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?

The V-regions of the Heavy (V_H) and Light (V_L) 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, V_H1-7), 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 (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.

BACKGROUND:

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