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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5366_Библиотеки_им_академика_М_И_Перельмана.pdf
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these commercial microarray-based platforms vary widely, lengths of up to 350nt
have been attained, more than doubling the ~150nt achieved a decade prior [22].
Error rates as low as 1in 2000nt are also possible with today’s synthesizers. The
oligo lengths achieved by modern oligo synthesis platforms, however, will inevitably plateau as phosphoramidite reaction efciencies approach 100%. Nevertheless,
synthesis density—how many oligos can be synthesized per chip—is expected to
grow with the continued miniaturization of synthesis platforms.
R. L. Nugent and A. K. Sato
3.3 Gene Synthesis
As discussed above, oligo synthesis methods, although substantially improved over
the years, still struggle to synthesize oligos longer than a few hundred nucleotides.
As a result, methods were developed to stitch short, chemically synthesized oligos
into dsDNA gene fragments, called gene synthesis. Although the rst successful
attempts at gene synthesis utilized T4 DNA ligase to stitch oligos together sequentially, the discovery of new enzymes (thermostable ligases) and technologies (PCR)
paved the way for one-pot gene syntheses in the subsequent decades. These strategies, subject to continued innovation within the context of array-based oligo synthesis, form the basis of modern commercial gene synthesis platforms.
3.3.1 Early DNA Assembly Methods
The rst synthetic gene took more than 5 years to synthesize. Working with limiting
synthetic chemistries, Khorana and colleagues generated the rst synthetic genes by
chemically synthesizing, annealing, phosphorylating, and ultimately ligating a
series of short (8–12nt) oligos [27, 28]. Without thermostable ligases, Khorana’s
group resorted to sequential ligation reactions to tack on each chemically synthesized oligo. One-pot, ligation-dependent gene synthesis was not demonstrated until
1998 [29]. Ligation-based DNA assembly methods have inherently low error rates,
unlike the PCR-based methods that have largely, but not completely, superseded
them. At least two commercial options for ligation-based gene synthesis exist today:
Blue Heron Biotechnology (acquired by Eurons Genomics in 2019) and Sloning
BioTechnology GmbH (acquired by MorphoSys AG in 2010).
The invention of PCR in the 1980s spawned multiple PCR-based methods for
DNA assembly. Yet, to this day, Willem Stemmer’s polymerase chain assembly
(PCA) method [30] remains the most common among them (Fig.3.2). PCA, also
called oligo shufing, harnesses the ability of DNA polymerases to ll in gaps in
complementary single-stranded DNA by PCR extension. DNA polymerases randomly form larger and larger assemblies during thermocycling, and the nal assembly, once formed, is ultimately amplied by PCR using primers unique to each end
of the fully assembled DNA fragment. Stemmer and colleagues leveraged this
3 Increasing the Scalability of DNA Synthesis and Its Key Role in Expanding…
61
Fig. 3.2 Classic gene synthesis. (Adapted from Stemmer etal. [30])
method to assemble as many as 134 chemically synthesized oligos into DNA fragments up to 2.7kb in length [30]. The numerous other PCR-based assembly methods have been reviewed in detail elsewhere [31] and will not be discussed here,
except to note that they largely involve a subassembly approach whereby smaller
fragments are joined rst into subassemblies, which are subsequently stitched
together into full-length gene fragments.
3.3.2 Array-Based Gene Synthesis
By necessity, early gene synthesis attempts used column-synthesized oligos as
building blocks. The advent of microarray-based oligo synthesizers promised
increased throughput while posing new challenges for gene synthesis. Compared to
column-synthesized oligos, array-synthesized oligos are produced as a complex
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R. L. Nugent and A. K. Sato
mixture of low-quantity (femtomolar) and relatively low-quality oligos. These features—complexity, quantity, and quality—have complicated gene synthesis using
array-synthesized oligos.
Current microarray oligo synthesizers can generate one million oligos on a single chip, with each oligo synthesized at the atto- or femtomolar scale. Although
these minute quantities reduce the overall cost of oligo synthesis by minimizing
reagent use, they are insufcient for most DNA assembly methods. Moreover, the
complexity of the synthesized oligo pools can lead to unintended hybridization of
oligos during assembly. Two general strategies have been deployed to solve these
concentration and complexity problems: amplifying oligos by PCR and miniaturizing the volume of the assembly reaction.
Amplifying oligos by PCR is the more common method for increasing the concentration of array-synthesized oligos for gene synthesis. Tian etal. [32] solved this
problem by attaching a common primer sequence and a nicking endonuclease recognition site to the 3′-end of synthesized oligos. Once synthesized, oligos could be
amplied from the 3′-end by primer extension and the resulting complementary
strands cleaved by a nicking endonuclease, effectively amplifying the arraysynthesized pool. The strategy, however, fails when applied to more complex (>1000
oligo) pools [33]. Kosuri etal. [34] conceived a barcoding strategy whereby primer
sites were appended to each end of a subset of oligos to enable their selective amplication. This strategy enabled multiple subpools to be amplied from the master
oligo pool. Although these PCR-based methods are straightforward, they are also
error-prone. Not only is PCR inherently error-prone, but extending the length of
oligo synthesis to incorporate primer sites can increase the error rate of the synthesis
process, too.
Miniaturizing the assembly reaction is a less common, but nonetheless effective,
solution to the concentration problem. It also addresses the complexity problem.
Kong etal. [35] demonstrated the feasibility of this approach by partitioning oligo
synthesis and assembly into 500nL microuidic reactors. Quan etal. [26] utilized
an inkjet printing approach to spatially control the synthesis of oligos into physically separated microwells for subsequent gene synthesis.
Overall, the success of these strategies has led to their commercialization, in
principle, by Gen9 (now Ginkgo Bioworks) and Twist Bioscience.
3.3.3 Error Correction andSequence Validation
If oligo synthesis were a perfect process, gene synthesis would be straightforward
and relatively quick. Unfortunately, it is not. Errors in oligo synthesis lead to errors
in gene synthesis, as error-containing oligos are assembled along with perfect
(error-free) ones. Traditionally, error-free gene fragments were identied by cloning
and sequencing individual clones. This laborious and expensive process serves as a
bottleneck in the discovery workow. Because biological features are encoded by
DNA, even minor errors can have an outsized impact on the functionality of the nal
3 Increasing the Scalability of DNA Synthesis and Its Key Role in Expanding…
63
product. The quality of array-synthesized oligos is generally lower than that of their
column-synthesized counterparts, necessitating methods for error ltration and
correction.
The process of array-based gene synthesis spans multiple error-prone steps.
Array-based oligo synthesizers can generate errors as a result of inefciencies in the
synthesis cycle, side reactions (i.e., depurination), and deviations in spatially controlled steps (as discussed in Sect. 3.2.3 Modern Oligo Synthesis Platforms). DNA
assembly methods, particularly the PCR-based ones in common use, are as errorprone as the DNA polymerases they use. To mitigate the impact of these error
sources on gene synthesis, two general strategies have been developed to lter errorfree oligos from those containing errors: those that use mismatch-binding/cleaving
proteins to remove errors and those that enrich for error-free DNA.These strategies
are discussed briey below; see Ma etal. [23] for an in-depth review on the topic.
Mismatch detection is commonly used to lter errors in synthetic genes (i.e.,
duplexes). This strategy involves using a mismatch-binding/cleaving protein to
either lter out or degrade, respectively, DNA assemblies containing errors.
Mismatches are generated by denaturing the DNA duplexes and reannealing them;
this process causes error-containing strands to randomly pair with error-free strands.
Once this is done, mismatch-binding proteins (e.g., MutS) [36] or mismatchcleaving enzymes (e.g., T7 endonuclease 1) [37, 38] can be used to selectively isolate or degrade error-containing duplexes, respectively. The choice of strategy
depends on the length of the gene fragments and how many errors they contain;
mismatch cleavage is generally preferred for both long and error-rich fragments [39].
Several groups have used next-generation sequencing (NGS) to lter errorcontaining gene fragments. Among them, the most accessible is dial-out PCR [40,
41], which involves appending primer sites to the ends of each gene fragment, iden-
tifying error-free fragments by sequencing, and selectively amplifying them by
PCR.This approach is highly cost-efcient but time-consuming. Other sequencingbased methods have been proposed, but they are either low-throughput, incompatible with some NGS platforms, or require specialized equipment [15].
3.4 New Discovery Bottleneck
Modern DNA reading (sequencing) and writing (synthesis) technologies allow bioengineering to be approached more systematically. This systematic approach,
referred to as the design-build-test-learn (DBTL) cycle, starts by modeling a bioprocess, including all the parts (e.g., oligos and gene fragments) and processes that will
be used to produce and evaluate a bioengineered product; proceeds through the
building and testing of the bioengineered product; and ends with a learning phase,
where insights gained are used to inform the next iteration of the cycle. Traditionally,
low-throughput oligo synthesis has bottlenecked the cycle at the build phase. Now,
with modern gene synthesis platforms like Twist Bioscience’s, which can synthesize nearly 10,000 gene fragments (or >one million oligos) on a silicon chip the size
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R. L. Nugent and A. K. Sato
of a 96-well plate, the bottleneck has been shifted to the testing and learning phases
of the cycle.
In the next section, we discuss how modern gene synthesis technologies have
transformed the antibody discovery process by enabling the fabrication of massive
synthetic antibody libraries.
3.4.1 Evolution ofAntibody Discovery
Antibodies represent the largest class of biopharmaceuticals on the market. The rst
therapeutic monoclonal antibody (mAb), muromonab-CD3 (Orthoclone OKT3;
Janssen-Cilag), entered the market in 1986 and was developed using the traditional
hybridoma method [42]. The rst therapeutic human mAb came a decade and a half
later in 2002 with the approval of adalimumab (Humira; Abbott), a feat that was
made possible by the invention of phage display technology [43]. Spurred by the
concurrent development of molecular biology methods (e.g., molecular cloning,
oligo synthesis, and gene synthesis), mAb-generating technologies have matured
over the past half century. In this section, we discuss how modern gene synthesis
platforms have expanded and hastened the therapeutic antibody discovery process.
3.4.1.1 Hybridoma Technology
The year 1975 saw the emergence of a breakthrough technology for the facile production of mAbs: hybridomas [44]. George Köhler invented the technology in César
Milstein’s lab by fusing mortal B-cells with transformed ones (i.e., myelomas) [45].
Unlike the myeloma–myeloma fusions attempted by Milstein previously, Köhler’s
hybridomas produced highly specic antibodies of a single specicity. Hybridomas
unleashed mAbs onto the world, contributing to their pervasiveness in the laboratory and clinical settings.
Hybridomas continue to be used to this day. As of 2020, greater than 90% of
therapeutic mAbs that have received regulatory approval were generated using
hybridoma technology. Despite this clear success, using hybridomas to produce
therapeutic mAbs can be time-consuming and costly. Also, the technology cannot
be used to generate mAbs against toxic or nonimmunogenic antigens. Cellular stability and lack of direct access to antibody genes are additional challenges faced by
hybridoma technology [46].
3.4.1.2 Phage Display Technology
The invention of molecular technologies such as PCR and recombinant DNA gave
rise to the eld of antibody engineering in the late 1980s. Skerra and Pluckthun [47]
were the rst to express recombinant antibody fragments in Escherichia coli.
3 Increasing the Scalability of DNA Synthesis and Its Key Role in Expanding…
65
Shortly after, Orlandi etal. [48] demonstrated that antibody domains could be isolated using PCR without a priori knowledge of their coding sequences. The rst
antibody libraries followed, generated via PCR by Ward etal. [49] and then by Huse
etal. [50]. These studies demonstrated that antibody genes could be isolated and
screened for antigen binding in a library format, laying the foundation for the invention of phage display technology by McCafferty etal. [51] a year later. The invention spurred the development of hundreds of antibody libraries over the next decade
and a half. By 2006, over 30 companies were using phage display in their discovery
platforms [52].
The invention of phage displays substantially increased the throughput of antibody discovery. Unlike the laborious process of screening individual hybridoma
clones, phage display enables the screening of naïve, immunized, or synthetic repertoires en masse. Phage display involves fusing the genes encoding antibody fragments and phage coat proteins together. This process results in the display of
antibody fragments on the coat protein surface of phages. The resulting phages can
be subsequently screened for antigen binding by iteratively enriching them on
antigen- immobilized plates. The process is highly amenable to automation, much
more so than hybridoma technology [53]. It is also easily interfaced with highthroughput gene synthesis technologies.
3.4.1.3 Synthetic Antibody Library Construction
The diversication processes used to generate diverse antibody libraries mimic the
natural processes that occur in situ [54]. The natural process starts with the combinatorial recombination of V, D, and J gene segments in B-lymphocytes and ends
with afnity maturation via somatic hypermutation in response to antigenic stimulation. Similarly, the recombinant process involves combinatorial shufing of cloned,
semi-synthetic, or synthetic gene fragments within established antibody frameworks to obtain an antibody library. Subsequent afnity maturation occurs during
phage display. The afnity of antibody candidates can be matured further by generating a secondary library based on candidates identied from an initial library,
although this is not necessary. Indeed, antibody fragments with picomolar afnities
are routinely isolated from antibody libraries by phage display without the need for
additional afnity maturation [53].
Unlike natural and immunized antibody libraries, synthetic antibody libraries
incorporate synthetic sources of diversity in their complementarity-determining
regions (CDRs). Synthetic diversity can be sourced from degenerate primers, synthetic trinucleotides, and oligo pools of discrete sequences. The application of these
methods to the construction of semi- and fully synthetic antibody libraries is outlined below.
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R. L. Nugent and A. K. Sato
Semi-Synthetic Libraries
The rst semi-synthetic antibody libraries obtained their CDR diversity from degenerate primers [55, 56]. These libraries were constructed by shufing a bank of germline heavy chain variable regions (VH), which spanned CDRs 1 and 2; adding a third,
diversied CDR (CDR3) by PCR amplication using NNK/NNS degenerate primers (N=any nucleotide; K=G/T; S= G/C); and cloning the resulting VH and a
human light chain (VL) as scFv fragments (Fig. 3.3). Later degenerate antibody
libraries improved upon these initial libraries by minimizing the diversity of certain
residues to that found in natural antibodies [57] or by leveraging favorable antibody
frameworks [58]. These early libraries were relatively modest in size, at least by
present-day standards, at ~108 clones each. Although degenerate primers provide a
straightforward and cost-effective method for diversifying CDRs, the use of degenerate codons limited the diversity that could be obtained at each position within the
CDR and this diversity often did not exactly match the diversity seen in natural
human sequences [46].
Fully Synthetic Libraries
The rst fully synthetic antibody (and fully human) library was actually a set of
libraries called the Human Combinatorial Antibody Libraries (HuCAL), constructed
by Knappik etal. [59] using the trinucleotide phosphoramidite method (also known
Fig. 3.3 Semi-synthetic libraries created by NNK
3 Increasing the Scalability of DNA Synthesis and Its Key Role in Expanding…
Fig. 3.4 High-diversity antibody libraries created by overlap PCR
67
as trinucleotide-directed mutagenesis [TRIM]). Instead of using PCR to diversify
CDR3, Knappik et al. [59] sourced diversity from oligos synthesized from trinucleotide phosphoramidites. The nal libraries were generated by stitching
together synthetic oligos by overlap-extension PCR (Fig.3.4). Although the original
HuCAL design diversied only HCDR3 and LCDR3, a later iteration (HuCAL
GOLD) synthetically diversied all six CDRs [60]. HuCAL and HuCAL GOLD
comprised 2×109 and 3.6×1010 clones, respectively. HuCAL and HuCAL GOLD
are commercially available for antibody discovery via MorphoSys AG, who developed them.
Sloning BioTechnology GmbH (since acquired by MorphoSys AG) debuted a
new gene synthesis strategy—a ligation-based technology called Slonomics—in
2008 [61], providing a new method for antibody library construction. The method
uses a set of common building blocks to synthesize DNA sequences three nucleotides at a time by iterating a series of ligation, immobilization, and cleavage steps.
The rst library generated by this approach, a Fab library, was as massive as HuCAL
GOLD (3.6×1010) and exhibited more favorable expression in phages than other,
similarly diverse synthetic libraries [62]. Compared to the synthetic diversication
methods that preceded it, Slonomics offers more control over the amino acid composition at each residue position while avoiding the introduction of PCR-related
errors. The length of gene fragments synthesized by this method, however, is capped
at 462bp. Gene synthesis occurs in parallel using 96-well plates.
Innovations in microarray-based gene synthesis and antibody library technologies have converged to enable the construction of fully synthetic antibody libraries
at unprecedented scales. Twist Biopharma, a division of Twist Bioscience, has capitalized on the massive throughput of the Twist synthesis platform to create the largest collection of fully synthetic antibody libraries to date, totaling 15 distinct
libraries of ~1010 size each, with more libraries on the horizon. Collectively, these
libraries span multiple antibody formats (scFv, Fab, VHH), diversication strategies, and target classes, providing rich sources of unique antibodies for antibody
drug development.
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R. L. Nugent and A. K. Sato
3.5 Perspectives
Modern gene synthesis platforms have ushered in a new era of biopharmaceutical
development by enabling the construction of unprecedented antibody libraries. Our
goal in writing this chapter was not to comprehensively review the development of
gene synthesis and antibody screening technologies. For those interested, many
such reviews have been cited within this chapter. Rather, our goal was to describe
how select technological achievements led to the expansion of the biopharmaceutical discovery process to its current scale. The methods that contributed to the rst
successes in biopharmaceutical development have been superseded by arguably
superior and denitively more expansive technologies.
Recently, the combination of gene synthesis, automated experimentation, and
faster and cheaper sequencing technologies has led many in the eld to take advantage of machine learning to generate discoveries from ever-growing datasets. In one
example, Mason and colleagues [63] developed a neural network from library
screening and site-directed deep-sequencing data obtained from mammalian cells.
They subsequently used this model to predict the specicity of trastuzumab variants
to human epidermal growth factor receptor 2 (HER2). By narrowing down the original unltered antibody library to HER2-specic binders, Mason etal. were able to
greatly reduce the cost of antibody screening while generating a more successful
library. The success of this approach bodes well for the marriage of computational
and gene synthesis techniques in antibody discovery, a combination we believe
offers nearly limitless potential. We believe the continued co-development of these
technologies will spur a renaissance in biopharmaceutical discovery.
Still, there is much to learn when it comes to gene synthesis and antibody discovery. High secondary structure, repetitive sequences, and unknowingly toxic
sequences continue to complicate the fabrication of genetic parts for discovery
workows [13]. Moreover, our ability to generate antibodies of diverse formats,
specicities, and naturalnesses has outstripped our ability to understand how these
new engineered biomolecules behave in complex biological systems such as the
human body. Developability—ensuring that antibody candidates meet basic requirements for clinical utility, including expression, stability, and solubility—also
remains a challenge, albeit one that has been partially addressed by the advent of
synthetic antibody libraries. Moving forward, biopharma will need to leverage
insights gained from the latest synthetic antibody libraries to inform the development of the next generation of antibody therapeutics.
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