Deprecated: Hook custom_css_loaded is deprecated since version jetpack-13.5! Use WordPress Custom CSS instead. Jetpack no longer supports Custom CSS. Read the documentation to learn how to apply custom styles to your site: in /home/customer/www/ on line 6078
Antibody – In Scientio, Veritas

Tag: Antibody

MAb Therapeutics – New Technologies – a primer

As research into safer and more efficacious options for the therapeutic use of monoclonal antibodies (MAbs) goes on, interesting and innovative technologies have been discovered and refined by scientists, which represent the next level of accomplishment in the overall effort to develop “fully human” MAbs (which I have touched upon in the THIRD PART).

The Fully Human MAbs are generally made in one of two ways; either, naïve human B-cells are processed (via phage-display; gene-transfer to transgenic mice; or fusion of human B-cells) in order to, say, generate libraries of Immunoglobulin (Ig) V-regions. Screening techniques are utilized, at various levels thereafter, to select clones bearing desirable antibodies that are specific to the target antigen. In other words, the MAbs are made antigen-specific ex post facto.

Alternatively, memory B-cells are generated by training the immune system via immunization. For example, humanized transgenic mice are immunized with the target antigen, allowing them to generate specific, antigen-recognizing human antibodies. These B-cells are isolated and processed. However, in this process, there are several important caveats: (a) the antigen is still recognized in vivo in a murine context, (b) the human V-region incorporation into mouse genome may be restricted, and therefore, have limited diversity, and (c) there is the possibility that V(D)J rearrangements inside the murine B-cells may not be adequate. These difficulties – which may lead to the generation of low affinity, low frequency human antibodies, requiring extra steps of in vitro affinity maturation – are known issues in the development of human MAbs using transgenic mice.

To bypass this problem, a technology that incorporates immunological and genetic principles has been described [1] for rapid generation of MAbs specific to a vaccinating antigen, where immunized and/or infected-recovered donors – whose serum, therefore, contains the specific antibody – provide B-cells; these B-cells are sorted into separate enclosures, cultured and screened. From those B-cells that are secreting the desirable specific antibodies, the heavy and light chain genes are isolated and cloned into a human cell line, Human Embryonic Kidney (HEK)-293, which produces the human antibodies in culture. Using this technique, diagnostic and immunotherapeutic MAbs that can recognize and neutralize Anthrax have been generated [2].

Exton, PA-based company, Morphotek, using a similar technology [3], isolated B-cells from healthy individuals with serum antibodies to Staphylococcal entertoxin B (SEB), cultured them in vitro and fused with a mouse/human heteromyeloma cell line. The clones that produced the desired MAbs were identified, and their light and heavy Ig chain genes were sequenced and cloned into a mammalian cell line (Chinese Hamster Ovary, CHO, cells) for expression; CHO cells are known to be able [4] to produce the recombinant MAb protein molecule with the appropriate glycosylation (a process by which carbohydrate moieties are attached to the antibody molecule, which is crucial for the antibody function [5]). Using this technique, Morphotek has successfully generated human MAbs that are capable of neutralizing high doses of SEB both in vitro and in vivo.

However, some of the technologies used for the in vitro manipulation of the primary structure of the antibodies possibly introduces a degree of immunogenicity (as evidenced from human anti-human antibody or “HAHA” reactions), and may change the safety, tolerability, and in vivo performance characteristics of these therapeutic MAbs. For example, chimeric MAb Infliximab and transgenic-mouse-derived Fully Human MAb Adalimumab, both recognizing TNFα and used to treat Rheumatoid Arthritis, have been reported [6] to give rise to antibody response that neutralizes them and reduces their efficacy greatly. And this undesirable immunogenicity of the therapeutic MAbs themselves – according to a 2010 study [7] – resides, in part, in the CDR or V-region, the domain of the antibody molecule that is directly responsible for recognizing its cognate antigen! The good news is that, as the study contends, it is possible, via molecular genetics tools, to modify these CDRs such that immunogenicity of the MAb is reduced while retaining its biological activity. Neat, huh!

A combination of these approaches has been taken by an Austin, Texas-based biopharmaceutical company, XBiotech, which considers its human MAbs to be a generation ahead of the existing ‘Fully Human’ MAbs, and therefore, has designated its technology to be True Human™. But before I describe this technology, here is a little primer in Immunogenetics.

Remember the antibody structure I had talked about in the FIRST POST of this series, how heavy and light chains had antigen-recognizing Variable (V) regions and Constant (C) regions?

Immunoglobulin G

The V-regions of the Heavy (VH) and Light (VL) chains, that form the CDR or antigen-binding site, are produced in the B-cell by an interesting process, called VDJ recombination (a.k.a. somatic recombination). In the naïve B-cell, from the moment of its formation, there are 3 distinct type of gene segments (Variable, Diverse, and Joining segments) in the chromosome, present in what is known as a ‘germline’ configuration; for example, the area on chromosome 14, responsible for human Ig Heavy Chain (a.k.a. Heavy Chain locus), contains a total of 44 V-genes [8] (classifiable into 7 different families, VH1-7), 27 D-genes (of which 25 are involved in antibody generation), and 6 J-genes [9]. Prior to the generation of the antibody protein molecules, there occurs a VDJ recombination event, a nearly random rearrangement of the germline gene segments, eventually bringing together one of each, from which the final protein molecule is made. The Light chains (of which there are two types, λ and κ) similarly have V and J recombination (lacking the D segment). The randomness of the segment selection process is responsible for the generation of massive diversity in the antigen-recognition capability. To illustrate:

VDJ Recombination Schema

VDJ Recombination (Image courtesy: Wikimedia Commons)

In addition to this recombinatorial process that occurs in the germline gene, another process introduces further diversity to the antigen-recognition capabilities of the antibody, once the B-cell has encountered an antigen. This secondary process is known as Somatic Hypermutation; antigen-recognition by a naïve B-cell stimulates its proliferation, during which the chromosomal locus responsible for antibody generation mutates (mostly point mutations, in form of single base substitutions) extremely rapidly (100,000-1 million fold higher rate) to create a high degree of variation in primary structure. These mutations occur mostly at DNA “hotspots” known as hypervariable regions that correspond to CDRs, and result in enhanced affinity for the target, cognate antigen.

Somatic Hypermutation is followed by another process known as Clonal Selection; hypermutated B-cells are presented with the cognate antigens by a type of cells known as the follicular dendritic cells (FDCs), present in the germinal centers of lymphoid tissue. The B-cell clones/progeny with the highest affinities for the presented antigen are selected to survive, whereas other clones bind antigen with lower affinity are destroyed. The Clonal Selection process thus effectively increases the affinities of secreted antibodies for the target antigen. Taken together, Somatic Hypermutation and Clonal Selection are known as in vivo Affinity Maturation of the antibody.

The antigen that stimulates this process may be extrinsic, such as a vaccine antigen, or antigen from an infecting microbe, or intrinsic/autologous, such as a component of the host’s own body. While the former leads to a beneficial immune response, it is not hard to imagine that the latter may cause debilitating ‘autoimmune’ disorders if host’s own antibodies recognize and attack internal physiological components. The immune system has a checkpoint to prevent this, a process called Central Tolerance, that – for B-cells – occurs in the bone marrow and regulates autoreactive B-cells in several ways, including receptor editing (gene rearrangements to change reactive Ig molecules on the B-cell surface to non-reactive ones), clonal deletion (antigen-induced loss of cells from the B-cell repertoire via programmed cell death/apoptosis), and the induction of anergy (a.k.a. non-responsiveness). An additional checkpoint for those mature self-reacting B-cells that may survive this process and reach the peripheral lymphoid tissues is the requirement of T-lymphocyte signals. B-cells need co-stimulatory signals from T-cells in presence of the cognate antigen in order to proliferate and produce antibodies; therefore, mature B-cells recognizing self-antigens are denied T-cell help in the periphery and eliminated by apoptosis or anergy – the process being known as Peripheral Tolerance.

One important fact to remember is that the Central Tolerance requires B-cells to be exposed to self-antigens during maturation, i.e. antigens present in primary lymphoid organs. In the bone marrow, this exposure is naturally limited to bone-marrow specific antigens either present in the bone-marrow, or imported by circulation. Therefore, in a relatively healthy host, there may be a small proportion of mature B-cells that escaped these checkpoints and continue to secrete low-affinity antibodies to endogenous substances. For example, a 1996 paper [10] reported that naturally-occurring IgG and IgA autoantibodies specific for the pro-inflammatory cytokine Interleukin (IL)-1α occur in the sera of 5-25% healthy individuals. The other important concept to remember in the context of this discussion is that therapeutic MAbs produced in vitro do not undergo these in vivo checkpoints to eliminate self-reactive antibodies.

This also leads to the point where I found XBiotech’s True Human™ antibody technology and innovation to be interesting. Let me explain. As I have been informed, this company has access to a library of blood samples derived from normal individuals with functioning immune systems and no evidence of disease. They use a proprietary screening methodology to identify in those blood samples native human antibodies that recognize the target antigen they desire. Consider what that means; it means that the original B-cells that secreted these antibodies were already primed by antigen-exposure, and consequently, had already undergone somatic hypermutation of its genes, followed by clonal selection. Via XBiotech’s proprietary technology, they can thus find even low titer antibodies that recognize some endogenous substance as antigen with high affinity. They isolate the corresponding mature B-cells (with their full complement of ready-to-secrete antibodies) and use proprietary cloning strategies to clone the already-rearranged, ready-to-be-used Ig genes into CHO cells, and generate MAbs.

The beauty of this system? The in vivo B-cell selection and maturation process undergoes in the endogenous human microenvironments (germinal centers in bone marrow, lymph node or spleen); in addition, since the variable regions, including the CDRs (which are responsible for undesirable immunogenicity, as I mentioned above), are derived from native antibodies, True Human™ MAbs show no significant immunogenicity in the human host. Indeed, with their current True Human™ lead antibody, MAb-p1 (which recognizes human IL-1α), has been administered in Phase 1/2 studies to over 145 patients, in 457 doses, and the patient with the longest treatment has been over 785 days. They have observed no dose limiting toxicities and no evidence of any deleterious HAHA response (Data source: John Simard, Founder, President and CEO, XBiotech; see disclaimer below). It should be very interesting to follow the progress of the applications of this therapeutic technology.

This concludes the series on antibody therapeutics. Previous parts: FIRST POST, SECOND, and THIRD PART.

DISCLAIMER: This post describes proprietary technology in a purely informative manner, partly based on information provided by the inventor of this technology, a company called XBiotech. This does not constitute a comparative analysis of competing technologies, in whichever form they may exist, nor an endorsement of this technology I am describing. CONFLICT OF INTEREST: I declare that I have no financial interest whatsoever in writing about this technology. My interest in XBiotech and its technological achievements is purely academic. However, in the interest of transparency, I declare that this company, via its representative, made some of their data available to me at a time when it was embargoed.


  1. Smith et al. (2009) Rapid generation of fully human monoclonal antibodies specific to a vaccinating antigen. Nature Protocols, 4(3): 372–384; doi: 10.1038/nprot.2009.3; BACK TO TOP
  2. Smith et al. (2012) Human monoclonal antibodies generated following vaccination with AVA provide neutralization by blocking furin cleavage but not by preventing oligomerization. Vaccine, 30(28):4276-83. BACK TO TOP
  3. Drozdowski et al. (2010) Generation and characterization of high affinity human monoclonal antibodies that neutralize staphylococcal enterotoxin B. Journal of Immune Based Therapies and Vaccines, 8:9; doi: 10.1186/1476-8518-8-9 BACK TO TOP
  4. Sheeley et al. (1997) Characterization of monoclonal antibody glycosylation: comparison of expression systems and identification of terminal alpha-linked galactose. Analytical Biochemistry, 247(1):102-10. BACK TO TOP
  5. Zheng et al. (2011) The impact of glycosylation on monoclonal antibody conformation and stability. MAbs, 3(6):568-76; doi: 10.4161/mabs.3.6.17922. BACK TO TOP
  6. Radstake et al. (2009) Formation of antibodies against infliximab and adalimumab strongly correlates with functional drug levels and clinical responses in rheumatoid arthritis. Annals of Rheumatic Disease, 68(11):1739-45. BACK TO TOP
  7. Harding et al. (2010) The immunogenicity of humanized and fully human antibodies: residual immunogenicity resides in the CDR regions. MAbs, 2(3):256-65. BACK TO TOP
  8. Matsuda et al. (1998) The Complete Nucleotide Sequence of the Human Immunoglobulin Heavy Chain Variable Region Locus. Journal of Experimental Medicine, 188(11): 2151–2162. BACK TO TOP
  9. Li et al. (2004) Utilization of Ig heavy chain variable, diversity, and joining gene segments in children with B-lineage acute lymphoblastic leukemia: implications for the mechanisms of VDJ recombination and for pathogenesis. Blood, 103(12):4602-9. BACK TO TOP
  10. Garrone et al. (1996) Generation and characterization of a human monoclonal autoantibody that acts as a high affinity interleukin-1 alpha specific inhibitor. Molecular Immunology, 33(7-8):649-58. BACK TO TOP

Monoclonal Antibodies in Therapeutics – a primer

Let me start with a quick re-cap of how Monoclonal Antibodies (MAbs) are made in the laboratory.

Mouse MAb production

The journey of MAb therapeutics to its current state has been interesting, with several important iterations.

Murine: These were the first generation MAbs, in which immunized mouse splenocytes (spleen cells, including B-lymphocytes) were fused with a myeloma partner to form a hybridoma. The successful hybridomas would be screened for clones that would recognize the specific antigen. The resultant monoclonal antibody would have both heavy and light chains of mouse origin.

The first ever therapeutic MAb, described in 1986 and licensed by the US FDA in 1992, was a murine monoclonal against CD3 surface protein of T-lymphocytes; named ‘Muromonab-CD3’, this MAb of the class IgG2a was indicated for use to prevent heart, liver or kidney transplant rejection. In the World Health Organization’s International Nonproprietary Names (INN) system, a murine antibody is identified by the pre-stem -o- in its INN; muromonab, around since before INN existed, is the only exception. One example is edrecolomab, approved in Germany since 1995), a mouse IgG2a against a protein antigen called EpCAM (Epithelial Cell Adhesion Molecule), carried on the surface of many tumor cells.

Theoretically, a related species of MAbs may come from rats, one in which both chain types are of rat origin, and a rat antibody would be identified by the pre-stem -a- in its INN. However, currently there are no purely rat therapeutic MAbs in the market. There is a rat-mouse hybrid MAb, engineered with binding sites for two different antigens (class known as ‘trifunctional antibodies’) with the substem -axo- in its INN, called catumaxomab, but more about that later; by design, this MAb qualifies as the next-generation of therapeutic MAbs, the ‘Chimeric’ antibodies.

Murine MAbs, although in use, are considered to have limited therapeutic value. Recognized by the human body as foreign substances, the murine MAbs often elicit a human immune response (known as HAMA or “human anti-mouse antibody” response) which neutralizes the foreign antibodies, prevents their ability to engage immune cells as intended, and accelerates their removal. This, obviously, limits the effectiveness of murine MAbs, and may even result in deleterious side effects reminiscent of the ‘serum sickness‘ observed during the days of serum therapy, such as fever, chills, arthralgia and life-threatening anaphylaxis. The Chimeric MAbs came off the efforts to solve this problem.

Chimeric: These were the second-generation MAbs; a chimeric antibody is encoded by genes from more than one species, technically a fusion molecule, produced by genetic engineering methods. Remember the basic antibody structure from the FIRST PART of this series? For the heavy and the light chains, a chimeric chain contains a foreign Variable (VH and VL) domain (originating from one species other than human, or synthetic) which is connected to a Constant region (CH and CL) of human origin. The heavy chain constant region comprising the Fc portion is recognized by the immune effector cells for binding and bringing about many of the antibody’s cell-mediated effects, such as opsonophagocytosis, ADCC and so forth. Therefore, in a chimeric MAb, while the foreign variable regions confers epitope specificity for the intended therapeutic target, the human constant domain – introduced by genetic engineering to replace the analogous foreign constant domain – eliminates most of the potential immunogenicity of (i.e. the possibility of HAMA response against) the MAb without altering its specificity. Therapeutic chimeric antibodies are identified by the pre-stem -xi- in its INN; for example, abciximab, infliximab and the widely successful rituximab. There are other examples in the SECOND PART of this series.

The above mentioned Catumaxomab, which I considered ‘chimeric’, is a different type of hybrid; one half of the antibody (one heavy IgG2a chain and one kappa light chain) is of mouse origin, recognizing tumor-associated EpCAM, whereas the other half (one heavy IgG2b chain and one lambda light chain) is of rat origin, recognizing CD3 on the surface of T-lymphocytes. The hybrid Fc segment of this engineered antibody is capable of binding to immune effector cells like any antibody. Therefore, this MAb binds to a tumor cell via one arm, to a T lymphocyte via the other arm, and to an Fc-receptor bearing immune effector cells, such as macrophage, a natural killer cell or a dendritic cell, via its Fc portion, triggering an immunological reaction against the cancer cell. It has been approved for malignant ascites in the European Union, and currently undergoing phase II/III clinical trials in the US for gastic and ovarian cancers.

Some chimeric MAb may still have issues. For instance, cetuximab, approved by the US FDA for metastatic colorectal cancer, is known to give rise to HAMA IgEs.

Although beyond the scope of this description, here is a quick reference to other non-antibody chimeric proteins in therapeutic use. Etanercept, for example, is a TNFα blocker biologic drug, to make which the gene for human tumor necrosis factor receptor (TNFR) was recombined via genetic engineering with the gene for human IgG1 Fc segment. In the resultant chimeric protein, the TNFR part provides specificity for the target, and the antibody Fc segment ensures stability and localized drug delivery. Etanercept has US FDA approval to treat rheumatoid arthritis, psoriatic arthritis, plaque psoriasis and ankylosing spondylitis.

Humanized: These were the third generation therapeutic MAbs, designed to be an improvement upon Chimeric MAbs. In the “humanization” process, the antibody protein sequence is genetically modified to increase its similarity with human antibodies, by interspersing human sequences within the murine complementarity determining region (CDR) segments in the epitope-specific Variable domain; the goal is to further reduce the change of an unwanted HAMA response. A humanized therapeutic MAb is identified by the pre-stem -zu- in its INN; for example, trastuzumab and alemtuzumab. Other examples are in the SECOND PART. Nevertheless, induced antibody responses against humanized therapeutic mAbs (termed ‘HAHA’, human anti-human antibody response) have also been observed, giving rise to the concern that other factors, such as the nature of the antigen, the disease process treated and the schedule of administration, may contribute to the unwanted immunogenicity of the MAb.

Human or ‘Fully Human’: This represents the fourth generation of therapeutic MAbs. A human antibody is one of which both chain types, and the J chain in the case of polymeric antibodies, are of human origin. A human antibody is identified by the pre-stem -u- in its INN; for example, adalimumab and ustekinumab. Other examples are in the SECOND PART. Different technologies were devised and studied to aid in production of human MAbs.

  • Phage Display: ‘Phage’ or filamentous bacteriophage is a virus that can infect and replicate within bacteria. It offers an elegant and incredibly powerful technique to produce antibodies in vitro. In short, the genes for the Variable (V) regions of antibody heavy and light chains can be inserted separately into these phages, and they display the proteins on the surface. These genes can come from (a) naïve B-cells, or (b) immunized, antigen-specific memory B-cells. The proteins may be selected by screening against the antigen of choice, and the desirable proteins may be produced and secreted by bacteria infected by the specific phage. The secreted proteins can be reassembled in vitro to make functional antibodies. This technology is still developing and has great potential, but it is cumbersome, requiring multiple different steps. The antibodies made with this technology may also suffer from random heavy and light chain pairing, reducing the antibody efficacy. An example of fully human phage display derived therapeutic MAb is adalimumab.
  • Transgenic Mice: In early 1990s, transgenic mouse strains, in which mouse Ig genes were replaced by human Ig genes, were developed. This mouse strain contains human genes and produces human antibodies, and murine-murine hybridomas – which immortalize antigen-activated B-cells – can produce large quantities of antigen-specific human MAbs; examples are golimumab and ustekinumab. However, the antibody diversity (in epitope recognition) is created in a mouse strain environment, where the antibodies undergo affinity maturation, as opposed to a in human environment. Although this gave rise to the concern of limited germline repertoire engagement, now there are other mouse strains with larger human Ig transgenes to address that issue. Moreover, the results of clinical trials have now started to come in and should permit better analysis of the efficacy of these transgenic MAbs.
  • Primarily human system:This, of course, is the best-scenario for MAb therapeutics – the generation of human MAbs in a human system, thereby eliminating unwanted immunogenicity of the therapeutic agents. However, for human MAbs, the generation of human hybridoma cell lines of acceptable stability has posed a particularly galling challenge. For one, antigen-primed human B-lymphocytes needed for fusion are difficult to obtain on a routine basis; therefore, several alternative strategies have been devised for that purpose – which, nonetheless, has not been without problems.
    1. Mouse myeloma cell line transfected with human immunoglobulin genes – secrete low levels of human antibody.
    2. Fusion of human B cells with a murine myeloma partner – barring a few exceptions, mouse-human hybridomas cease human immunoglobulin production due to disturbances in gene expression; also, the peculiar phenomenon of selective loss of human chromosomes is well known; for some reason, mouse-human hybrids seem to preferentially retain human chromosomes 14 (H chain) and 22 (λ-L chain), and lose chromosome 2 (κ-L chain).
    3. Fusion of human B cells with a mouse-human hybrid ‘heteromyeloma’ – designed to solve the problem of chromosomal loss, but are unstable and secrete low levels of antibody.
    4. Transformation (and consequently, immortalization) of antigen-primed human B-cells with lymphotrophic (i.e. preferentially infecting lymphocytes) Epstein-Barr virus (EBV, a.k.a. human γ-herpesvirus 4) – moderately successful technique, but these transformed cells show unstable growth and secrete low levels of antibody, ceasing production altogether after a variable time period.
    5. Fusion of human B cells with a human lymphoblastoid cell line (LCL) – unfortunately, such hybridomas tend to secrete antibody molecules derived via permutation of the antibodies from both the fusion partners.
    6. Fusion of an EBV-transformed human B-cell line with a murine or human myeloma or a human LCL – secretes large amount of antibody. Because of the chromosomal loss in murine-human hybrids, human-human hybrids are considered more preferable. EBV transformation is beneficial because it causes polyclonal activation of B-cells, generating a greater V-region diversity, and makes the B-cell more competent for hybridization. These hybrids are stable and more efficient, and can be made to secrete specific antibodies.

In the next post, I shall focus on some very recent developments in antibody therapeutics and their applications. Stay tuned!

Sources for further reading

  1. KOZBOR, et al. (1982) Human hybridomas constructed with antigen-specific Epstein-Barr virus-transformed cell lines. Proc. Natl. Acad. Sci. USA, 79: 6651-5.
  2. TENG, et al. (1983) Construction and testing of-mouse-human heteromyelomas for human monoclonal antibody production. Proc. Natl. Acad. Sci. USA, 80: 7308-7312.
  3. WINTER, et al. (1994) Making antibodies by Phage Display technology. Ann. Rev. Immunol., 12:433-455.
  4. LONBERG et al. (1994) Antigen-specific human antibodies from mice comprising four distinct genetic modifications. Nature, 368: 856-859 (human antibody technologies from transgenic mice, as described by Medarex, Inc.).
  5. GREEN et al. (1994) Antigen-specific human monoclonal antibodies from mice engineered with human Ig heavy and light chain YACs. Nat Genet, 7: 13-21 (human antibody technologies from transgenic mice, as described by Abgenix, Inc.).
  6. KARPAS, et al. (2001) A human myeloma cell line suitable for the generation of human monoclonal antibodies. Proc. Natl. Acad. Sci. USA, 98: 1799-1804.
  7. LONBERG (2005) Human antibodies from transgenic animals. Nature Biotech., 23: 1117-25.
  8. BERNETT, et al. (2010) Engineering Fully Human Monoclonal Antibodies from Murine Variable Regions. J. Mol. Biol., 396: 1474–1490 (human antibody technologies from transgenic mice, as described by Xencor, Inc.).
  9. NECHANSKY (2010) HAHA – nothing to laugh about. Measuring the immunogenicity (human anti-human antibody response) induced by humanized monoclonal antibodies applying ELISA and SPR technology. J. Pharm. Biomed. Anal., 51: 252–254.

This is the THIRD PART of a multipart series on exciting new developments in the world of antibodies.

ACKNOWLEDGEMENTS with gratitude: The images of the mouse and rabbit have been modified from the product listing page of Charles River laboratories; the images of the myeloma cell and hybridoma cell have been modified and adapted from Figure 5 of Karpas et al. (PNAS USA 2001, 98:1799).

Antibody Therapeutics – a primer

Consideration of antibodies as a therapeutic modality isn’t new. Ever since the discovery of anti-toxin antibodies and the observation of their efficacy against certain bacterial toxins (e.g. tetanus toxin) in the early 1890s, the subsequent 40-odd years saw a rise in the therapeutic usage of antibody preparations (under the umbrella term ‘serum therapy’) for various infectious diseases (N.B. informative reviews at References 1, 2 and 3) – the name indicating the origin of these preparations: polyclonal serum collected from immunized animals (such as sheep) or pre-exposed immune human donors. Serum therapy worked, no doubt, but also gave rise to various side effects related to the presence of a complex foreign protein substance (e.g. animal serum in a human body), a condition known as ‘serum sickness’, accompanied by fever, chills, joint pain, allergy, and in extreme cases, anaphylaxis. Although this challenge was somewhat resolved by the 1930s through better-purified antibodies, the introduction of vastly superior, potent, and better tolerated antimicrobial chemotherapy (‘antibiotics’, starting with sulphonamides and then the β-lactam, penicillin) around 1935 sounded the death-knell for serum therapy. Within 5 years, antibacterial serum therapy fell into disuse, leaving only a niche area for those conditions that were not amenable to antibiotic treatment, namely, venoms, toxins (such as diphtheria and tetanus), and viral infections (such as, hepatitis A and polio) (1).

Continue reading

Antibodies – a primer

Antibodies (singular: ‘antibody’; officially known as ‘immunoglobulin’ or ‘Ig’) are glycoprotein (i.e. proteins containing carbohydrate moieties on them) molecules present in the serum (the non-cellular part of blood); these molecules, with diverse functions, play a very important role in defending the body against a variety of microbial pathogens, their deleterious by-products, or other foreign substances. These foreign substances, a part or the whole of which is capable of eliciting an immune response from the body, are referred to as ‘Antigens‘.

Immunoglobulin G

Each basic antibody molecule is composed of two larger proteins (called ‘Heavy chains’) and two smaller proteins (called ‘Light chains’), which are connected to each other via a chemical structure containing two sulfur atoms (a.k.a. disulfide linkages). The portions of the heavy and light chains involved in binding to the antigen are called Variable (V) regions, VH and VL respectively. The remaining portions of the protein molecules are called Constant (C) regions of each chain. The heavy chain constant regions (CH) constitute the Fc fragment of the antibody molecule, which is responsible to binding to other immune cells; the upper parts of the antibody molecule, involved in antigen recognition, constitute the Fab fragment. Based on the structure of the CH region, mammalian antibodies are grouped into five classes: IgM, IgD, IgG, IgE and IgA, each with closely related but different functions.

Now, antibodies are produced in the body by a special type of white blood cells (‘leukocytes’) called B-lymphocytes, or simply B-cells. When an antigen enters the body, B-cells will get to see it either alone, or in conjunction with other types of immune cells. In this event, B-cells undergo a rearrangement in their genes to produce antibody molecules which are capable of interacting with the antigen in a highly specific manner. Through such interactions, antibodies are able to:

  1. neutralize toxic substances (‘toxins’) of microbial origin;
  2. agglutinate (i.e. cause to clump) bacteria and other microbes;
  3. coat the microbes (a process called ‘opsonization’) such that certain immune effector cells (such as macrophages, dendritic cells and neutrophils) can recognize and destroy the microbes by internalizing (via a process called ‘phagocytosis’) and breaking them down;
  4. induce certain immune effector cells (such as neutrophils, basophils, eosinophils, mast cells and cytotoxic T and Natural Killer lymphocytes) to release potent antimicrobial molecules (via a process called ‘degranulation’) that can destroy invading microbes;
  5. directly kill certain microbes by interfering with their uptake of essential substances, or damaging their outer membrane (a few examples here); and
  6. activate certain serum proteins (called ‘complements’) to form a complex that can poke holes into the membrane of certain bacteria, causing them to lyse (i.e. be destroyed by dissolution).

Certain beneficial antibodies, in addition, are known to influence the overall host immune response (a process called ‘immunomodulation’) to benefit the host by restricting the collateral damage of host tissue and cells caused during the fight between host immune cells and the pathogen (an example here). All in all, pretty useful things to have around, right?

The different biological effects of antibodies

The different biological effects of antibodies.
Image ©Nature; Used with permission. Source: Casadevall, et al. (2004) Nature Reviews Microbiology 2: 695-703; doi:10.1038/nrmicro974

In fact, this unique feature of the physiological defence system is responsible for the benefits of the prophylactic (i.e. ‘preventive’) treatment known as immunization or vaccination, first tested in the eighteenth century by Edward Jenner, and elucidated by experimental work of Louis Pasteur. This treatment introduces a relatively harmless, attenuated or inactivated substance, a microbe or its product, into the body, and the immune system generates a relatively mild response. However, B- and T-lymphocytes retain the information about the antigen in their genetic codes (a phenomenon referred to as ‘immunologic memory’); the next time the body encounters the actual microbe or microbial product (as during an infection), the B-cells are able to quickly produce a rapid, strong and prolonged antibody response that adequately defends the body.

There is another interesting aspect of the antibody response. When the body encounters an antigen via inoculation/immunization or disease, various different populations of B-cells come across the same antigen, and produce antibodies that specifically recognize different parts of the antigen structure. The specific chemical structure  – the bare molecular structure (‘primary’) as defined by arrangement of atoms, or more complex secondary, tertiary or quaternary structures in three dimensions – of a part of the antigen, that is recognized by an antibody, is referred to as an ‘epitope’ of that antibody. Therefore, a normal B-cell response may produce antibodies against various epitopes of the same antigen; this is known as a polyclonal response, where each epitope-specific antibody comes from a genetically identical (i.e. ‘clonal’) subset of the total B-cell pool. This works out well, because this way the body manages to produce high titer (a measure of concentration) antibodies with high affinity for the target antigen. Such antibodies to specific antigens, produced via immunization in various laboratory animals (such as mice, rats, guinea pigs, rabbits, pigs, goats, sheep and horses), are used extensively for biomedical research as well as disease diagnostics. The presence of multiple epitopes offers more robust detection, as well as utility when the exact nature of the antigen is unknown.

However, in another application of the same principle, it is possible to generate in vitro Monoclonal antibodies, antibodies from identical B-cells which are clones of a unique parent B-cell. Naturally, all such antibodies recognize the exact same epitope. This technique, which has been around since early 1970s (and led to the 1984 Nobel Prize in Physiology/Medicine to Georges Köhler, César Milstein, and Niels Kaj Jerne), involves selection and isolation of a single clone of B-cells from a polyclonal pool, and fusing cells of that clone to a myeloma partner (a cancerous B-cell that has lost its ability to secrete antibodies). This makes the hybrid B-cell clone (now called a ‘hybridoma’) immortal – just like its cancer cell partner – and it can be grown indefinitely in artificial medium in which the unique and homogeneous monoclonal antibodies would be secreted. Purified monoclonal antibodies are immensely important as laboratory reagents because their high antigen-specificity helps reduce background noise and cross-reactivity in immuno-assays.

Given these interesting and useful properties of antibodies, it is not a surprise that scientists would consider their usage in a therapeutic manner, too. Indeed, Antibody Therapeutics is a fascinating field of study, which I shall discuss in the next post.

This is the FIRST PART of a multi-part series on exciting new developments in the world of antibodies.