and further understanding gives every right to be optimistic that sooner rather than later, the dream in its totality would be actualized.
29
9.0
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