Monoclonal antibodies (mAb) are protein molecules that are

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Alexander J. Howard
Biol 509
Dr. Mourad
IPFW Biology
November 5, 2010
Monoclonal antibodies isolated without screening by analyzing the variable gene repertoire
of plasma cells.
Sai T. Reddy, Xin Ge, Aleksandr E. Miklos, Randall A. Hughes, Seung Hyun Kang , Kam Hon
Hoi, Constantine Chrysostomou, Scott P. Hunicke-Smith, Brent L. Iverson, Philip W. Tucker,
Andrew D. Ellington & George Georgiou. Nat. Biotechnol. 28:9, 965-969 (2010)
Introduction & Objective:
Monoclonal antibodies (mAb) are protein molecules that are microscopically identical with
respect to their polypeptide sequence and consequently their ability to bind with high affinity to a
specific antigen. Preparations of high purity mAb have proven invaluable in a diverse number of
applications, including medicine, biochemistry, chemical engineering, and as diagnostic reagents
in clinical & molecular biology research. Currently there are a plethora of mAb drugs available
for treatment of autoimmune disorders like Crohn’s disease & rheumatoid arthritis (infliximab,
adalimumab), acute organ rejection (basiliximab, daclizumab), cancers like B cell leukemia, nonHodgkin’s lymphoma & squamous cell carcinomas (alemtuzumab, rituximab, nimotuzumab &
cetuximab), and as anticoagulation and antiviral agents (abciximab, palivizumab). The use of
mAb as catalytic enzymes has numerous potential applications in both medicine (antibody
directed enzyme prodrug therapy) and materials technology. Lastly, the importance of mAbs in
general molecular biology research is overwhelming, as they allow for detection,
characterization, and quantification of specific substances of interest (ELISA, western
immunoblotting).
The objective of the current study was to demonstrate a novel method for mAb production
without the need for affinity screening at the antibody-antigen level. The authors eliminate the
physical screening process entirely by taking advantage of the highly efficacious B cell selection
processes of affinity maturation and somatic hypermutation that occur naturally in mammalian
immune responses.
Early approaches to antibody production involved the immunization of animals with antigen
followed by recovery and purification of immunoglobulins from the serum. With the advent of
hybridoma technology in 1975 by Köhler & Milstein it was possible to create immortalized B
cell lines for proliferation in vitro. Once high affinity Ab secreting hybridomas were identified
and selected, the subsequent clonal populations of cells derived from them all express the same
identical Ab. In this sense, the antibody produced is monoclonal because all cells are clones of
one original B cell hybridoma.
Isolation of high affinity antibody producing B cells is laborious because a large number of
potentially useful B cells must be isolated and individually exposed to antigen to determine
binding affinity. Although recombinant antibody discovery methods such as phage display
libraries solve the problem of immortalizing B cell clones in vitro, they still rely on direct
interactions between receptor molecules and antigen to isolate and enrich high affinity genetic
material. Techniques like these consume time, labor, and materials, mostly due to the large
number of variables involved in the screening process.
Experiments & Results:
The general experimental strategy is best summarized in six steps. In the first step, female
Balb/C mice are immunized with antigens (human complement serine protease, C1s; human Bcell regulator of IgH transcription, Bright; chicken egg ovalbumin, OVA), and after six days
plasma cells (CD45R- CD138+) are extracted and isolated. Second, mRNA is isolated and V
gene cDNA is synthesized from highly expressed transcripts using reverse transcription. In the
third step the VL and VH genes are amplified from the cDNA generated in step two. In the fourth
step, high throughput DNA sequencing is used to generate frequency distributions for the most
abundant complementarity determining region 3 (CDR3) sequences. This bioinformatic analysis
of previously obtained V gene sequence data is reveals the most common CDR3 sequences using
a simple frequency rank system (Supplementary Fig. 3). In the fifth step, the highest ranking
CDR3 sequences are used to construct synthetic mAb genes, which are then cloned into suitable
vectors for expression in mammalian and bacterial cells. Finally, in step six, bacterial and
mammalian cells are transformed with the recombinant mAb genes (using vectors generated in
the previous step) and expressed as either single-chain variable fragments (scFvs) in E. coli or
full size recombinant IgG in HEK293F mammalian cell lines. Finally, antigen binding assays are
performed to evaluate the binding affinity of recombinant mAbs.
The sequence data obtained from VL and VH cDNA library preparations relied on homologies to
conserved framework regions to identify the most highly represented CDR3s for each
immunized mouse BMPC repertoire. Heavy chain CDRs (CDRH3) were found to be more
diverse than light chain CDR3s (CDRL3), most likely as a result of an additional heavy chain
gene recombination event (V-D-J vs V-J). In all immunized mice, the top four most highly
represented CDRH3 sequences on average represented 10-20% of the total repertoire. Further, to
avoid the problem of heavy and light chain pairing, and the process of screening all together, the
authors hypothesized that VL and VH sequences from the same mouse that are represented at
similar frequencies arise from the same plasma cell. To test this hypothesis, VL and VH sequences
were paired in this manner and used as templates for automated gene synthesis. They were then
expressed as scFvs in E. coli, and the bacterial cell lysates were analyzed by ELISA. The results
indicated that ~78% of scFvs created in this manner were highly antigen specific (21 of 27
antibodies generated from the six immunized mice). High specificity binding was indicated in
samples demonstrating more than threefold higher ELISA signal on antigen coated wells
compared to wells coated with an unrelated protein antigen. Mouse C1s-2 showed the highest
serum antibody titers; antibodies from this animal were selected for biophysical characterization
by surface plasmon resonance. Certain pairings of VL and VH genes from this animal generated
an IgG (expressed in HEK293F cells) with subnanomolar binding affinities.
Conclusion:
This series of experiments presents a novel method for mAb production, with results attesting to
the full functionality of mAbs created in this manner. The most distinguishing feature of the
research strategy is best illustrated in the context of the central dogma of molecular biology. The
antibody coding genetic material undergoes several conversions; the most outstanding step in
this series is when the physically “real” cDNA is converted into pure information in the form of
sequence data. Using computers, this data is analyzed and manipulated with ease, and then used
as a schematic for the creation of new biological material in the form of synthetic genes. Put
simply, this method converts biological materials into intangible information, and then back
again to tangible biological materials.
There are several advantages to this approach for mAb production. Firstly, although
sophisticated automation, software, and robotics are use in several steps to facilitate sequencing
and gene reconstruction, the overall reagent and labor costs are small compared to other
methods. Thus, for a dedicated mAb producing facility, the improved efficiency will lower the
price of services while helping pay for start-up costs. Secondly, the method could potentially be
used in primates, including humans. This would allow for the production of fully humanized,
highly specific mAb instead of chimeric recombinant antibodies. Third, this technology may
prove valuable for antibody discovery using complex antigens like bacterial and viral pathogens.
Lastly, the potential future applications of this technology are virtually endless; the bioinformatic
analysis step can be expanded and modified to create custom engineered multivalent antibodies
that may be hybrid protein molecules with respect to their function. For example it may be
possible to create mAbs with multiple enzymatic properties, and the ability to act as allosteric or
direct activators and/or inhibitors of other proteins, DNA, and RNA.
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