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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5886_Библиотеки_им_академика_М_И_Перельмана.pdf
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A. K. Sato and S. Rife
There is currently signicant interest in the development of single-domain
heavy-chain (VHH) biologics owing to their antibody-like properties and smaller
size (affording them distinct pharmacokinetics relative to full-length antibodies).
Assembling VHH libraries can be challenging with trimers due to the potential for
truncation. Truncated oligonucleotides can then lead to a diminished percentage of
full-length VHH candidates and the need for increased screening to ensure that all
full-length variants are tested.
With trimers, it is alsodifcult to avoid liability motifs that can result in instability of biologics following post-translational modications or isomerization. In order
to avoid motifs with trimers, certain amino acids would need to be avoided to eliminate the possibility of unwanted amino acid pairings that would create an undesirable motif.
Direct synthesis, on the other hand, has enabled a much higher degree of precision and efciency. Through solid-phase synthesis along with miniaturized chemistry, companies like Twist Bioscience have made it possible to synthesize
oligonucleotides without randomization. By deliberately adding every nucleotide in
a growing strand, direct synthesis can be programmed to generate combinatorial
variant libraries containing uniform variant representation and prespecied amino
acid ratios (Fig.2.4).
The improved accuracy and efciency gained through direct synthesis has
enabled researchers to focus their resources on generating and testing millions of
rationally designed antibody variants. In so doing, direct synthesis has improved the
scale of antibody discovery by allowing parallelization—rather than only creating a
Fig. 2.4 Amino acid distribution after synthesis with NNK, Trimer, and direct synthesis. When
compared to NNK and Trimer synthesis, libraries created with direct synthesis technology (Twist)
showed less than 1% deviation from the designed amino acid frequency (y-axis). (Data produced
by Twist Bioscience)
2 Synthetic Biology inDrug Development andBeyond
41
small handful of desired antibodies at a time, millions can be rapidly produced and
screened in parallel.
Importantly, such libraries need not be full-length antibodies. As researchers are
primarily interested in identifying antibodies with a specic and strong afnity for
their target of interest, resources can be conserved by limiting synthesis to antibody
fragments that contain CDR loops, such as VHH, single-chain variable fragments
(scFv), and fragment antigen-binding regions (Fabs). This approach opens the door
to high-throughput screening opportunities.
As with other synthesis methods, direct synthesis is not without its hurdles.
Highly parallel direct oligonucleotide synthesis requires substantial infrastructure
and automation. As such, these methods are often more expensive than degenerate
methods and, like trimers, must contend with nucleotide coupling efciencies to
ensure uniform representation. Therefore, careful design and quality control (often
through next-generation sequencing) is needed, further driving up costs. However,
direct synthesis has greatly decreased the costs associated with DNA synthesis and
will likely continue to do so as the technology advances [54].
Phage Display
Converting synthetic DNA libraries into screen-ready antibody libraries requires
cellular machinery. Therefore, synthesized libraries must be inserted into a chassis
organism whose internal machinery can be taken advantage of to produce antibody
fragments.
Following DNA synthesis, antibody sequences can be assembled in plasmid or
phagemid vectors and transduced into a display system. In these systems, antigenbinding fragments are heterologously expressed in a chassis organism such as a
bacteriophage, yeast, or bacteria cell. Importantly, these antibody fragments are
fused to a native surface protein within the chassis, enabling surface expression of
the fragment [55, 56].
Phage display is the most commonly used display system in antibody discovery
and development owing to its ease of use and scale. This system utilizes bacteriophages—typically M13 bacteriophage—that selectively infects E. coli. For M13
bacteriophage, antibody fragments can be assembled into phagemid vectors as
fusion proteins with one of the phage’s ve surface proteins. Importantly, the
phagemid vector contains genetic elements that enable it to be enclosed in the viral
capsid during replication [55].
The resulting bacteriophage will express antibody fragments on its surface while
also containing phagemid vectors within. Selection is then performed by rst immobilizing puried antigens on solid surfaces, such as nitrocellulose membranes, polystyrene plates, magnetic beads, or column matrices. Then, a phage library
corresponding to the synthesized antibody library can be exposed to the immobilized antigen. Successive cycles of washing will eliminate all unbound phages, leaving just those phages that express high-afnity antigen-binding fragments (Fig.2.5).
High-afnity binders can then be recovered with the use of myriad elution buffers. Because the eluted phages each contain a phagemid, they can be transduced
into E. coli and subsequently sequenced to determine which antibody sequences
correspond to high-afnity binders.
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A. K. Sato and S. Rife
Fig. 2.5 Phage display for antibody discovery. Workow for the discovery and optimization of
antibodies using phage display. Helper phages may be needed to ensure production of engineered
bacteriophages. (Figure adapted from “Phage Display Panning,” by BioRender.com [2022].
Retrieved from https://app.biorender.com/biorender- templates)
Phage display screens can be used to test libraries as large as 1010 unique antibody fragments, enabling researchers to rapidly home in on antibody sequences
with desired binding properties [55, 56]. In contrast to immunization approaches,
invitro display systems can be used to develop biologics against highly toxic and
non-immunogenic targets.
Though phage display has clear advantages over hybridoma technology, antibodies discovered through phage display often require substantial optimization.
Replicating invitro the immune system’s ability to rapidly optimize antibodies for
given antigens is difcult. Without comprehensive optimization, phage-derived
antibodies may be prone to invivo aggregation, low levels of expression, and other
issues that limit the antibody’s clinical utility [57]. However, invitro optimization
2 Synthetic Biology inDrug Development andBeyond
43
systems are advancing and may reduce developability concerns stemming from
phage display in the near future.
In the meantime, phage display remains a powerful discovery tool. As of August
2020, 70 phage-derived antibodies had advanced to clinical trials, 14 of which have
already been approved [56].
Similar display systems have been developed with other chassis organisms, such
as S. cerevisiae [55]. Herein, synthetic DNA encoding antibody fragments are trans-
fected into the yeast where they integrate with the yeast’s cell wall, akin to the
integration that occurs in bacteriophages. These eukaryotic display systems can be
useful as they replicate post-transcriptional modications that may affect antibody
performance in human cells, such as glycosylation. While yeast glycosylation patterns may differ from what is observed in humans, yeast cells can be engineered to
express human glycosylation machinery. Useful as it is, yeast display suffers from
decreased efciencies that limit its scale to approximately 108 antibody fragments
per experiment.
There are many more display systems worth diving into, and we encourage you
to read Mahdavi etal.’s excellent overview in the International Journal of Biological
Macromolecules to learn more [55].
2.6 CAR-T Cell Therapies
One of the most recognized therapeutic applications of synthetic biology is in the
development of cell therapies broadly and chimeric antigen receptor (CAR) presenting T-cells specically. Conceptually, cell therapies aim to endow an organism
with enhanced immune cells, whether those are stem cells lacking pathogenic mutations or, in the case of CAR-T cells, differentiated cells with specic and potent
pathogen targeting abilities. In contrast to the transient nature of antibodies and
small molecules, cell therapies have the potential to help patients indenitely, providing lasting protection against recurrent malignancies or viruses like HIV [58–60].
Among the many different cell therapies, CAR-T cell technology has seen
remarkable advancement in recent years, driven largely by improvements in synthetic biology (Fig.2.6).
T-cells are a class of lymphocytes that play a critical role in adaptive immunity
[60]. Born in the thymus, naive T-cells roam the body awaiting activation. Unlike
B-cells, which recognize pathogens through antibodies, T-cell activation is initiated
when a unique cell surface protein—the T-cell receptor (TCR)—recognizes a major
histocompatibility complex (MHC) protein displaying an antigenic peptide. This,
combined with cascading signals from costimulatory factors, prompts T-cells to
release a slew of cytokines that, depending on the type of T-cell, are often proinammatory [61–63].
T-cells have the ability to direct adaptive immune cells against specic pathogens and thus have signicant potential in therapeutic applications. Like antibodies,
the human TCR repertoire is extremely diverse owing to V(d)J recombination. But
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Fig. 2.6 CAR-T cell therapy overview. Workow for CAR-T cell therapy. T-cells are isolated
from donor or patient blood and engineered to express a chimeric antigen receptor targeting tumorcell- specic antigens. After successive rounds of growth, the engineered T-cells can then be reinfused into the patient to affect therapeutic benets. (Reprinted from “CAR T Cell Therapy
Overview,” by BioRender.com [2022]. Retrieved from https://app.biorender.com/biorender-
templates)
A. K. Sato and S. Rife
while a T-cell may have the ability to recognize antigens, it must do so when the
antigenic peptide is in complex with an MHC protein [63]. Because of this, researchers developing cell therapies based on T-cells would need to match highly antigenspecic T-cells against specic antigen-presenting cells together to enable a potent
therapy. Or a simpler approach is to engineer therapeutic T-cells using synthetic
biology.
In the late 1980s, researchers at multiple universities began engineering T-cells
to express a synthetic fusion protein that combined antibody variable domains with
T-cell receptor signaling domains [64, 65]. The resulting chimeric protein could
thus recognize antigens with antibody-like specicity and, upon binding, elicit
immunogenic activity from the host T-cell. These early experiments are broadly
considered the rst demonstrations of CAR-T technology [66].
Importantly, the inclusion of antibody variable domains in CARs enables antigen
recognition without the involvement of MHC proteins; and, the addition of a
costimulatory factor (CD3ζ) enables the chimeric protein to elicit T-cell activation
without MHC complex recognition on antigen-presenting cells [66]. Collectively,
these modications make it possible to aim T-cells at specic antigens, such as proteins uniquely present on the surface of cancer cells, which can then independently
mount an immune response.
The power of CAR-T cells was recognized around the world in 2012 when
6-year-old Emily Whitehead became the rst child to receive CAR-T therapy [67].
2 Synthetic Biology inDrug Development andBeyond
45
Emily had undergone 16months of intermittent chemotherapy, but the acute B-cell
lymphoblastic leukemia that owed through her veins resisted treatment. With no
other options, Emily was enlisted in a clinical trial to test the effect of a new CAR-T
therapy, known as tisagenlecleucel (also referred to as Kymriah). T-cells from
Emily’s body were removed and transduced with genes carrying a synthetic CAR
that targeted the cell surface protein CD19. It has been more than 10years since her
treatment, and Emily remains cancer free.
While both promising and inspiring, it is also a warning: following treatment,
Emily’s body responded with a cytokine storm that nearly killed her. Cell therapies
come with a substantial risk of inducing a potentially lethal condition known as
cytokine release syndrome (CRS). The cause for critical side effects in cell therapies
can be multifaceted. CRS can be induced through an overwhelming CAR-induced
on-target, on-tumor inammatory reaction. Careful design and validation of the
CAR for a given target and therapeutic indication is key for development of safe and
efcacious therapies.
Beyond designing the synthetic receptor, the cellular vehicle that the CAR is
inserted into plays a major role in drug development. While autologous therapies
focus on engineering a patient’s own cells, allogeneic therapeutics offer the ability
to build therapies with broader accessibility by using engineered cells from donors
(discussed further below). However, such an option raises additional requirements
in terms of design, engineering, and functional evaluation of cells. In contrast to
autologous therapies, allogeneic therapies must take into consideration graft- versushost disease (GVHD)—a condition wherein donor immune cells attack healthy host
tissues [68]. Similarly, the engineered cells are at risk of being rejected by the
host’s body.
The development of safe and effective CARs thus requires careful and extensive
optimization to ensure both safety, specicity, and efcacy. Fortunately, many of the
tools that characterize synthetic biology can be leveraged during CAR development.
Antibody display systems, for example, enable large-scale and high-throughput
screening of potential antigen-binding domains. Once identied as specic for the
target antigen, DNA coding for binding domains can be synthesized and assembled
into a CAR gene.
Subsequent optimization will be required, wherein variations in antigen-binding
domains, hinge and transmembrane domains, and costimulatory factor combinations are tested. This is because cellular response to CAR-antigen binding is highly
context specic, and alterations in the exibility of transmembrane domains, as well
as the intracellular environment, can alter downstream signaling. Therefore,
researchers will need to generate and express combinatorial variant CAR libraries
for iterative rounds of optimization [69, 70].
As with antibody discovery, the precise and large-scale production of synthetic
DNA and the combinatorial assembly of variants can accelerate CAR-T development (Fig.2.7).
In some allogeneic therapies, even when CARs are optimized to be highly specic, patients are still at risk for GVHD.This is because T-cells are trained to recognize foreign cells through mismatches in MHC proteins (the human equivalent of
46
Fig. 2.7 Creation of chimeric antigen receptor and T-cell receptor libraries via combinatorial
assembly. Through combinatorial assembly, user-dened combinations of gene fragments can be
shufed together to create highly uniform screening libraries that enable comprehensive screening
of the variant space and the discovery of CAR formats with novel functionalities
A. K. Sato and S. Rife
HLA proteins). It is thus important for donors and patients to carry similar HLA
proles [71].
One approach to avoid GVHD is to use the patient’s own cells, as in the case of
Emily Whitehead. This is known as autologous cell therapy. However, the patient’s
immune cells may not be suitable for engineering due to the effects of previous
therapeutics or the pathogen/malignancy affecting the patient.
In these situations, physicians may opt for allogeneic CAR-T cell therapy
wherein T-cells are collected from a healthy donor, engineered to express the synthetic CAR, and then transfused into the patient. The risk of GVHD is signicant in
allogeneic CAR-T cell therapy. And while supplementary chemotherapies are being
explored to mitigate the risks of GVHD, and HLA matching is possible, the
risks remain.
Another option is to use gene editing technology to engineer T-cell stocks that do
not express HLA proteins, or else express specic HLA proles. These allogeneic
T-cells could then serve as a repository, off-the-shelf option for researchers and
physicians to access when needed. Research into this possibility is ongoing and
holds signicant promise. Currently, CAR-T cells are generated rapidly, providing
little time for cells to be optimized. However, if T-cell stocks can be made, researchers may have ample time to engineer CAR-T cells that target multiple pathogenic
antigens by expressing multiple different CARs [72].
The CAR-T therapy that saved Emily Whitehead’s life was approved by the FDA
for the autologous treatment of adult patients with relapsed or refractory follicular
lymphoma after two or more lines of therapy [73]. While a substantial landmark for
synthetic biology, much more research is needed to develop this technology. For
2 Synthetic Biology inDrug Development andBeyond
47
instance, CAR-T cells are not yet durable enough to provide lifelong immunity, and
the intense immunological side effects will need to be contended with.
To this end, the success of CAR-T cell therapy has spurred interest in similar cell
therapies [74]. One such therapy leverages natural killer (NK) cells—a type of
peripheral leukocyte with potent cytotoxic abilities. Unlike T-cells, NK cells do not
become activated against targets based on T-cell receptor-antigen binding. Instead,
NK cells are under a dynamic state of intracellular signaling that suppresses activation. However, when exposed to a myriad of contextual signals in the cell’s microenvironment that decrease suppression and enable cell-killing, cells that
downregulate self-identifying HLA proteins, for example, are more likely to be targeted by NK cells [75, 76].
Interest in the use of NK cells for CAR therapy stems from a growing body of
evidence that shows CAR-NK cells are less likely to induce CRS and GVHD (owing
to a different cytokine release prole relative to T-cells), are capable of potent ontumor cell-killing, and may affect cytotoxicity independent of CAR-antigen binding
(through natural activation by the tumor microenvironment) [76]. With a reduced
risk of CRS and GVHD, CAR-NK cells may prove to be better suited for off-theshelf, allogeneic cell therapy development.
As with CAR-T cells, CAR-NK cell therapy faces several challenges related to
transduction efciency, ex vivo expansion of engineered cells, tumor evolution
toward the loss of CAR-targeted antigens, and more [76]. As such, development of
CAR-NK will similarly require extensive preclinical optimizations in CAR design
and engineering methods.
Nonetheless, development of both CAR-T and CAR-NK therapies represents
shining examples of synthetic biology and its application in therapeutic development. It is likely that the number of clinical trials testing either CAR-T and CAR-NK
will continue to increase as advances in synthetic biology continue to expand the
scale and throughput of preclinical development.
Notably, there are many additional cell therapies that are worth exploring. The
reader is encouraged to read more on cell therapies in a recent review by Wang
etal. [74].
2.7 Conclusion
Synthetic biology is an expansive eld, one that aims to solve many of the world’s
problems through the engineering of biological systems. Though relatively new, the
eld has already made a substantial impact on modern drug discovery and the
advancement of biologics research. In the preceding sections, we have attempted to
provide a brief overview of this impact, highlighting choice contributions where
possible. We have undoubtedly left many details and areas of interest out of this
discussion, not for a lack of importance but rather in service of brevity.
While there is still much to be covered, there are some key concepts that we have
tried to emphasize. When precisely synthesized, synthetic DNA can be a versatile
48
A. K. Sato and S. Rife
tool in the synthetic biologist’s toolkit. Novel genes can be rapidly assembled and,
through cloning, inserted into a chassis organism. This process represents a synthetic dogma that drives modern drug development in a myriad of ways.
CRISPR-Cas systems are an excellent example. The CRISPR-Cas complex is a
synthetic creation, modied from a bacterial immune system and used extensively
for gene editing. Among its many uses, CRISPR-Cas affords researchers the ability
to carry out functional genomic screening on a large scale, such that individual
genes can be systematically perturbed across the entire genome within a single
screen [77]. Such a study can reveal key genes involved in disease progression and
thus potential therapeutic targets. To do this, synthetic DNA coding the CRISPRCas complex will be synthesized, assembled, and inserted into the cell line of interest for high-resolution study.
Beyond target identication and validation, synthetic biology is greatly advancing our ability to discover therapeutic small molecules and biologics. Secondary
metabolites, for example, are produced in myriad organisms and have proven to be
powerful sources of therapeutic small molecules. However, screening of secondary
metabolites has been severely limited. With complex structures and often low natural concentrations, secondary metabolites can be difcult to detect and harder to
screen. As such, discovery efforts are largely limited to metabolites produced in
large quantities or in organisms that can be easily cultured in the laboratory setting.
Synthetic biology offers a way to greatly expand the scope of these efforts. Once
described, the molecular pathways—often identied in biosynthetic gene clusters—
that produce secondary metabolites can be reconstituted in chassis organisms and
leveraged for the large-scale production of diverse secondary metabolite libraries.
Such libraries have already been used to surface promising small molecules.
DNA synthesis, cloning, and expression in chassis organisms drives therapeutic
antibody discovery, as well. Advances in DNA synthesis technology have made it
possible to precisely and rapidly engineer millions of unique DNA sequences. These
in turn can be assembled into synthetic antibody libraries whose diversity extends
beyond natural repertoires, providing researchers with a unique opportunity to
design, discover, and optimize novel therapeutic antibodies.
In short, it is clear that synthetic biology is a versatile eld and one that is integral to modern drug development.
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