SBE SPECIAL SECTION:

BIOMOLECULAR
ENGINEERING

30 S
 BE Update: The Many Applications
of Biomolecular Engineering
32 Vaccines as Therapies
40 Cancer-Fighting T Cells
47 Single-Cell Analytics
52 Computational Protein Engineering
58 Plant Metabolic Engineering
SBE SPECIAL SECTION:
BIOMOLECULAR ENGINEERING
The Many Applications of
Biomolecular Engineering
T he discipline of biomolecular engineering focuses on the
interface between molecular biology, biophysical chemis-
try, and chemical engineering — with the goal to translate our
fundamental understanding of molecular biology into useful
therapies, devices, and processes. Applications of this disci-
pline include clinical medicine and developmental biology.
To support the community of scientists and engineers
working at the forefront of this emerging discipline, SBE
holds an annual International Conference on Biomolecular
Engineering (ICBE). The 7th ICBE, which was held in Janu-
ary 2017, is the basis for the articles in this special section.
With such a broad and exciting discipline, there were
many topics to choose from in designing this special
section. Our guest editor, Jennifer Maynard, an associate
professor and Laurence E. McMakin, Jr. Centennial Fac-
ulty Fellow in the McKetta Dept. of Chemical Engineering
at the Univ. of Texas at Austin, sought to create a collec-
tion of interesting, as well as promising, developments to
invoke excitement around this emerging discipline. The
topics covered also convey the breadth and depth of emerg-
ing areas of biomolecular engineering.
In the first article, “A Materials Approach to Vaccines
as Therapies” (pp. 32–38), Christopher Jewell and Neil
Dold of the Univ. of Maryland discuss the application of
biomolecular engineering tools to develop vaccines that treat
— not just prevent — infectious diseases. The authors begin
their discussion by describing how a healthy mammalian
immune system responds to threats — providing a basis to
discuss what happens when the immune system fails and
how engineering approaches can be used to get it back on
track. Two pervasive examples of a failing immune system
are autoimmune disorders and cancer. Autoimmune diseases
occur when immune cells inappropriately target one’s own
cells as if they were foreign or infected. Cancer, on the other
hand, is a disease in which mutated self-cells lose metabolic
control and divide in an unrestrained manner.
Jewell and Dold show how designing vaccines with
biomaterials enables new approaches to treating cancer and
autoimmune disorders, such as multiple sclerosis (MS) and
Type I diabetes. They discuss several emerging develop-
ments, including more-efficient vaccine-delivery mechanisms,
implanted scaffolds to recruit immune cells, and microneedle
arrays for the delivery of vaccines with local precision.
Building on the ideas presented in the first article, Yvonne
Chen and Eugenia Zah introduce a new treatment for can-
cer — adoptive T-cell therapy (ACT) — in their article,
“Engineering Cancer-Fighting T Cells” (pp. 40–46). Unlike
30  www.aiche.org/cep  October 2017  CEP
traditional therapeutics such as chemo­therapy, radiation, and
surgery, ACT harnesses a patient’s own immune cells as a liv-
ing drug to treat previously incurable cancers. This treatment
involves tumor-targeting T cells obtained by either isolating
T cells that naturally target tumors or genetically modifying
T cells to express a transgenic tumor-targeting receptor.
In the third article, “There’s Plenty of Room at the Bottom
of a Cell” (pp. 47–51), Angela Wu and Lei Yu of Hong Kong
Univ. of Science and Technology provide an overview of
single-cell sequencing — a technique used to simultaneously
profile all expressed genes within a single cell. They high-
light several applications of this technique, including gaining
unprecedented understanding of the embryonic development
process, cancer treatment, and the discovery of new cell types.
Switching gears somewhat, the next article, “Computa-
tional Protein Engineering” (pp. 52–57), focuses on compu-
tational tools to design proteins. Robert Pantazes of Auburn
Univ. explains how protein-based catalysts have the potential
to be less expensive, safer, and more effective than traditional
catalysts. However, proteins have not reached their wide-
spread potential because they are too difficult and expensive
to engineer for a particular purpose. Computational protein
engineering overcomes some of these limitations, Pantazes
explains, by providing a means to explore protein folds not
observed in nature and designing novel proteins from scratch.
The topic of the final article is metabolic engineer-
ing. There are comparatively fewer studies on plants than
microbes by metabolic engineers. Although plants are the
ultimate source of many of our chemicals and fuels, the
large engineering gap between microbes and plants limits
the power of much of synthetic biology. In the article, “Plant
Metabolic Engineering for Chemicals, Fuels, and Precursors”
(pp. 58–62), Timothy A. Whitehead of Michigan State Univ.
and Sean Cutler and Ian Wheeldon of the Univ. of California,
Riverside, describe the engineering divide between plants and
microorganisms, and discuss ways to narrow this gap.
As the community and the field of biomolecular engi-
neering are maturing and branching out, SBE has launced
a second conference series on biomolecular engineering in
ASIA (ICBE ASIA) with the next ICBE ASIA scheduled for
January 8-10, 2018 in Singapore. Join us and the chairs of
the conferences to stay abreast of the continual research and
cutting-edge impacts of this evolving field.
Gabriel Levesque-Tremblay, Sr. Engineering Specialist
Society for Biological Engineering (SBE)
An AIChE Technological Community
Copyright © 2017 American Institute of Chemical Engineers (AIChE)
 ABSTRACT SUBMISSION DEADLINE
EXTENDED TO OCTOBER 16

ICBE Asia 2018 | JANUARY 8-10, 2018 | GRAND HYATT SINGAPORE, SINGAPORE

The International Conference on Biomolecular Engineering - ICBE CONFERENCE CO-CHAIRS:

Asia 2018: Engineering Living Matter will present researchers at the
■ Matthew Chang
forefront of biological engineering the opportunity to showcase cutting
National University of Singapore
edge ideas to other experts in the field, as well as allow for discussion
amongst researchers and exhibitors about the implementation of new
■ Poh Chueh Loo
expertise in Asia and globally.
National University of Singapore

CONFERENCE TOPICS INCLUDE: ■ Julie Champion

Georgia Institute of Technology
■ Foundational Technologies for Biomolecular Engineering.
New tools and platforms developed to enable engineering
ORGANIZING COMMITTEE:
of molecules, cells, and multi-cellular entities. Technologies
for molecular design or synthesis, functional screening and ■ Zhen Xie
selection, integration into living systems. Tsinghua University, China

■ Biomolecular Programming - from DNA to Community. ■ Sierin Lim

Programming approaches across single to multiple lengthscales. Nanyang Technological
Sustainable programming for bioprocess engineering, University, Singapore
microbiome programming, and molecular networks.
■ Wilson Wong
■ Translational Biomolecular Engineering. Boston University, USA
Biomolecular engineering applications for the benefit of
society. Translational approaches from production of high value ■ Inchan Kwon
chemicals, to cellular immunotherapies, to catalytic protein Gwangju Institute of Science
materials. and Technology, South Korea

■ Wenjun Zhang
University ofCalifornia
SUBMIT YOUR ABSTRACT BY OCTOBER 16 Berkeley, USA

Karen Polizzi
For more information about ICBE Asia 2018 ■

ImperialCollege London, UK
visit www.aiche.org/icbeasia

© 2017 AIChE 1954_17 • 09.17 FOLLOW US: @ChEnected

SBE SPECIAL SECTION:
BIOMOLECULAR ENGINEERING

A Materials Approach to
Vaccines as Therapies
Neil Dold The widespread adoption of vaccines over
Christopher M. Jewell
Univ. of maryland the last two centuries has transformed
medicine by preventing infectious disease.
In the future, so-called therapeutic vaccines
could be used as effective treatments for
cancer and autoimmune disorders —
ushering in a new era of medicine.

V
accines are traditionally viewed as a technology that
arms the immune system before the body encoun-
ters an infectious disease. This preventive approach
has helped fight chickenpox, measles, influenza, and other
pathogens. Vaccines have also profoundly reduced the
incidence of polio, with only 37 confirmed cases reported in
2016 worldwide (1), and they are responsible for the global
eradication of smallpox.
Advances in the use of vaccines to prevent infection
have radically improved health around the globe, but there
is an equally provocative wave of emerging research in
developing selective, therapeutic vaccines designed to treat
— not just prevent — noninfectious diseases such as cancer
and autoimmune disorders. These vaccine-like therapies
aim to specifically target tumors, which normally evade the
immune system, and to stall autoimmune diseases such as
multiple sclerosis (MS) and Type I diabetes — diseases in
which the immune system erroneously attacks one’s own tis-
sues. Several recent advances in this area harness the unique
capabilities of polymers and other biomaterials.
This article discusses some of the challenges facing
therapeutic vaccines for cancer and autoimmune diseases,
and the potential of new biomaterial-based technologies
that target these diseases. Therapeutic vaccines fit into the
broader scheme of immunotherapies that try to correct or
enhance the immune system’s behavior; some of the other
exciting approaches, such as cell therapy, are highlighted
elsewhere in this special section. This is meant as an intro-
ductory article; Refs. 2–6 provide more in-depth discussions
of biomaterial-based vaccines for infectious disease, cancer,
and autoimmune disorders.
The healthy immune response
Vaccines for infectious diseases equip the immune system
to fend off safe mimics of a particular pathogen (e.g., protein
fragments or a killed virus), enabling the body to quickly rec-
ognize and destroy this same pathogen during an actual infec-
tion in the future. The body’s pathogen-specific responses
are faster after vaccination, because vaccines create immuno­
logical memory that can last for decades. The immune sys-
tem must be able to recognize and quickly destroy pathogens
with molecular precision, while avoiding attack of self-cells
and tissues through a programmed state known as tolerance.
Mammalian immune systems have evolved non-specific
(i.e., innate) responses that form a first line of defense
against foreign invaders, along with highly specific (i.e.,
adaptive) responses that target distinct pathogens and gener-
ate memory against these pathogens (Figure 1).

32  www.aiche.org/cep  October 2017  CEP Copyright © 2017 American Institute of Chemical Engineers (AIChE)
 Innate immune cells — including macrophages and
dendritic cells (DCs) — can recognize biological pat-
terns that occur in pathogens and are typically absent
in normal human tissues (e.g., lipopolysaccharides that
comprise bacterial membranes, DNA, or RNA associated
with viruses). Within minutes to hours of detecting the
pathogen-associated patterns, innate immune cells mount
responses to directly kill the infected cells, engulf patho-
gens, and release signaling molecules called cytokines
to signal danger and recruit more immune cells. These
processes underpin the inflammation associated with tissue
sites containing infection or wounds.
Innate immune cells do not possess the capacity for
memory, so they share information with cells of the adap-
tive immune system by digesting and presenting the peptide
fragments — called antigens — from the digested pathogens.
Antigen-presenting cells (APCs), such as macrophages and
DCs, present antigens alongside co-stimulatory signals to
activate T and B lymphocytes, which are key players of
the adaptive immune response. While APCs can encounter
lymphocytes throughout the body, APCs eventually migrate
to lymph nodes and the spleen to present the antigen to T and
B cells residing in these immune tissues. Free antigen can also
drain to the lymph nodes and spleen, where it is processed by
APCs and then presented to T and B cells.
Each B cell or T cell has a receptor for a specific anti-
gen, which, when activated in the lymph nodes or spleen,
only binds with specific antigen molecules — enabling a
molecule-specific immune response. The activation process
significantly enhances the ability of the B or T cells to rec-
ognize a given antigen. Activated B cells mature and secrete
p Figure 1. A healthy immune system involves a nonspecific innate response that arises quickly but lacks memory and a highly specific adaptive response
that requires time to develop initially but creates long-lasting immunological memory. These responses work together to successfully clear pathogens.
Copyright © 2017 American Institute of Chemical Engineers (AIChE)
antibodies that can neutralize extracellular pathogens or
identify pathogens for destruction by other immune mecha-
nisms. T cells have several functions. One type of activated
T cell, a cytotoxic T cell, directly seeks out and destroys
host cells infected with viruses to prevent the virus from
spreading. B cells require other types of activated T cells for
activation — demonstrating the interconnectivity between
cells in the body’s immune mechanisms.
A fraction of activated T cells and B cells remains in
the body after a pathogen is cleared. These persistent cells
provide the immune system with memory of the pathogen,
enabling lymphocyte populations to have a strong and
rapid response to that same pathogen in the future. Gener-
ating this protective memory — the goal of most traditional
vaccines — is not always efficient or long lasting, which is
why multiple immunizations may be required over a short
period of time (e.g., hepatitis vaccines) and why adults
receive periodic booster shots for certain diseases (e.g.,
tetanus). References 7–9 provide more detailed information
on immune responses.
When the immune system goes wild, or mild
Unfortunately, the normal immune processes can fail
— cancer and autoimmune disorders are two pervasive
examples. Autoimmune diseases occur when immune
cells inappropriately target one’s own cells as if they were
foreign or infected (Figure 2a). Some of the most common
autoimmune diseases are MS, Type 1 diabetes, rheumatoid
arthritis, and lupus. The immune system of someone with
MS degrades the myelin sheath around neurons in a process
called neurodegeneration. In patients with Type 1 diabe-
CEP  October 2017  www.aiche.org/cep  33
SBE SPECIAL SECTION:
BIOMOLECULAR ENGINEERING
tes, the immune system destroys pancreatic islet cells that
produce insulin; without enough insulin, the patient’s blood
glucose levels are not properly regulated. Therapies that
direct the immune system to stop attacking specific “self”
molecules — termed tolerance — could stop or reverse auto-
immune diseases without causing the broad immune system
suppression characteristic of many existing treatments.
Cancer, on the other hand, is a disease in which unhealthy
self-cells lose metabolic control and divide in an unrestrained
manner (Figure 2b). Cancer cells are able to avoid attack
by the immune system by secreting suppressive signaling
molecules and presenting those molecules to immune cells.
Therefore, even if a T cell is activated against a cancer anti-
gen and infiltrates a tumor, the tumor may signal the T cell
to ignore the tumor (tolerance), or the tumor may develop
new self-antigens not recognized by the infiltrating immune
cells. Moreover, cancer vaccines that generate tumor-specific
T cells are risky, because those lymphocytes could recog-
nize noncancerous tissues. Effective cancer vaccines must
therefore program the immune system to selectively recog-
nize tumors and overcome the tumor’s immunosuppressive
microenvironment.
Regulating the immune system’s overzealous mistakes
p Figure 2. Dysregulation of biological signals can cause immune cells to attack one’s own tissues in autoimmune diseases (a) or to ignore unhealthy
cancerous tissues that need to be cleared by the immune system (b).
34  www.aiche.org/cep  October 2017  CEP
in autoimmunity and its lack of action toward cancerous
cells presents a multitude of opposing challenges. The next
section discusses new approaches to tackle cancer and
autoimmune diseases with therapeutic vaccines that combine
biomaterials with insight gleaned from conventional vac-
cines against infectious disease.
Biomaterials in conventional vaccines
To generate memory against a particular pathogen, vac-
cines for infectious disease use attenuated pathogens (e.g.,
modified by chemicals or heat) or specific antigens isolated
or derived from a pathogen. Most vaccine formulations con-
tain antigens along with additives known as adjuvants (10).
It has only been in the past few decades that researchers
have taken an in-depth look at adjuvant material properties
(e.g., particle size, surface charge, specific interactions with
innate immune cells) to understand how they influence vac-
cine performance (11).
Perhaps not surprising then, biomaterial-based adju-
vants were previously thought of as inert carriers; recent
studies, however, reveal that many biomaterials exhibit
intrinsic physiochemical features (e.g., shape, charge,
chemical functionality) that engage immune signaling
Copyright © 2017 American Institute of Chemical Engineers (AIChE)
pathways. For example, polymers such as poly(lactide-
co-glycolide) (PLGA), poly­styrene, polyanhydrides, and
poly(β-amino esters) are all capable of stimulating innate
immune responses (12–14). In addition to stimulating
the immune system, biomaterials also enable the use of
well-known drug-delivery and tissue-engineering technolo-
gies that are difficulat to achieve with traditional vaccines,
including co-delivery, cargo protection, controlled release,
and targeting.
All of these features can be harnessed for therapeutic
vaccines. For example, one new biomaterial-based approach
for treating autoimmune disorders involves delivering
immunosuppressive molecules together with self-antigens
to restore tolerance against those antigens. New strategies
to fight cancer are exploiting biomaterials for personalized
vaccines that maintain antitumor immunity within immuno­
suppressive tumors, while avoiding unregulated responses
that could damage healthy tissue.
The following sections highlight four delivery
approaches — particulate formulations, implanted scaf-
folds, micro­needles, and self-assembly of immune signals
— that are revealing new potential for next-generation
therapeutic vaccines.
Particulate formulations for delivery
Most conventional vaccines are delivered in a poorly
localized or largely soluble dose. The physicochemical fea-
tures of particles can be exploited to develop more-efficient
delivery approaches. However, few rational design methods
have been implemented for either conventional or biomaterial-
based vaccines and immunotherapies.
Because particulate materials are readily internalized by
immune cells, loading vaccine cargos into particles enables
co-delivery of multiple compounds and the controlled release
of these cues (Figure 3). In the area of immune tolerance to
combat autoimmunity, one approach involves co-loading
nanoparticles with myelin peptides (self-molecules that are
attacked in patients with MS) and a regulatory ligand. The
goal is to reprogram T cells during presentation of the self-
antigen and direct the myelin-recognizing cells to become
regulatory T cells that specifically control the inflammatory
Co-Delivery of Antigen with Combinations of
Stimulatory or Regulatory Immune Cues
Tumor Antigens with Self Antigen with Regulatory
Stimulatory Cues for Cancer Cues for Tolerance
p Figure 3. Biomaterials can be used to deliver multiple components,
including antigens and regulatory immune cues.
Copyright © 2017 American Institute of Chemical Engineers (AIChE)
Several features of biomaterials
can be harnessed for the development of
therapeutic vaccines.
immune cells attacking myelin (15, 16). This approach has
demonstrated promising therapeutic effects in stopping or
reversing disease in mouse models of MS, allergies, and other
inflammatory states.
Another platform being developed involves encapsulat-
ing peptides and tolerizing cues in larger microparticles (17).
These particles, which can be injected directly into lymph
nodes, are designed such that they are too large to drain away.
Instead, the particles create a local depot that reprograms the
microenvironment of lymph nodes and trains differentiat-
ing T cells to become regulatory. In one noteworthy study, a
single lymph-node treatment permanently reversed disease in
a mouse model of MS (17).
An important opportunity arising from such approaches
is the ability to create therapies with vaccine-like specificity;
such therapies could control autoimmune disease without
compromising the patient’s immune system — a common
side effect of existing therapies.
Other particle-based strategies under development
could change how self-antigens are processed to selectively
restore tolerance (18–21). These approaches involve deliv-
ering the self-antigen, in distinct particulate forms, to APCs
expressing scavenger receptors or other features associ-
ated with clearance of naturally dying self-cells (e.g., red
blood cells). Nano­particles are being used to co-opt natural
regulatory pathways — an important theme in the immune
engineering field. Several new studies in this area seek to
define the specific design features needed for tolerance.
In one study, monodispersed quantum dots decorated
with self-peptides were delivered to mouse cells while the
MS was active (i.e., mouse model of MS). The researchers
found that delivering many quantum dots, each with a low
density of peptide, is more effective in promoting tolerance
than delivery of fewer higher-peptide-density quantum dots
(Figure 4) (20). Santamaria and his group have devel-
Control Over Presentation of Antigen to Alter
Processing and Immune Cell Differentiation
vs.
High Density, Few Carriers Low Density, Many Carriers
p Figure 4. Researchers found that delivering many quantum dots, each with
a low density of peptide, is more effective in promoting tolerance than delivery
with fewer quantum dots, each displaying a high density of peptide coverage.
CEP  October 2017  www.aiche.org/cep  35
SBE SPECIAL SECTION:
BIOMOLECULAR ENGINEERING
oped an approach to directly control how a self-antigen is
presented to T cells using iron nanoparticles displaying self-
peptides in natural antigen presentating protein complexes
(22). Research has revealed that the dose of self-antigen
presentating complexes determines the extent of expansion
of antigen-specific T cells, while the density of these com-
plexes determines how effectively the differentiation of
T cells is directed toward regulatory T cells (23). Funda-
mental studies that provide this level of molecular insight
will be increasingly important to support the rational and
efficient design of more complex therapies.
An important emerging trend in the fight against cancer
is the use of biomaterials to create candidates for personal-
ized cancer vaccines (Figure 5). Kuai and colleagues have
designed a cancer vaccine from nanoscale discs, which
are composed of synthetic high-density lipoproteins and
decorated with stimulatory adjuvants and multiple peptide
tumor antigens (24). These cancer vaccines effectively
expanded the number of T cells that targeted model tumor
peptides in mice, and could also be assembled using differ-
ent tumor peptides identified through sequencing of tumor
cells from individual mice. In two challenging mouse can-
cer models, personalized nanodiscs were delivered in com-
bination with gold-standard immunotherapies. Strikingly,
the treatment produced complete tumor regression (i.e.,
no tumor present), whereas treatment with the immuno­
therapies and a soluble version of the vaccine produced
less than 50% regression.
The ability to use biomaterials as flexible platforms for
quickly customizing cancer vaccines and immunotherapies
(e.g., by simply switching peptide antigens or other com-
Modular Platforms for Personalized Medicine
Antigen
Adjuvant with
with Linker Cholesterol
Nanodisc Personalized Vaccine
p Figure 5. Biomaterials are being used to create candidates for personalized
cancer vaccines.
Recruitment of Immune Cells to Scaffolds for
Engineering of Cell Function or Differentiation
T B T B
DC DC
From Tissue Scaffold with Signals for Back to Tissue
and Blood Recruitment and Modulation and Blood
p Figure 6. Scaffolds can be implanted in the body to recruit immune cells.
36  www.aiche.org/cep  October 2017  CEP
ponents), along with the ability to co-deliver defined ratios
of immune components, has significant implications for
personalized medicine.
Implanted scaffolds recruit
immune cells and program responses
Scaffolds can be implanted in the body to recruit
immune cells and act as a classroom of sorts. This is similar
in some ways to lymph nodes or the spleen, where innate
and adaptive immune cells interact (like classmates) to
promote a desired function or phenotype (Figure 6). Recent
studies have revealed the role of adaptive immune cells
in responding to implants (25) and key immune signaling
pathways that help determine how the body will respond to
polymers or other foreign bodies (26).
One early example of engineered scaffolds to promote
and control the immune response involved a porous PLGA
matrix loaded with material obtained from killed tumor
cells, a molecule to recruit immune cells, and a stimulatory
DNA molecule as an adjuvant. In mice, the scaffold success-
fully recruited DCs to capture the tumor cell fragments (i.e.,
antigens); these DCs subsequently migrated to the lymph
nodes and initiated T-cell responses that conferred prolonged
immunity against tumor models that are often fatal (27).
More recently, researchers have prepared spontane-
ously assembling scaffolds with larger pores to recruit
innate immune cells (28). Recruitment was largest in scaf-
folds assembled with the greatest aspect ratios, which cor-
related to larger pore sizes to accommodate the recruited
cells. Moreover, scaffolds loaded with a model antigen
and other immune signals were able to release these com-
ponents in a controlled manner over a period of weeks. In
mice with tumors expressing the model antigen, 90% of
vaccinated mice were still alive when all untreated mice
had succumbed.
This example demonstrates the integration of multiple
emerging findings from the immune engineering field to
provide better control over immune cell recruitment and
function. Other research teams are using scaffolds to directly
deliver and sustain, rather than recruit, immune cells for
anti-tumor activity — an approach that combines materials
with the idea of adoptive cell therapies (29).
With a goal of restraining immune function, rather
than activating it, one recent approach sought to treat
auto­immunity in a mouse model of Type I diabetes using
a layered scaffold constructed from a 5-mm dia. PLGA
disc (30). Before implantation, the scaffold was seeded
with functional pancreatic islet cells (i.e., cells attacked
during diabetes) to produce insulin and restore control of
blood glucose. However, immune tolerance is required to
prevent these new islet cells from meeting the same fate
as the original islets in the host mouse — destruction by
Copyright © 2017 American Institute of Chemical Engineers (AIChE)
malfunctioning self-reactive immune cells. Thus, the outer
layers of the disc were porous to support islet cell seeding,
while an inner layer of solid PLGA contained transforming
growth factor-beta 1 (TGF-β1). TGF-β1 reduces antigen
presentation by innate cells and promotes differentiation of
T cells into tolerogenic regulatory T cells.
Islets in the mice that received scaffolds loaded with
TGF-β1 had longer functional lifetimes and lower inflamma-
tory cytokine secretion than the mice that received scaffolds
without TGF-β1. This approach achieves control over local
inflammation at the site of transplantation, paving the way
for additional therapeutic vaccine strategies that deliver
other signals locally to control the immune environment in
and around transplant sites.
Microneedles deliver vaccines with local precision
Microneedle arrays are alternatives to hypodermic
needles that deliver cargo loaded in or on the surface of
dozens of polymeric microscale needles, each several
hundred microns high (Figure 7). At this size scale, drug
delivery is nearly painless because the microneedles reach
few pain receptors. Their small size also confers other
unique benefits. Microneedles can efficiently deliver cargo
intradermally to skin-resident immune cells. A recent
first-in-humans Phase I clinical trial demonstrated the use
of 650-µm dissolvable microneedle arrays to effectively
deliver the inactivated influenza vaccine (31). Of note, these
arrays can be stored in unrefrigerated conditions, self-
administered by patients, and discarded following use more
safely than traditional needles.
Researchers are also exploring the use of microneedles
for cancer vaccination and combination immunotherapies.
For example, Zhen Gu and colleagues at the Univ. of North
Carolina at Chapel Hill fabricated microneedle patches for
the sustained delivery of anti-PD1, an antibody that blocks
the immunosuppressive PD-1 pathway that tumors activate to
suppress anti-tumor response (32).
Another approach employs robotic automation to coat
microneedle arrays with up to 128 nanoscale layers juxta-
posing tumor antigens and stimulatory nucleic acid adju-
vants (33). In mice, this co-delivery promoted the expansion
of T cells specifically reactive to the tumor antigen. This
New Tools for Efficient Delivery to Immune Cells
with Improved Efficacy, Compliance, and Stability
Microneedle Patch for Self-Administration of Vaccines
p Figure 7. Microneedle arrays are alternatives to hypodermic needles.
Copyright © 2017 American Institute of Chemical Engineers (AIChE)
example of microneedle delivery of tumor antigens high-
lights the ability to combine features from different ends
of the design space — automated manufacturing and co-
delivery, for example — to control immune responses more
precisely. Together, the advances discussed in this section
show that microneedles are already finding clinical utility
in infectious disease, while holding exciting potential for
fighting cancer and autoimmune disorders through themes
of stimuli responsiveness, manufacturing advantages, and
co-delivery.
Self-assembly of immune cues
into structured materials to simplify design
Synthetic biomaterials and existing vaccine excipients
may affect the immune system in ways that are complex or
difficult to characterize. The intrinsic immunostimulatory
effects of biomaterials discussed previously represent one
important example, since the carrier itself can impact the
response of the immune system to the other vaccine com-
ponents. Furthermore, avoiding unanticipated inflammation
is especially important in autoimmunity — where inflam-
mation might make the disease worse — and in avoiding
off-target effects of immunotherapies. Thus, materials made
entirely of well-defined immune signals with attractive
features of biomaterials, such as co-delivery and tunable
loading, provide an opportunity for more modular design
strategies that also eliminate confounding intrinsic effects.
Self-assembly is well-suited for this goal, owing to sponta-
neous, molecularly defined processes.
Our group has developed an approach that involves
electro­static self-assembly of immunological polyelectrolytes
such as peptide antigens and immune-modulating nucleic
acids on a dissolvable particle template (Figure 8) (34).
The template is subsequently removed to create carrier-free
immune polyelectrolyte multilayer (iPEM) capsules. The size
of the iPEM capsules can be tuned, the absolute and relative
loadings of the antigens and nucleic acids can be controlled,
and multiple components can be delivered.
In mice, iPEM capsules containing model tumor antigens
and a stimulatory nucleic acid confered anti-tumor immu-
Improved Rational Design through Simplification
and Self-Assembly of Immune Signals
---
-- -- - --- - -
+++++ +
+ +++ + - --
- -
Peptide Stimulatory or Regulatory Carrier-Free
Antigen Nucleic Acid Ligands iPEM Capsules
p Figure 8. Immunological polyelectrolytes such as peptide antigens and
immune-modulating nucleic acids can be electrostaticly self-assembled on a
dissolvable particle template. The template is subsequently removed to create
carrier-free immune polyelectrolyte multilayer (iPEM) capsules.
CEP  October 2017  www.aiche.org/cep  37
SBE SPECIAL SECTION:
BIOMOLECULAR ENGINEERING
nity. By exploiting the modularity of this platform, we have
extended this system to autoimmunity (35). The iPEMs
built from myelin peptide and a regulatory nucleic acid that
blocks distinct inflammatory signaling pathways eliminated
disease in a mouse model of MS. These iPEM capsules also
reduced inflammatory cytokines in samples from human MS
patients, while iPEMs formed from myelin and scrambled
nucleic acid ligands did not.
Self-assembled immunological cues can also borrow
directly from pathogens. Virus-like particles (VLPs) are
constructed from protein subunits of viruses, but they do
not include components that can make viruses infectious.
These VLPs rely on the intrinsic immunogenicity of viral
proteins and can be harnessed in the context of cancer
vaccination to combat cancer by efficiently co-delivering
tumor antigens in the VLP.
VLP vaccines are sometimes ineffective if the patient
has previously been exposed to the virus proteins, because
immunity is not directed against the cancer antigen. Several
research groups are investigating the use of viral proteins
that humans do not commonly encounter, such as those
derived from bacteriophages (36) and plant viruses (37). In
one example, a carbohydrate tumor antigen that is normally
a difficult target for the immune system was attached to
the surface of a bacteriophage subunit (36), and in another
example, the antigen was attached to a plant virus particle
carrier to substantially enhance survival in a mouse model
of lymphoma (37).
Finally, Hudalla, et al., engineered protein moieties that
can self-assemble into nanofibers while exposing mul-
tiple different proteins along the length of the fibers (38).
This approach was successful in inducing high antibody
responses against the engineered protein fibers.
Although all of these strategies for self-assembly of
immune signals are still in the preclinical phase, they
represent an exciting frontier for exerting exquisite control
over immune function without the worry of unanticipated
effects of excipients.
Outlook
The complexity of the immune system and the diver-
sity of biomaterials available for vaccine development are
enormous. Yet, we are only in the early stages of understand-
ing how materials can be engineered in a rational way to
control specific functions of immune cells and tissues; this
is the same trial-and-error challenge facing the vaccine and
immunotherapy field more broadly. Understanding these
fundamental interactions will be important in enabling a
new generation of materials that actively program immune
response. Interdisciplinary partnerships are also needed to
combine immunology expertise with engineering expertise
in materials design and characterization. In addition, the
38  www.aiche.org/cep  October 2017  CEP
The complexity of the immune system and
the diversity of biomaterials available for
vaccine development are enormous.
experience of process and quality engineers will be vital
because many of the strategies employed in preclinical stud-
ies will require significant resources to scale up in a manner
that meets applicable regulatory standards for human use.
Perhaps the greatest challenge for those working on
biomaterial vaccines is the balance between understanding
immunological mechanisms and eliciting a prophylactic
or therapeutic benefit. Do we need to characterize new and
existing adjuvants more fully before we champion their
use? Or, should striking results that shrink tumors or reverse
autoimmune symptoms be a focus, even with unchecked
cost or inefficient processes? Of course, the answers to these
questions will need to equilibrate somewhere in between;
more rigorous analysis in basic studies, in translation, and
in manufacturing will be useful in assessing the risks and
rewards of new therapeutic vaccines.
Nevertheless, materials-based vaccination and immuno­
therapies are breaking new ground in treating pathogenic
and noninfectious disease, and we will continue to learn
more about the exciting potential and most prominent chal-
lenges in the coming years CEP
NEIL DOLD is a graduate fellow in the Fischell
Dept. of Bioengineering at the Univ.
of Maryland, College Park (neildold@
umd.edu). He received a BS in biomedical
engineering at Bucknell Univ. in 2012 and
worked in the medical device industry for
several years before beginning his graduate
work in Christopher Jewell’s lab in 2015. His
graduate research explores the ability of
polymers and self-assembled constructs to
enhance immune responses in the context of cancer. He is supported by
an NIH Graduate Fellowship from the National Cancer Institute.
CHRISTOPHER M. JEWELL, PhD, is an associate
professor in the Fischell Dept. of Bio­
engineering at the Univ. of Maryland and a
Damon Runyon-Rachleff Innovator (Email:
cmjewell@umd.edu). He is an associate
scientific advisor for Science Translational
Medicine and holds appointments as a
non-clinical scientist at the Baltimore
Veterans Administration and as a full
member of Greenbaum Cancer Center. He
has published more than 60 papers and patents using materials to study
and control immune function, and to enable drug and gene delivery. He
has received many awards for research and education, including the
NSF CAREER Award, and was selected as a 2016 Cellular and Molecular
Bioengineering Young Innovator, and as a Young Investigator of the
Melanoma Research Alliance and the Alliance for Cancer Gene Therapy.
He was selected as the state of Maryland’s Outstanding Young Engineer
by the Maryland Academy of Science, the state’s highest honor awarded
to an engineer under age 36. After his doctoral work, Dr. Jewell worked at
the Boston Consulting Group before completing his postgraduate train-
ing at MIT and Harvard.
Copyright © 2017 American Institute of Chemical Engineers (AIChE)
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CEP  October 2017  www.aiche.org/cep  39
SBE SPECIAL SECTION:
BIOMOLECULAR ENGINEERING

Engineering
Cancer-Fighting T Cells
Eugenia Zah Adoptive T-cell therapy is a personalized cancer
Yvonne Y. Chen
Univ. of California, Los Angeles treatment that has the potential to cure previously
untreatable malignancies. Researchers are applying
techniques in biomolecular engineering and
synthetic biology to the development of
next-generation therapeutic T cells with improved
safety and efficacy against a variety of cancers.

A
doptive T-cell therapy (ACT) is a new treatment
that holds promise for the safe and effective treat-
ment of cancer. Unlike traditional thera­peutics
such as chemotherapy, radiation, and surgery, ACT har-
nesses a patient’s own immune cells as a living drug to
treat previously incurable cancers.
The treatment involves tumor-targeting T cells (a type
of white blood cell) that are obtained by either isolating
T cells that naturally target tumors or genetically modify-
ing T cells to express a transgenic tumor-targeting receptor.
These receptors could be natural T-cell receptors (TCRs)
or synthetic receptors known as chimeric antigen receptors
(CARs). The tumor-targeting T cells are then expanded and
reinfused into the patient (Figure 1).
The leading ACT candidates are T cells that have been
engineered to express CARs targeting CD19, which is a
pan-B-cell marker found on the majority of cancerous B cells.
In multiple clinical trials, 90% of advanced B-cell leukemia
patients treated with CD19 CAR-T cells experienced com-
plete remission (1). These results garnered immense excite-
ment among researchers and the general public.
In July 2017, the first CD19 CAR-T cell product,
CTL019 (tisagenlecleucel) from Novartis, received unani-
mous support from the U.S. Food and Drug Administration
(FDA) Oncologic Drugs Advisory Committee for approval
to treat B-cell acute lymphoblastic leukemia (B-ALL) in
pediatric and young-adult patients (2). Several companies
are now engaged in fierce competition to bring ACT to
the market. This drive toward commercialization of ACT
provides ample opportunities for chemical and biomolecular
engineers to apply their expertise to the manufacturing and
development of cellular products.
This article provides an overview of ACT cancer
treatment and highlights the major milestones in this new
therapy’s development. It identifies limitations and chal-
lenges associated with ACT, as well as strategies under
consideration and developments that aim to address such
challenges. Finally, it explores opportunities for engineers to
make a difference in this burgeoning field.
Commercialization of ACT
In the past five years, several companies specializing in
ACT as a novel cancer treatment have emerged. According
to Coherent Market Insights, the global CAR-T cell mar-
ket is valued at approximately $72 million in 2017 and is
expected to reach $8.5 billion by 2028 (3).
Three companies at the forefront of the ACT market —
Juno Therapeutics, Novartis, and Kite-Pharma — have all
received “breakthrough therapy” designation by the FDA
for their CD19 CAR-T cell products (4, 5). Juno Therapeu-
tics’ JCAR015 therapy received breakthrough designation
in 2014, but it experienced a major setback after several
patients being treated with it died from cerebral edema (i.e.,
swelling of the brain) — forcing the company to terminate

40  www.aiche.org/cep  October 2017  CEP Copyright © 2017 American Institute of Chemical Engineers (AIChE)
its clinical trial in March 2017 (6). Since then, Kite Pharma
has also reported one patient death due to brain toxicity,
but the company remains optimistic, while Novartis has not
(yet) reported any fatalities (6). Currently, Novartis and Kite
Pharma are competing to become the first to successfully
commercialize their drugs, CTL019 and KTE-C19, respec-
tively. Both Novartis and Kite Pharma expect to receive
FDA approval for their drugs in 2017.
Although ACT has had substantial clinical success
in the treatment of B-cell leukemia and lymphoma, signifi-
cant concerns about the safety and efficacy of this novel
therapy remain to be addressed. The development of new
tools in biomolecular engineering and synthetic biology
has enabled the engineering of T cells with safeguards
against toxic and potentially deadly side effects of the
treatment. In addition, T cells with better anti-tumor capa-
bilities are being developed for the treatment of cancers
beyond B-cell malignancies.
ACT: A personalized treatment for cancer
Conventional T cells play a central role in the adaptive
immune system and are naturally capable of detecting and
killing bacterially and virally infected cells, making them
ideal candidates for cell-based immunotherapy. In ACT, the
T cells’ cytotoxic capabilities are redirected against tumor
cells expressing specific antigens (7). This redirection is
accomplished by either naturally occurring, tumor-targeting
TCRs or by transgenic receptors that are introduced into
T cells via genetic engineering.
Steven A. Rosenberg and his team at the National
Institutes of Health (NIH) were the first to demonstrate that
cell-based immunotherapy could be used to treat cancer.
They showed that the adoptive transfer of patient-derived,
tumor-infiltrating lymphocytes (TILs, or T cells with innate
tumor-recognition capabilities)
could effectively treat patients with
metastatic melanoma (8). Of the 93 Excise Tumor
melanoma patients — many with
severe tumor burdens and visceral
disease (characterized as stage IV
cancer) — who were treated with
TIL-ACT, 20 patients experienced T-Cell
complete regression, and 19 expe- Isolation by
rienced ongoing complete regres- Apheresis
tive against melanoma — a uniquely immunogenic cancer
where high numbers of TILs are present in the tumors — it
has not been as successfully or widely applied to other
types of cancer.
Engineering T cells to target cancer
To overcome limitations of TIL-ACT, scientists have
engineered T cells to express transgenic receptors, which
recognize tumor cells that are otherwise undetectable by the
immune system (Figure 1). In this strategy, peripheral T cells
are isolated from a patient and then genetically modified —
using viruses that encode the genes of interest — to express
receptors that recognize cancer-associated antigens. The
ability to convert any T cell into an anti-tumor effector cell
(i.e., lymphocytes able to eliminate tumor cells) significantly
broadens the applications of ACT for the treatment of other
cancers beyond melanoma.
T cells can be modified to express two types of receptors,
natural and synthetic. All T cells naturally express TCRs,
which recognize and bind to peptides (i.e., antigens) derived
from pathogens. These pathogen-derived peptides are dis-
played on the cell surface by major histocompatibility com-
plex (MHC) molecules; MHC molecules bind to the peptides
and then display them on the surface for TCR recognition.
All nucleated cells and platelets express MHC Class I mol-
ecules, which present fragments of cellular proteins on the
cell surface. Cells infected by bacteria or viruses can present
bacterial or viral protein fragments, thereby alerting T cells
to the presence of infection. Tumor cells that express mutated
proteins can similarly present tumor-associated protein frag-
ments via MHC molecules, thus allowing T cells that express
cognate TCRs to recognize tumor cells. The binding of TCRs
to the peptide-MHC complex on tumor cells activates the
T cells and triggers an anti-tumor immune response.
Lymphodepleting
Therapy
TIL Isolation TIL Expansion Infusion
TCR TCR-T Cells
Viral
Transduction
Expansion
CAR
and Infusion

sions 3–7 years after treatment (9). Virus Encodes

Tumor-Antigen-
An important pre­requisite for Specific Receptor CAR-T Cells
TIL-based therapy, however, is
that the patient’s own immune sys- p Figure 1. ACT uses autologous tumor-infiltrating lymphocytes (TILs) or T cells expressing a tumor-targeting
tem must contain tumor-targeting T-cell receptor (TCR) or a chimeric antigen receptor (CAR). In TIL-ACT, TILs are isolated from excised tumors
and subsequently expanded. In TCR/CAR-ACT, peripheral T cells are isolated from patient blood, genetically
T cells that can be isolated and
modified to express either a TCR or a CAR molecule, and subsequently expanded. Prior to T-cell reinfusion,
expanded from resected tumors. patients typically undergo a lymphodepleting regimen (chemotherapy that depletes existing lymphocytes in the
While TIL-ACT has been effec- patient), which has been demonstrated to improve ACT’s treatment efficacy.
Article continues on next page

Copyright © 2017 American Institute of Chemical Engineers (AIChE) CEP  October 2017  www.aiche.org/cep  41
SBE SPECIAL SECTION:
BIOMOLECULAR ENGINEERING
Scientists have identified genes that encode TCRs to
target a variety of cancer antigens (9). In an early example,
adoptive transfer of T cells that were genetically modi-
fied to express a TCR that targets melanoma-associated
antigen 1 (MART-1) induced the regression of advanced
melanoma (10). TCR-ACT has also been applied in the
treatment of other epithelial cancers (9).
Despite its therapeutic potential, however, TCR-ACT
still has several limitations. First, TCRs can only recognize
tumor cells via peptides presented in the MHC complex. To
avoid detection by TCRs, tumor cells can downregulate (i.e.,
reduce) their surface MHC expression, thereby rendering the
TCR-ACT treatment ineffective. Furthermore, MHC exists
in several different subtypes (known as human leukocyte
antigens, or HLAs) in humans, and the specific subtypes
expressed vary among individuals. TCRs must be matched
to specific HLA subtypes, so different TCR-based treatments
would need to be developed to match each patient’s HLA
type. Finally, identifying high-affinity TCRs for cancer-
associated antigens is still a challenge. Because most cancer
types are not caused by infectious agents, tumor cells fre-
quently do not express non-self antigens that can be recog-
nized by TCRs — often making it impossible to isolate TCRs
that specifically recognize tumor cells.
The development of CARs has overcome some of the
major limitations of TCR-ACT. CARs were first conceptual-
ized in the late-1980s by Zelig Eshhar, an immunologist at
the Weizmann Institute of Science. The first CARs, which
were engineered to treat malignancies including ovarian and
renal cancer as well as neuroblastoma, achieved little clinical
success (11). It took more than 20 years to optimize CAR
molecules for therapeutic applications (see timeline below).
CARs are modular fusion proteins that can, in prin-
ciple, be engineered to target any tumor antigen of inter-
est. Importantly, CARs are capable of binding to antigens
independent of MHCs. The CAR molecule consists of an
extracellular ligand-binding domain (most commonly an
antibody-derived single-chain variable fragment, or scFv)
fused to an extracellular spacer, transmembrane domain,
and one or more cytoplasmic signaling domain(s) that gen-
erally include the CD3z chain (12, 13). Second-generation
CARs, which contain a co-stimulatory domain in addition
to the CD3z chain, have been most effective in clinical
trials thus far. Specifically, CAR-ACT utilizing T cells that
1976 1988
Identification of T-cell growth Earliest trials of ACT using TILs
factor interleukin-2 (IL-2) enables for the treatment of metastatic
culture of T cells in vitro melanoma
42  www.aiche.org/cep  October 2017  CEP
target the pan-B-cell marker CD19 has achieved remarkable
clinical success in the treatment of advanced B-cell malig-
nancies (7). Multiple clinical trials have reported >80%
response rate to CD19 CAR-ACT in patients with relapsed
B-ALL and a 50–80% response rate in patients with refrac-
tory B-cell lymphoma (1). Several obstacles limiting the
widespread implementation of CAR-ACT, however, still
need to be addressed.
Strategies to improve the efficacy of CAR-ACT
CARs are single-input receptors, i.e., they identify target
cells based on the expression of one particular antigen. In
the presence of CAR-T cells, tumors face a strong selective
pressure that drives them to downregulate surface expression
of that cognate antigen. This process by which tumor cells
becomes undetectable to CAR-T cells via antigen down­
regulation is termed antigen escape, and it has been observed
as a frequent cause of cancer relapse in patients treated with
CAR-ACT. In CD19 CAR clinical trials for the treatment
of B-ALL, several patients who initially achieved com-
plete remission eventually relapsed due to the outgrowth of
CD19-negative tumors (14, 15).
Tumor antigen escape. A potential solution to antigen
escape is to engineer receptors to target multiple antigens
such that tumor cells would need to downregulate more
than one surface antigen to successfully avoid detection.
To combat the problem of antigen escape with CD19 CAR,
researchers have engineered T cells to express two recep-
tors targeting different antigens in one cell, e.g., CD19 CAR
paired with CD123, CD22, or receptor tyrosine kinase-like
orphan receptor 1 (ROR1)-specific CARs (16).
Alternatively, scientists have generated single-chain bispe-
cific CARs by fusing an additional scFv to the extracellular
end of the second-generation CAR (17). A single-chain recep-
tor is more genetically compact than a double receptor and
can be integrated into T cells more efficiently. The feasibility
of this approach was first demonstrated in the construction of
a bispecific CAR targeting human epidermal growth factor
receptor 2 (HER2) and CD19. Subsequently, our group and
others have engineered clinically relevant CD19/CD20 and
CD19/CD22 bispecific CARs to prevent antigen escape in
the treatment of B-cell malignancies (18, 19). In addition, sci-
entists have developed a HER2/IL13Rα2 receptor to prevent
antigen escape in glioblastoma (16).
1989 2002
Construction of first Melanoma patients
chimeric receptors treated with TIL-ACT
for T cells with exhibit complete
antibody-directed durable responses
specificity
Copyright © 2017 American Institute of Chemical Engineers (AIChE)
 Tumor microenvironment. Despite the clinical success of
CAR-ACT against B-cell cancers, multiple obstacles have
limited the success of CAR-ACT in the treatment of solid
tumors. First, solid malignancies may reside in areas of the
body that are difficult for CAR-T cells to access. For example,
for cancers of the brain and spinal cord, CAR-T cells must be
able to cross the blood-brain barrier to reach the tumor site. In
addition, solid tumors are often characterized by an immuno-
suppressive micro­environment in which inhibitory cytokines
such as transforming growth factor beta (TGF-β) and inter-
leukin (IL)-10 are present at high levels, thus inactivating any
immune cells that may reach the tumor site (20). Furthermore,
tumor cells can express inhibitory ligands on their surfaces to
impede the immune response and induce T-cell exhaustion.
T-cell homing, function, and persistence. Various strate-
gies to improve T-cell homing, function, and persistence
in the tumor microenvironment have been developed. For
example, T cells that express transgenic chemokine recep-
tors have been engineered to improve T-cell homing to
tumor sites; clinical trials to evaluate the expression of the
chemokine C-X-C motif receptor 2 (CXCR2) and nerve
growth factor receptor (NGFR) in TILs for ACT are in
progress (20). The overexpression of co-stimulatory ligands,
such as CD80 and 4-1BBL, can also enhance the expansion
and persistence of engineered T cells (12). (Two signals —
antigen-specific and non-antigen-specific — are required
to activate T cells. Co-stimulatory molecules provide the
non-antigen-specific signal.) Finally, T cells can be armed to
secrete proinflammatory or proliferative cytokines, such as
IL-2, IL-7, IL-15, and IL-21, to aid the anti-tumor response.
In addition to enhancing the primary (effector) function
of T cells to destroy tumor cells, the therapeutic efficacy
of T cells can be improved by enabling T cells to resist
defense mechanisms employed by tumor cells. For exam-
ple, a dominant-negative TGF-β receptor (DNR) has been
developed to reduce T-cell susceptibility to the immuno­
suppressive effects of tumor-secreted TGF-β. TGF-β is a
cytokine that regulates cell growth and differentiation. In
the context of solid tumors, TGF-β is known to be a potent
immunosuppressant that inhibits T-cell growth and effector
functions. The DNR is a truncated version of the TGF-β
receptor chain 2 that is missing its cytoplasmic signaling
domains. The expression of DNR can reduce T-cell suscep-
tibility to TGF-β-mediated growth inhibition because the
2006 2010
ACT clinical trial using First report of CD19
genetically modified T CAR-ACT successfully
cells (MART-1 TCR) treating a B-cell
showed partial lymphoma patient, who
response in 2 out of had partial remission
15 patients following therapy
Copyright © 2017 American Institute of Chemical Engineers (AIChE)
receptor would still bind to TGF-β but it would not signal.
As another example, a synthetic IL-4/IL-7 fusion recep-
tor was made by fusing the extracellular domain of the
anti-proliferative IL-4 receptor to the cytoplasmic signaling
domain of the proliferative IL-7 cytokine signaling domain.
This receptor converted an inhibitory input (i.e., binding of
IL-4) to an activating output (i.e., IL-7 signaling) (20). The
expression of this receptor was shown to improve the prolif-
erative capacity of T cells in IL-4-rich environments.
T-cell exhaustion. Recently, T-cell exhaustion has emerged
as a major factor limiting the efficacy of ACT. Chronic
exposure to antigen stimulation can exhaust T cells, leav-
ing them unable to mount an effective anti-tumor response.
Checkpoint inhibitors, such as cytotoxic T lymphocyte-
associated antigen 4 (CTLA-4) and programmed cell death
protein 1 (PD-1), play important roles in regulating the
immune response to prevent T-cell overstimulation, but they
can also contribute to T-cell exhaustion (20). The binding
of these receptors to their ligands — CD80 and CD86 for
CTLA-4, and programmed death ligand 1(PD-L1) for PD-1
— triggers downstream signaling events that inhibit T-cell
function. Consequently, the increased expression of CTLA-4
and PD-1 during T-cell activation, as well as the upregulated
expression of PD-L1 on tumor cells, can substantially worsen
T-cell function in the tumor microenvironment.
Monoclonal antibodies that block the interaction between
inhibitory receptors and their ligands, known as checkpoint-
inhibitor therapies, have shown promise in improving the
potency of cancer treatments. For example, the FDA has
approved CTLA-4 inhibitor ipilimumab and PD-1 inhibitor
pembrolizumab for the treatment of melanoma (20).
However, while clinically effective, checkpoint-inhibitor
therapies can also cause toxic side effects, because the
antibodies can interfere with the regulation of other immune
cells in the body, not just the tumor-targeting T cells (21). To
improve safety associated with checkpoint inhibiting thera-
pies, new gene-editing technologies have enabled scientists
to selectively knock out PD-1 or CTLA-4 in T cells so that
checkpoint blocking is specific to the tumor-targeting cells.
The safety of PD-1 knockout T-cell therapy is currently
being evaluated under clinical conditions.
Finally, a major obstacle limiting applications of ACT
is the lack of suitable tumor-specific antigens to target (22).
Since cancer cells are derived from healthy tissue, finding
2014 2017
CD19 CAR-ACT receives Termination of clinical trial
FDA breakthrough status evaluating JCAR015 (Juno
recognition Therapeutics)
CEP  October 2017  www.aiche.org/cep  43
SBE SPECIAL SECTION:
BIOMOLECULAR ENGINEERING

tumor-exclusive antigens is a challenge. One strategy is to Strategies to improve safety of CAR-ACT

target antigens that are expressed on cancer cells as well as
nonessential cells. For example, the CD19 CAR recognizes
CD19 antigens expressed on both cancerous and healthy
B cells. As a consequence, one common side effect of CD19
CAR-ACT is B-cell aplasia, or the depletion of healthy
B cells. Fortunately, B-cell aplasia can be clinically man-
aged with the periodic administration of immunoglobulin.
However, such on-target, off-tumor toxicities limit the pool
of potential targets to a very small number of antigens whose
off-tumor targets are nonessential to human survival.
Another approach is to target antigens that are over­
expressed on cancer cells (e.g., MART-1, carcinoembryonic
antigen (CEA), and HER2). This approach, however, can
cause severe toxicity problems, since T cells can mount pow-
erful attacks even on target cells with low antigen expression
levels (22). The deadly side effects of on-target, off-tumor tox-
icity have been observed in clinical trials of the HER2 CAR
and CEA-targeting TCR. In the case of the HER2 CAR trials,
patients suffered from respiratory stress when CAR-T cells
targeted lung epithelial cells that expressed low levels of
HER2 (5). Patients treated with T cells expressing a transgenic
CEA TCR developed severe colitis when the adoptively trans-
ferred T cells attacked cells in the colon (21).
A potentially safer alternative is to target antigens that
are unique to cancers. These include cancer-specific muta-
tions that give rise to neoantigens, cancer-testes antigens
(which are restricted to cancer cells and cells in early devel-
opmental stages), viral antigens on cancers with viral ori-
gins, or components of the tumor stroma. However, it is still
a challenge to identify TCRs and to design CAR molecules
that can effectively recognize these proteins.
Safety remains a critical concern as ACT marches
toward FDA approval and subsequent commercialization.
As mentioned earlier, a major safety concern is on-target,
off-tumor toxicity, which occurs when engineered T cells
designed to target cancer cells also kill normal, healthy
cells expressing the same antigen. Toxicity can also occur
when CAR-T cells cross-recognize incorrect-but-similar
antigens expressed on healthy cells. In a clinical trial to
evaluate T cells expressing a transgenic TCR that targets
melanoma-associated-antigen-3 (MAGE-A3), two patients
died from cardiac failure within 5 days of T-cell infusion;
subsequent analysis revealed that the TCR cross-reacted
with the protein titin, which is expressed in heart muscle
cells (21). In another trial that involved a different MAGE-
A3 TCR, patients experienced toxicity in the brain, high-
lighting the unpredictability of side effects associated with
imperfect T-cell specificity (11). Thus, there is an unmet
need for general strategies that can minimize toxicity
caused by imperfect tumor-targeting specificity.
In response to this challenge, scientists have engineered
next-generation CARs capable of multiple-input logic func-
tions to address on-target, off-tumor toxicity (Figure 2). One
strategy involves CARs engineered such that they are only
activated when they detect the correct combination of tumor
antigens. In another system, a CAR containing only the CD3z
signaling domain is paired with another CAR containing
only co-stimulatory chains (17). Each receptor recognizes a
different tumor-associated antigen, and only in the presence
of tumor cells that express both targeted antigens would both
signaling chains be activated and a maximum T-cell response
be observed (AND-gate signal integration). In this strategy,

Second-Generation CAR Next-Generation CARs

OR Gate AND Gate AND-NOT Gate

scFv#1 scFv#2

scFv#2 scFv#1 scFv#2 scFv#1 scFv#2

scFv 4-1BBcyto
Spacer Truncated
Tm CD3ζ
Notch
Co-Stim scFv#1
CD28cyto PD-1 or
CD3ζ Co-Stim CD3ζ Co-Stim
TF CTLA-4
CD3ζ 4-1BBcyto <>=
CAR CD3ζ
cyto

p Figure 2. The second-generation CAR, which has been the most successful of the CAR treatments in clinical trials, consists of a single-chain fragment
(scFv), extracellular spacer, transmembrane domain, and two cytoplasmic signaling domains (a co-stimulatory domain, typically cytoplasmic domains of
CD28 or 4-1BB fused to a CD3z chain). To improve CAR-T-cell efficacy, a second scFv is fused to the second-generation CAR structure — creating a single-
chain bispecific CAR with OR-gate logic function. To prevent off-tumor toxicity, researchers have developed receptors exhibiting AND-gate or AND-NOT logic
function. In one AND-gate strategy, a CAR containing only the CD3z chain is paired with a second receptor containing two co-stimulatory domains. In another
strategy, the first receptor is a synthetic notch receptor that releases a transcription factor when it binds to a cognate antigen. The released transcription
factor subsequently drives the expression of a second-generation CAR molecule, which can respond to a second antigen. In both cases, T cells are only
activated if they recognize two antigens. In the AND-NOT gate system, a second-generation CAR is paired with an inhibitory CAR that contains an scFv fused
to the signaling domains of PD-1 or CTLA-4. CAR signaling occurs only in the presence of a tumor-associated antigen in the absence of a healthy antigen.

44  www.aiche.org/cep  October 2017  CEP Copyright © 2017 American Institute of Chemical Engineers (AIChE)
the binding of one antigen is insufficient to fully activate the
CAR-T cell and elicit an immune response.
In an alternative AND-gate strategy, antigen binding to a
constitutively expressed synthetic Notch (synNotch) recep-
tor induces the expression of a CAR molecule (17). The
synNotch receptor consists of an extracellular scFv fused
to the Notch transmembrane domain, which is linked to a
cyto­plasmic transcription factor. The pulling force exerted by
the binding of surface antigens on the target cell to synNotch
receptors on the T cell causes the synNotch receptor to cleave.
During cleavage, the intracellular transcription factor is
released into the nucleus, where it drives the expression of a
CAR from a cognate promoter. The synNotch system requires
the sequential presentation of two antigens by target cells.
In a third example, a conventional CAR targeting a
tumor-associated antigen is paired with an inhibitory CAR
that triggers an overriding negative signal when bound to
an antigen that is expressed on normal tissue (17). In such a
system, the T cells would not target healthy cells that express
both the tumor-associated antigen and an antigen charac-
teristic of normal tissue. In essence, the CAR-T cell would
perform an AND-NOT-gate signal computation through the
combination of conventional and inhibitory CARs.
In addition to safety issues related to targeting specificity,
cytokine release syndrome (CRS) has emerged as a major
side effect of ACT. CRS arises when transferred immune cells
become overstimulated and produce excessive amounts of
inflammatory cytokines, inducing high fever, organ failure,
and even death. Immunosuppressive agents such as cortico­
steroids can be used to control CRS, but these steroids can
also compromise the potency of ACT (11). More-targeted
inhibitors such as tocilizumab, a monoclonal antibody that
blocks IL-6 receptor alpha (IL-6Rα), can often control CRS
without suppressing the efficacy of transferred T cells, but the
ideal treatment window can be difficult to identify (11).
As an additional safety measure to prevent potential
adverse side effects of ACT, researchers have engineered
CAR-T cells to express suicide genes that can be induced to
rapidly eliminate transferred cells. Two examples of suicide
genes are herpes simplex virus thymidine kinase (HSV-TK)
and a synthetic, monomeric caspase-9 that can be activated by
the dimerization-inducing drug AP1903 (21). T cells express-
ing HSV-TK are susceptible to cell death via ganciclovir
administration. Similarly, exposure to AP1903 can induce
apoptosis in T cells that express the synthetic caspase-9.
AP1903 dimerizes caspase-9, which becomes active in the
dimeric form and can induce apoptosis of transferred T cells.
CAR-T cell manufacturing considerations
ACT is a quintessentially personalized cancer treat-
ment, since the T cells are typically isolated from the patient
undergoing treatment (23). The use of patient-derived, auto­
Copyright © 2017 American Institute of Chemical Engineers (AIChE)
logous T cells minimizes the risk of graft-versus-host disease
(GVHD) and rejection of transferred cells. However, the need
to generate a personalized product for each individual patient
also increases the complexity of this treatment strategy. To
qualify for ACT treatment, a patient must be able to provide
a sufficient number of isolated T cells. Consequently, auto­
logous ACT is limited to those patients who are able to pro-
duce enough T cells for modification. Furthermore, the need
to manufacture patient-specific products currently restricts
the availability of this therapy to specialized treatment cen-
ters, imposes significant costs, and subjects each patient to
potential variations in cell product quality.
Scientists have used gene-editing techniques to demon-
strate that knocking out one or both endogenous TCR chains
in donor T cells is an effective strategy for generating univer-
sal CAR-T cells that are less susceptible to GVHD toxicity
(21). Recently, donor-derived, TCR-knockout T cells were
used to treat two infants with relapsed leukemia, patients who
otherwise would have not been able to produce enough T cells
to undergo ACT treatment (24). However, both patients
eventually exhibited GVHD symptoms that were attributed to
incomplete TCR knockout in the infused cells, highlighting
this strategy’s remaining risks and room for improvement.
As CD19 CAR-T cells race toward FDA approval, the
development of cost-effective and robustly reproducible
means to manufacture the cells is of growing importance. To
support a successful commercial product, the ideal manufac-
turing process must not only yield consistently high-quality
products for each patient, but must also be sufficiently robust
and simple that cell production can be performed at local
EUGENIA ZAH is a PhD candidate in the Dept.
of Chemical and Biomolecular Engineer-
ing at the Univ. of California, Los Angeles
(Email: ezah@ucla.edu). After receiving
her BSE in chemical engineering from
Princeton Univ., she went on to pursue
her doctoral degree at UCLA under the
supervision of Yvonne Chen. In graduate
school, her research focuses on engineer-
ing next-generation chimeric antigen
receptors to improve the efficacy of adop-
tive T-cell therapy and developing robust
design-build-test platforms for new CARs.
YVONNE Y. CHEN, PhD, is an assistant professor
in the Dept. of Chemical and Biomolecular
Engineering at the Univ. of California, Los
Angeles (Email: yvchen@ucla.edu). Her
laboratory focuses on applying synthetic
biology and biomolecular engineering
techniques to the development of novel
mammalian cell systems, particularly for
cell-based cancer immunotherapy. She
received a BS from Stanford Univ. and a PhD
from the California Institute of Technology,
both in chemical engineering. She was a Junior Fellow at Harvard Univ.
and has been a recipient of the NIH Director’s Early Independence
Award, the Hellman Fellowship, the ACGT Young Investigator Award in
Cell and Gene Therapy for Cancer, and the NSF CAREER Award.
CEP  October 2017  www.aiche.org/cep  45
SBE SPECIAL SECTION:
BIOMOLECULAR ENGINEERING
medical centers, rather than only at a handful of special-
ized production sites that may be far away from the cancer
patient. In addition, the manufacturing process must be able
to produce treatments in a timely manner. This challenging
process-design problem presents exciting opportunities for
chemical and biomolecular engineers.
Closing thoughts
Engineers and scientists have hailed cell-based immuno-
therapy as the latest major breakthrough in cancer treatment.
However, the right balance between treatment safety and
efficacy needs to be achieved before widespread imple-
mentation of ACT can become a reality. Recent fatalities in
clinical trials by Juno Therapeutics and Kite Pharma serve as
reminders that ACT, despite its curative potential, can also
have severe and potentially deadly side effects. Therefore,
safety and efficacy concerns surrounding ACT still need to be
thoroughly addressed. The engineering strategies discussed
in this article highlight potential contributions that chemical
and biomolecular engineers can make in this exciting area of
Literature Cited
1. Fesnak, A. D., et al., “Engineered T Cells: The Promise and Chal-
lenges of Cancer Immunotherapy,” Nature Reviews Cancer, 16 (9),
pp. 566–581 (2016).
2. McGinley, L., “FDA Panel Recommends Approval of CAR T-Cell
Therapy,” Washington Post, www.washingtonpost.com/news/to-
your-health/wp/2017/07/12/novel-cancer-treatment-wins-endorse-
ment-of-fda-advisers/?utm_term=.1b05d093c063 (July 12, 2017).
3. Coherent Market Insights, “Global CAR T Cell Therapy Market,”
www.coherentmarketinsights.com/market-insight/car-t-cell-therapy-
market-102 (Feb. 2017).
4. Brower, V., “The CAR T-Cell Race,” The Scientist, www.the-
scientist.com/?articles.view/articleNo/42462/title/The-CAR-T-Cell-
Race/ (Apr. 1, 2015).
5. Chustecka, Z., “CAR T Cells Motoring to Market — By Next
Year?,” Medscape, www.medscape.com/viewarticle/872765#vp_1
(Dec. 5, 2016).
6. Adams, B., “Investors Spooked by Kite CAR-T Death, but Biotech
Remains Confident,” FierceBiotech, www.fiercebiotech.com/
biotech/investors-spooked-by-kite-car-t-death-but-biotech-remains-
confident (2017).
7. Rosenberg, S. A., “Raising the Bar: The Curative Potential of
Human Cancer Immunotherapy,” Science Translational Medicine,
4 (127), pp. 127–127 (2012).
8. Dudley, M. E., et al., “Cancer regression and Autoimmunity in
Patients after Clonal Repopulation with Antitumor Lymphocytes,”
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9. Rosenberg, S. A., “Cell Transfer Immunotherapy for Metastatic
Solid Cancer — What Clinicians Need to Know,” Nature Reviews
Clinical Oncology, 8 (10), pp. 577–585 (Aug. 2, 2011).
10. Morgan, R. A., et al., “Cancer Regression in Patients after Transfer
of Genetically Engineered Lymphocytes,” Science, 314 (5796),
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11. Johnson, L. A., and C. H. June, “Driving Gene-Engineered T Cell
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46  www.aiche.org/cep  October 2017  CEP
research and toward the clinical implementation of ACT.
ACT has generated much excitement and press coverage
because of its potential to provide a treatment for previ-
ously incurable malignancies, but its success so far has been
largely limited to B-cell malignancies. To improve therapeu-
tic potency against other cancer types, multiple clinical trials
are underway to evaluate the combination of ACT with other
cancer treatments, such as checkpoint-inhibitor therapies,
vaccines, radiation, and chemotherapy. Another approach is
to combine ACT with oncolytic viruses, which has shown
promise in preclinical tests. These strategies, however, must
be explored with caution, because toxicities may be exacer-
bated when treatments are combined.
Finally, it will be interesting to see how ACT continues to
evolve in the coming years with the development of new syn-
thetic biology and biomolecular engineering tools. Whether
ACT will be able to live up to the expectations set forth by the
public and the scientific community is a difficult, yet exciting,
challenge to which chemical and biomolecular engineers have
much to contribute. CEP
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for Cancer Therapy and Beyond,” Trends in Molecular Medicine,
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Sustained Remissions in Leukemia,” New England Journal of
Medicine, 371 (16), pp. 1507–1517 (2014).
15. Gardner, R. A., et al., “Intent-to-Treat Leukemia Remission by
CD19 CAR T Cells of Defined Formulation and Dose in Children
and Young Adults,” Blood, 129 (25), pp. 3322–3331 (2017).
16. Jaspers, J. E., and R. J. Brentjens, “Pharmacology and Therapeu-
tics Development of CAR T Cells Designed to Improve Antitumor
Efficacy and Safety,” Pharmacology and Therapeutics, 2 (2017).
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Immune Cells to Treat Cancer,” Cell, 168 (4), pp. 724–740 (2017).
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meric Antigen Receptors Prevent Antigen Escape by Malignant B
Cells,” Cancer Immunology Research, 4 (6), pp. 498–508 (2016).
19. Qin, H., et al., “Preclinical Development of Bispecific Chimeric
Antigen Receptor Targeting Both CD19 and CD22,” Blood,
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for Solid Malignancies,” Clinical Cancer Research, 21 (15),
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Line,” Science Translational Medicine, 7 (280), pp. 1–8 (2015).
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to Cancer Gene Therapy,” Cancer Gene Therapy, 21 (2), pp. 45–47
(2014).
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Translational Medicine, 9 (374) (2017).
Copyright © 2017 American Institute of Chemical Engineers (AIChE)
 SBE SPECIAL SECTION:
BIOMOLECULAR ENGINEERING

There’s Plenty of Room

at the Bottom of a Cell
Angela R. Wu Single-cell RNA sequencing is giving engineers
Lei Yu
Hong Kong Univ. of Science and scientists the ability to better understand and
and Technology manipulate the smallest unit of a living organism —
the single cell. This technique has already ushered
in breakthroughs in biology and medicine. However,
challenges and opportunities abound.

S
ingle-cell RNA sequencing (scRNA-seq) is a tech-
nique used to simultaneously profile all expressed
genes within a single cell (i.e., transcriptome-wide
profiling). It involves various biological, chemical, and
physical techniques to manipulate a minuscule amount of
RNA from a single cell. The ability to obtain comprehensive
gene-expression profiles at single-cell resolution has enabled
researchers to rapidly identify and characterize novel and
rare cell types, which in turn provides a foundation for the
development of new therapeutic avenues — revolutionizing
the pace of biological discovery and clinical innovation.
This article provides an overview of scRNA-seq technol-
ogy and discusses various techniques for performing this
type of sequencing. It also discusses emerging develop-
ments, opportunities, and challenges from an engineer’s
perspective throughout.
Genomics at single-cell resolution
In 1959, when Richard Feynman first gave his famous
lecture “Plenty of Room at the Bottom,” he conjectured that
if we could understand and manipulate matter at the atomic
scale, it would enable a myriad of previously unimaginable
discoveries and innovations. Today, similarly profound
advancements are happening in the life sciences field.
The single cell is the smallest functional unit of living
beings, and bioengineers and biologists have long studied
life at the single-cell level. But most approaches used in the
past to characterize single cells — from physical observa-
tions of single cells to single-neuron electrophysiology mea-
surements — have focused on just a few features, properties,
or genes. Cellular RNA levels, which is a well-accepted
proxy for cellular identity and behavior, could be measured
for a panel of chosen genes by techniques such as micro­
array analysis, but the ability to measure all gene expres-
sion in a cell and provide systems-level insights has, until
recently, remained elusive.
The arrival of next-generation nucleic-acid-sequencing
technologies drastically improved cellular readout (i.e.,
results) — from measuring selected gene targets to profiling
all genes, or producing so-called whole transcriptomes (i.e.,
maps of all RNA transcripts for a given sample) (1). Initially,
whole transcriptomes were generated from tissue samples,
with all of the cells in that tissue blended together into a
smoothie of sorts. But since each tissue in the body contains
a multitude of cell types, each with diverse functions, a pro-
file of a “tissue smoothie” is informative to a limited extent.
The recent breakthroughs in transcriptome amplification,
coupled with next-generation sequencing technology, culmi-
nated in the creation of highly sensitive whole-transcriptome
analysis methods for single cells (2, 3).
In the first published scRNA-seq study on mammalian
cells, Fuchou Tang and colleagues used scRNA-seq to
investigate mouse cells during early embryonic development
(4). The scientists used scRNA-seq to gain novel insights

Copyright © 2017 American Institute of Chemical Engineers (AIChE) CEP  October 2017  www.aiche.org/cep  47
SBE SPECIAL SECTION:
BIOMOLECULAR ENGINEERING
into the embryonic development process with unprecedented
clarity, and, in the process, showed that scRNA-seq can
overcome challenges related to analyzing small samples of
biological material. Developmental biology, especially early-
stage embryonic development, continues to be an area of
research in which scRNA-seq is proving to be an indispens-
able tool for discovery.
Cancer treatment is another area of research that is
benefiting greatly from scRNA-seq. Researchers have used
scRNA-seq to unequivocally demonstrate and investigate
the intratumoral heterogeneity in glioblastoma and colorectal
cancer — shedding light on previously unknown mecha-
nisms of cancer relapse and drug resistance, and exploring
fundamental questions such as the validity of the cancer
stem-cell model and the effects of immune interactions
within tumor microenvironments (5–8).
A third important application of scRNA-seq is in the dis-
covery and identification of new cell types, which has notably
given rise to the Human Cell Atlas Project — a consortium
established to characterize and catalog all distinct cell types
in the human body in healthy and various disease states (9).
The Human Cell Atlas Project, arguably the Human Genome
Project of this era, is poised to have an equal or even greater
impact on human health than its predecessor. The consortium
has already identified novel cell types in the intestines, lungs,
and many other tissue types, and has revised the taxonomy of
dendritic cells in the immune system (10–13).
Bulk RNA Single-Cell RNA
Sequencing Sequencing
Dissociation
Tissue
Homogenization
Single-Cell
Capture and
RNA-to-DNA
Conversion and Isolation
Amplification
RNA-to-DNA
Conversion and
Amplification
Sequencing
p Figure 1. Bulk RNA sequencing maps the genome from tissue samples
blended together into a bulk mixture. This method does not account for the
differences between cells within a single tissue. Single-cell sequencing, on
the other hand, sequences genetic material from one cell at a time, which
provides more information about the cells within tissue samples.
48  www.aiche.org/cep  October 2017  CEP
Many ways to skin a cat; many ways to sequence a cell
RNA sequencing, whether for millions of cells or a
single cell, follows a similar general procedure (Figure 1).
The main differences between the many-cells and
single-cell methods are in the sample preparation and the
RNA-to-DNA conversion chemistry. One of the challenges
in sequencing single cells is the limited quantity of available
RNA, which makes loss or damage of RNA during isolation
and extraction extremely detrimental to the results. Cell viabil-
ity is critical to the integrity of RNA-seq results, because cells
respond to stress with changes in gene expression, or worse,
RNA degrades if the cell begins to die. Therefore, dissociation
of tissue samples into single cells and isolation of these single
cells, without causing them too much trauma, are critical steps
to preserving RNA integrity in the scRNA-seq workflow.
Dissociation. A combination of mechanical agitation and
enzymatic digestion is typically used to release individual
cells from a tissue matrix. The unique composition and
structure of different tissue types make it essential to use tis-
sue dissociation protocols that are optimized for the sample
at hand; otherwise, cell viability and yield, and consequently
RNA-seq data quality, may suffer.
Single-cell isolation. After the sample is dissociated into
a single-cell suspension, individual cells can be isolated
using one of the many available techniques. These can be
classified into five types of methods (Figure 2): flow cyto­
metry or fluorescence-activated cell sorting (FACS)-based;
pipette- or capillary-based; laser-capture-microdissection
(LCM)-based; microfluidics-based; and microwell-based.
Each method type has benefits and downsides related to
versatility, labor-intensiveness, scale/throughput, and cost.
• Flow cytometry involves a complex system in which
cells in a suspended fluidic stream are sorted into different
tubes or wells based on the cell’s fluorescence intensity.
The speed and resolution of laser-based FACS systems are
sufficient to isolate individual cells. This type of method
is widely used because it can be combined with immuno­
staining or other fluorescent staining methods to selectively
isolate cells expressing certain biomarkers of interest and
discard cells that are not of interest (14, 15). Its ubiquity in
life science laboratories and its relatively low operating cost
makes this an easy method for biologists to adopt.
• Pipette/capillary-based methods are labor-intensive but
also versatile, as they can be adapted to almost any sample
type, even samples with a very low concentration of cells,
such as samples of early-stage embryos. For example, an
automated capillary isolation platform and special software
can be used to map a sample and direct a micropipette to pick
up the cells of interest. Mouth pipetting can be used to manu-
ally aspirate single cells one-by-one into tubes; archaic as this
method may seem, it offers an extremely high level of control
in picking the desired cell and has very low capital costs.
Copyright © 2017 American Institute of Chemical Engineers (AIChE)
 • LCM-based methods are similar to the pipette methods
in terms of labor intensity and versatility. This approach
uses a laser to isolate a single cell by ablating the bound-
ary between the cells. A built-in camera records the original
position of the isolated cell (16). One advantage of LCM
is that it can be applied without the cell-dissociation step,
which may be problematic in some cases. However, it has
limitations. It is not always possible to guarantee clean laser
dissection margins with this method, and LCM hardware is
expensive and cost-prohibitive for many laboratories.
• Microfluidics-based methods are burgeoning. These
offer the high-speed throughput of conventional automated
techniques in a miniaturized system. With this type of method,
the fluidic flow of the sample is controlled in a well-designed
device with micron-grade channels to isolate single cells. The
first commercial microfluidics device functioned by control-
ling microfabricated valves in soft polymer-based microchips,
thereby manipulating on-chip fluid flow to capture and isolate
single cells and achieve biochemical reactions in nanoliter-
volume chambers. Later, microfluidics droplet-based systems
emerged. These were designed to compartmentalize single
cells into thousands of nanoliter-sized aqueous droplets on
demand by adjusting the interaction between the aqueous
phase and the oil phase of the systems. Microfluidcs-based
platforms offer higher cellular throughput, on the order of
thousands of cells per experiment (17, 18). However, the
commercial platforms remain costly to buy and operate, and
the demanding requirements for chemistry and engineering
knowledge to build a system in-house make the barrier to
entry quite high for most biologists.
• Microwell-based single-cell separation technology
enables a few thousand cells to be analyzed simultaneously
in a simpler system than a microfluidics-based method. The
technique involves spreading the diluted cells into a care-
fully designed array of picoliter wells and allowing the cells
to randomly distribute by gravity (19, 20).
Cell lysis. Once individual cells have been separated and
isolated in their own compartments, whether those com-
partments are water-in-oil droplets, physical chambers in
a microchip, or microwells, the cells are broken apart (i.e.,
lysed) to release RNA.
RNA-to-DNA conversion. Next, the RNA is converted
into DNA and amplified by various biochemical methods to
produce large quantities of DNA. The Smart-seq2 protocol is
the most popular method, owing to the availability of easy-
to-use commercial kits, its straightforward biochemistry, and
the relatively short hands-on time required to use it. Smart-
seq2 amplification is a variation of polymerase chain reaction
(PCR), which amplifies DNA exponentially (21, 22). The
CEL-seq2 protocol uses an in vitro transcription chemistry,
which is quite different from PCR, and amplifies transcripts
linearly rather than exponentially, so less amplification bias is
Copyright © 2017 American Institute of Chemical Engineers (AIChE)
Barcode
Beads Oil
Pipette
Sample
Barcoded
Cells
ut Han
hp ds
ug -
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Dissociation
ro
nT
Th
ime
Mouth Pipette Microwells
Microfluidics FACS
Electro-
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Sample
Single Cells
p Figure 2. Several techniques are available to isolate single cells from the dis-
sociated cellular suspension. These include: fluorescence-activated cell sorting
(FACS); pipette- or capillary-based methods; microfluidics-based methods;
and microwells. Each method has benefits and downsides related to labor-
intensiveness and throughput. Source: Annual Reviews of Analytical Chemistry.
introduced into the transcriptome profile (23).
In addition to the myriad physical and biochemical
approaches that already exist, new scRNA-seq methods are
continually being adapted, providing versatility to vari-
ous applications, applicability to different tissue types, and
enabling different biological questions to be answered.
Example 1: Dissection of cellular regulation
CRISPR/Cas9-based genome editing technology is revo-
lutionizing scientists’ ability to efficiently change and probe
the relationship between genes, their function, and their
influence on cell behavior. In a CRISPR/Cas9 assay, dif-
ferent genes in a cell can be targeted and turned off simply
by changing the sequence of a guide RNA molecule in the
system. Several research groups have simultaneously tested
the function of thousands of different combinations of genes
by introducing different combinations of guide molecules to
individual cells (24–26). In their experimental setups, each
individual cell received a different combination of guide
RNA that turned off the corresponding combination of genes
via the CRISPR mechanism, and the response of each cell
to this genetic change was measured by scRNA-seq profil-
ing, which associates a transcriptome profile directly with a
genetic perturbation (Figure 3).
Each group profiled thousands of single cells using
microfluidic droplet-based methods to isolate the cells. In
one fell swoop, they identified genes that, when turned off,
affected cellular behaviors such as proliferation, drug resis-
CEP  October 2017  www.aiche.org/cep  49
SBE SPECIAL SECTION:
BIOMOLECULAR ENGINEERING
tance, and immune-function modulation. Without Cas9 or
scRNA-seq technology, such experiments would take years
of work using traditional genetic screening methods.
Example 2: Noninvasive cancer monitoring
Multiple myeloma (MM) is a cancerous growth of plasma
cells in the bone marrow. It remains incurable, and patients
suffering from MM often relapse, some more than once, and
eventually become resistant to therapy. Presently, patients
must undergo painful bone marrow biopsies to assess their
response to therapy and monitor for signs of resistance. But
since biopsies are invasive and carry high medical risk, they
cannot be performed regularly, thus relapse or emerging resis-
tance to therapy might not be detected until it is too late.
Recently, Lohr, et al., proposed the use of scRNA-seq
and single-cell DNA sequencing to profile the circulating
tumor cells (CTCs) isolated from patients’ blood as a pos-
sible replacement for bone marrow biopsying (27). They
performed genotyping and mutational analysis of single
CTCs, and compared the results to the same analysis of bulk
cells taken in a bone marrow biopsy. They found that the
mutations detected in each patient’s biopsy samples were
also detected in CTCs, and what’s more, the standard clinical
biopsy test missed mutations in two of three patients because
the biopsy did not yield enough material for a test. They then
used the scRNA-seq analysis for gene expression to verify
that the gene expression of CTCs was comparable to that of
the cells obtained by bone marrow biopsies, and they identi-
fied large chromosomal translocations, some of which are
important markers of clinical risk.
Although scRNA-seq is currently too costly for wide-
spread clinical use, Lohr, et al., demonstrated the techni-
cal feasibility of using a single-cell genomics approach to
achieve noninvasive, personalized monitoring of tumor
relapse and response to therapy for MM patients. With
efforts to reduce costs, it is foreseeable that in the future,
scRNA-seq could become a routine, life-saving clinical tool.
Guide RNA
Coding
Sequence Unique PolyA Tail
Unique PolyT Tail
Barcode Barcode
Guide RNA Cells
Cells Sorted into Droplets
p Figure 3. A library of guide RNAs, each tagged with a unique barcode that
targets specific genes, is introduced into a sample of suspended cells. Each cell
pairs with a guide RNA that turns off the corresponding combination of genes
via the CRISPR mechanism. Cells tagged with barcoded PolyT primers are then
sorted and isolated into droplets. ScrRNA-seq shows the cell’s response to the
genetic change (genes turned off). Source: George Retseck Illustration.
50  www.aiche.org/cep  October 2017  CEP
Challenges and opportunities abound
Several challenges and opportunities exist for the future
of scRNA-seq. Although it is useful and versatile, existing
scRNA-seq technology is limited in that it can only be used
to explore one aspect of cells — the cell’s RNA or gene
expression. The assay itself is destructive, so once the gene
expression of a cell is probed, the same cell can no longer
be probed for its protein expression or epigenetic profile.
One significant area of technology development in this field
will be the ability to simultaneously analyze multiple –omes
(e.g., genome, proteome, transcriptome, epigenome, and
metabolome) of a single cell (28, 29). For example, proto-
cols that simultaneously survey a single cell’s transcriptomic
and proteomic profile would allow scientists to identify
relationships between cellular identity and cellular behavior.
Cell-to-cell interaction is a critical facet of cellular behav-
ior that must be explored to understand the organ as a whole.
Existing scRNA-seq technology requires sample dissociation,
which eliminates the ability to determine spatial, cell-to-cell
information. Hence, a method that preserves spatial informa-
tion would advance the utility of scRNA-seq. Although a few
techniques have been developed that partially preserve spatial
context, such as the use of DNA barcodes in a spotted array to
label RNA with the approximate location within a tissue sec-
tion where they were found, the dearth of follow-up applica-
tions of these methods indicates the difficulty and limitation
of applying them (30, 31). We still lack a true single-cell,
scalable, spatially aware method for genomic profiling.
ANGELA R. WU, PhD, is an assistant professor
in the Div. of Life Science and the Dept. of
Chemical and Biological Engineering at
Hong Kong Univ. of Science and Technology
in Hong Kong (Clear Water Bay Rd., Clear
Water Bay, Kowloon, Hong Kong; Phone:
+852 3469-2577; Email: angelawu@ust.hk).
She is interested in developing enabling
technologies, such as microfluidic devices
and single-cell multiomic profiling methods,
to bridge gaps between engineering and life
sciences as well as to enable discoveries in
biology and medicine. Her research group collaborates with clinicians
to apply these new technologies in a translational setting. During
her postdoctoral work at Stanford Univ., she pioneered some of the
single-cell transcriptomics methodologies that are widely used today.
She is a founding member of the start-up company Agenovir Corp.,
based in South San Franscisco, CA. In 2016, she was named one of MIT
Technology Review’s Innovators Under 35 in Asia. She received a BS
in bioengineering from the Univ. of California, Berkeley, and an MS and
PhD in bioengineering from Stanford Univ.
LEI YU is a PhD candidate in the Div. of Life
Science at Hong Kong Univ. of Science and
Technology (Email: lyuah@ust.hk). After
completing his BS at Sun Yat-sen Univ.,
Guangzhou, China, in 2016, he started
his doctoral studies under the supervi-
sion of Angela Wu. His current single-cell
genomics research includes developing
new single-cell amplification methods and
applying those methods to further our
fundamental understanding of multiple myeloma.
Copyright © 2017 American Institute of Chemical Engineers (AIChE)
 Commercial single-cell genomics systems are still too
costly for most labs. Reducing the costs of single-cell isolation
and RNA processing remain opportunities for both business
and fundamental research. This will be particularly important
for clinical implementation of single-cell genomics.
Closing thoughts
As an emerging technique, single-cell genomics has
already provided exciting breakthroughs at the frontiers of
biology and medicine. Although technological challenges
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for certain desirable applications of scRNA-seq remain,
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plenty of room at the bottom for further understanding and
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blocks of life itself. CEP
of Lineage Fates and Cell Identity in Mid-gastrula Mouse Embryo,”
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Profiling of Individual Cells Using Nanoliter Droplets,” Cell, 161 (5),
pp. 1202–1214 (May 21, 2015).
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20. Fan, H. C., et al., “Combinatorial Labeling of Single Cells for
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scriptome Profiling in Single Cells,” Nature Methods, 10 (11),
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22. Picelli, S., et al., “Full-Length RNA-Seq from Single Cells Using
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CEP  October 2017  www.aiche.org/cep  51
SBE SPECIAL SECTION:
BIOMOLECULAR ENGINEERING

Computational
Protein Engineering
Robert Pantazes Proteins could transform the chemical process
Auburn Univ.
industries if they could be engineered to facilitate
the production of biofuels, molecular medicines,
and other specialty chemicals. This reality is nearing as
expanded knowledge of proteins and improvements
in computational tools are making it possible to design
specific protein structures and functions.

P
roteins are vital to life on Earth: They replicate and
repair DNA, ensuring the continuation of genetic
material. They form internal support structures for
cells and routes for intracellular transport of materials.
And, perhaps most importantly, they catalyze a vast array
of chemical reactions, speeding creation, organization, and
consumption of important molecules.
Proteins are the most common catalysts nature uses
to convert molecules from reactants to products. These
protein catalysts function at room temperature and pres-
sure and are often highly selective. Chemical engineers
are resourceful and creative and have devised numerous
methods of producing chemicals from feedstocks, yet
many processes using traditional catalysts require extreme
conditions that increase cost and reduce safety.
For many production processes, protein solutions have
the potential to be less expensive, safer, and more effective
than traditional catalysts. Although the utility of proteins
has been recognized for a long time, they are still far from
as prevalent as their potential suggests they could be. The
reason: It is often too difficult and expensive to engineer a
protein for a particular purpose.
Nearly all commercially meaningful efforts to engineer
proteins have relied on experimental methods, which are
limited to changing an existing protein. A sequence of
100 amino acids, for example, can be arranged into 20100
(~1.3×10130) proteins — significantly more variants than
the number of electrons in the universe (1). There are many
known proteins with more than 500 amino acids, making
the number of possible proteins exceedingly vast.
Nature has provided only a minuscule fraction of pos-
sible protein structures, and it is effectively impossible to
assemble a random protein with any function, let alone a
specific function. Because the requirements of industrial
applications are often far removed from natural conditions,
it may be impossible to identify a protein candidate that
can feasibly be modified.
Computational protein engineering overcomes some
of the limitations of experimental methods by exploring
protein folds not observed in nature and designing novel
proteins from scratch (i.e., de novo design). Designing
proteins at-will could be a powerful way to improve many
chemical engineering processes. Although computational
methods have had limited success, improved methods and
expanded computational resources are rapidly increasing
the areas in which these methods can be reliably applied.
This article discusses protein structures and computa-
tional engineering methods, provides examples to demon-
strate state-of-the-art technologies, and examines the future
of computational protein engineering.

52  www.aiche.org/cep  October 2017  CEP Copyright © 2017 American Institute of Chemical Engineers (AIChE)
Protein structures
Proteins are composed of 20 standard amino acids. The
amino acids all share a similar structure (Figure 1a) — an
amine group bound to a central carbon, to which a side-
chain and a carboxyl group are also attached. The side-
chains differentiate the amino acids.
A protein’s primary structure (Figure 1b) is the linear
order of its amino acids, which are connected to each other
by a peptide bond between the carboxyl group and the
amine group. The primary structure is considered to have
a direction, which is defined as starting with the amino
acid that has an unbound amine group and ending with the
amino acid that has an unbound carboxylic acid group.
Proteins do not exist as extended, linear conformations,
but fold into complex, thermodynamically favorable geom-
etries. Secondary structure elements (Figure 1c), such as
α-helixes and β-strands, are arrangements of amino acids
near one another in the primary structure.
The tertiary structure (Figure 1d) is the arrangement of
the secondary structure elements into the overall complex
fold. This involves one or more domains, which are stable
sets of secondary structure elements.
Exceptions to these general protein structure guide-
lines exist. Some proteins incorporate rare amino acids,
such as selenocysteine; some are modified by the addition
of carbohydrate, phosphate, or other groups that affect
their function or stability; and still others are intrinsically
disordered and have dynamic tertiary and secondary struc-
tures. In general, however, protein structures and functions
are determined by their primary, secondary, and tertiary
structures. All experimentally determined protein structures
are deposited in the Research Collaboratory for Structural
Bioinformatics (RCSB) Protein Data Bank (PDB), which
currently contains more than 130,000 structures (2).
(a) (b)
H
NH3 +
COOH TKKEAEKFAAILIKVFAEL
C NFDYTYTV
p Figure 1. All proteins are composed of a linear chain of amino acids. (a) All amino acids have the same backbone structure, but they have different side
chains bound to the central carbon. (b) A protein’s primary structure is the linear order of its amino acids. (c) Amino acids near one another in the primary
structure assemble into secondary structure elements, such as α-helixes and β-strands. (d) The secondary structure elements fold into the overall complex
protein fold, or tertiary structure, which brings into close proximity amino acids that may be far from one another in the primary structure.
Copyright © 2017 American Institute of Chemical Engineers (AIChE)
The complex structures of proteins are the result of
finely balanced interplays between enthalpic and entropic
forces. Computational methods use force fields to design
and predict protein structures. Force fields, such as Amber
(3), CHARMM (4), GROMACS (5) and Rosetta (6), are
mathematical functions that calculate forces and energies
between atoms, amino acids, and proteins. These calcula-
tions treat atoms as spheres of constant size and charge
with fixed parameters for energy functions. Typical terms
in the calculations include van der Waals forces, electro-
static forces, solvation effects, bond lengths, bond angles,
and dihedral bond angles. Statistical parameters for the
presence of naturally occurring features, such as length
and angles of hydrogen bonds and the frequency of certain
amino acids, are also used.
The most common types of calculations are molecular
mechanics (MM), which predict the energy minimum of
a protein structure, and molecular dynamics (MD), which
calculate the behavior of the protein over time. The fixed-
parameter approximations of MM and MD can cause inac-
curate predictions. Density functional theory and quantum
mechanics calculations do not involve these approxima-
tions and are sometimes used to improve accuracy, but
they require significantly more calculation time to produce
a prediction.
Early attempts at protein engineering aimed to create
new proteins by considering the rules that govern protein
structures and functions (7). Computational protein engi-
neering is an extension of this practice, utilizing enhanced
calculation abilities to aid the design process. Computa-
tional methods were originally modeled after experimental
methods and focused on redesigning existing proteins for
a new function, which is still a common practice. The real
promise of computational techniques was demonstrated in
(c) (d)
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SBE SPECIAL SECTION:
BIOMOLECULAR ENGINEERING
2003 with the design of Top7 (8) — a protein with a fold
that had previously never been observed in nature.
Computational protein engineering has continued to
advance and, as the following examples demonstrate, is
reaching a point where it can reliably design proteins with
specified structures and functions.
Predicting protein structures
Computational protein design attempts to predict how
a sequence of amino acids will fold and the resulting
structure it will create. The Critical Assessment of Protein
Structure Prediction (CASP) is a blinded, biennial assess-
ment of current techniques for predicting protein structures
that uses proteins with solved but unpublished structures
as targets. The most recent cycle was completed in 2016
(9); over the two-and-a-half decades since the first CASP,
the number of known protein structures has dramatically
increased, from several hundred to over 130,000 (2).
Protein structure prediction involves initial structure
prediction, which identifies a preliminary structure, and
then refinement of the preliminary structure. The initial
structure prediction employs as much known structural
information as possible. In cases where the target protein
has a high sequence similarity (i.e., homology) with a pro-
tein of known structure, the known structure can be used
as a template to predict the target protein’s structure (i.e.,
template-based methods). For proteins that lack homology,
it is often still possible to identify a template by consid-
ering the order of secondary structure elements in the
protein. In situations where no template can be identified,
methods have been developed to predict proteins entirely
from scratch or by using short fragments of structures (10).
Refinement of the predicted structure typically involves
MM and MD calculations to determine lower-energy
arrangements (9).
Recent CASP results indicate that the field of protein
structure prediction is maturing to a point of practical
utility, and the number of protein domains that can be
(a) (b)
p Figure 2. Computational methods have been used to design protein multimers, including a homodimer (a), a 24-subunit tetrahedron (b), and a 60-subunit,
25-nm icosahedron (c). Source: (14–16).
54  www.aiche.org/cep  October 2017  CEP
modeled successfully is increasing rapidly (9). Template-
based predictive methods (10–13) are consistently the most
effective and reliable approaches. Although many more
protein sequences than structures are known, the large
number of experimentally determined structures means
that good templates are now available for many proteins.
When a good template is not available, methods that start
from scratch are effective at predicting structures of small
proteins. Additionally, methods of assessing the accuracy
of predicted models are becoming increasingly helpful (9).
Computational protein engineering is still useful even
if it is not able to correctly design every possible protein.
It should, however, be sufficiently accurate for a diverse
set of protein structures with desired functions. Protein
structure prediction has reached a point where consistent
design of experimentally accurate proteins is now possible.
As structure prediction methods improve, computational
protein design will expand beyond natural proteins.
Designing self-assembling multimers
As the ability to predict protein structures has
improved, researchers have begun to design protein-protein
interactions. Many natural proteins exist as complexes of
homo­multimers (i.e., multiple copies of the same protein)
or hetero­multimers (i.e., one or more copies of multiple
proteins). Protein-protein interactions play important roles
in many life processes, including cell signaling, immune
response, and cell-cell adhesion. Designing protein-protein
interactions is complex, because it requires predicting
intermolecular interactions and their effects on protein
structure. However, creating these custom interactions
could enable the creation of complex systems.
Multimer proteins can be used in applications of
protein engineering less accessible to monomeric proteins.
Homodimers, heterodimers, homotrimers, and hetero­
trimers are common in cell signaling pathways and could
be used in drug design. Larger icosahedron structures
are nanometer-scale particles (14) that could be used for
(c)
Copyright © 2017 American Institute of Chemical Engineers (AIChE)
biomedical applications, such as carrying cargoes of drugs
and presenting antigenic epitopes as vaccines, as well as
serving as nanoparticles. Protein nanoparticles would have
very consistent size profiles, with nearly all particles being
of identical composition; therefore, chemical engineers
may find applications for them beyond biomedicine.
There are numerous examples of successful design
of multimer proteins. Mou, et al., designed an α-helical
homodimer by selecting a scaffold protein, predicting
numerous symmetric models, and then selecting a model
based on shape complementarity (15) (Figure 2a). King,
et al., designed 24-subunit cage-like proteins by identifying
potential building-block proteins, determining if they have
promising interfaces to replicate a tetrahedral geometry,
and using RosettaDesign to finalize the sequences (16)
(Figure 2b). Hsia, et al., built on this methodology to
design a 60-subunit, 25-nm icosahedron that was highly
stable to both chemical and thermal denaturation (14)
(Figure 2c).
Designing antibodies
Protein-based therapeutics are a very large segment of
the pharmaceutical market. Global sales of antibodies are
expected to be $125 billion per year by 2020 (17). Anti-
bodies are a promising target for computational protein
design because, like multimer proteins, antibodies bind
their target antigens by forming stable protein-protein
interfaces. In addition, because antibodies are so important
to the immune system, the physical and genetic features
that enable antibodies to bind to practically any molecule
have been extensively studied. Specifically, antibody
(a)
Target Peptides
Antibodies
p Figure 3. The OptCDR and OptMAVEn algorithms have been used to design nanomolar affinity antibodies against targeted peptide antigens. In the OptCDR
antibody (a), a peptide containing the FLAG epitope presents the epitope such that it sits in a deep pocket in the antibody’s complementarity determining
regions (CDRs). In contrast, the OptMAVEn antibody (b) binds a peptide that is in a more compact conformation. Although the peptides have different
orientations relative to their respective antibodies, both peptides are situated in the center of the antibodies’ binding pockets and form favorable interactions
with most CDRs.
Copyright © 2017 American Institute of Chemical Engineers (AIChE)
Antibodies are a promising target
for computational protein design
because they bind their
target antigens by forming
stable protein-protein interfaces.
genetics target mutational diversity to six complementarity
determining regions (CDRs), also known as hypervariable
regions, that are responsible for binding to target antigens.
There are numerous experimental methods to develop
antibodies that bind to an antigen. Historically, this
involved inoculating an animal with the antigen, waiting
for an immune response, and then isolating the antibodies.
Although effective at binding antigens, animal antibodies
are sufficiently different from human antibodies that they
can trigger an immune response to the antibodies them-
selves when used as a medicine.
Experimental and computational techniques attempt to
“humanize” animal antibodies, as well as to graft CDRs
from one antibody to another. The Bailey-Kellogg labora-
tory at Dartmouth College has developed the computation-
ally driven antibody humanization (CoDAH) method for
humanizing antibodies. CoDAH has been used to make
antibodies more human and improve thermostability, while
retaining binding affinity (18). Other researchers have
developed computational methods to humanize anti­bodies,
graft CDRs, and design CDRs to replicate the inter­
actions between an antigen and its native binding partner
protein (18–20).
The Maranas lab at Pennsylvania State Univ. has devel-
(b) Target Peptides
Antibodies
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SBE SPECIAL SECTION:
BIOMOLECULAR ENGINEERING
Given the pace of developments,
computational protein engineering
could be a mature and widespread field
in the next decade.
oped two algorithms to design antibodies from scratch to
bind any specified epitope. The Optimal Complementarity
Determining Regions (OptCDR) method designs CDRs
to be grafted into an antibody scaffold (21). OptCDR has
been used to design nanomolar affinity antibodies against
the FLAG epitope (Figure 3a) (22). The optimal method of
antibody variable region engineering (OptMAVEn) designs
the entirety of the domains in antibodies that are respon-
sible for antigen recognition (23). OptMAVEn is based
on a database of predicted structures for the human genes
that are used to create antibodies (24) and correspondingly
designs antibodies that are nearly or entirely human. It was
used to design antibodies that bind with nanomolar affinity
to a mannose-mimicking dodecapeptide (25) (Figure 3b).
These methods demonstrate that computational antibody
design is capable of creating properties close to those in the
immune system.
Designing transmembrane proteins
The expansion of experimentally determined protein
structures has helped to advance protein structure predic-
tion methods. However, because the primary experimental
methods for determining protein structures (e.g., X-ray
crystallography and nuclear magnetic resonance [NMR]
spectroscopy) are well-suited for soluble proteins but not
membrane-bound proteins, there are relatively few solved
transmembrane protein structures. In addition to the dearth
of known structures, force fields were initially parameter-
ized and optimized for aqueous environments, rather than
the hydrophobic environments necessary for computation-
ally designing transmembrane proteins. These are signifi-
cant limitations because transmembrane proteins are the
u Figure 4. Transmembrane
proteins represent a
significant challenge
for computational
protein design,
but some suc-
cesses are being
realized, such
as this four-helix
transmembrane
Zn2+ transporter.
The structure of four
α-helixes is represented
by space-filling spheres
so that the central pore used for
transporting zinc can be seen.
56  www.aiche.org/cep  October 2017  CEP
target of about half of all drugs (26).
Despite these challenges, the design of new trans­
membrane proteins has seen recent success. Joh, et al.,
designed a four-helix transmembrane protein that selec-
tively transports Zn2+, but not Ca2+, while antiporting
protons (27). The researchers developed a computational
algorithm called Rocker that mimics the mechanism many
natural transport proteins are thought to use, which involves
“rocking” between two states. That algorithm first designed
a symmetric coiled coil with two Zn2+ binding sites and
then designed the off-symmetry version by straightening
the helixes at one of the binding sites. Energy calculations
and MD simulation were used to screen a thousand possible
sequences. The final structure (Figure 4) was experimen-
tally verified and has the designed transport features.
Goparaju, et al., also designed a four-helix trans­
membrane protein that protein binds heme and other
porphyrins for electron transport (28). The researchers cre-
ated the protein using binary patterning of hydrophilic and
hydrophobic amino acids with α-helical propensities. Then
they placed histidine residues, which bind heme, at specific
positions to enable fast electron transfer rates. Experimen-
tal testing validated the heme binding and electron trans-
port features of the designed protein, but the final structure
was not experimentally resolved. This work demonstrates
that computationally designing transmembrane proteins
is possible.
Moving forward
The first structure of a computationally designed pro-
tein with a fold never observed in nature (i.e., Top7) was
published in 2003 (8). At that time, computational protein
design was an immature field. Predicting protein structures
was unreliable, computational methods were generally
limited to redesigning proteins with only modest successes,
and the regular design of a protein from scratch with a
specified function was unattainable.
Over the last 14 years, computational protein engineer-
ROBERT PANTAZES, PhD, is an assistant profes-
sor of chemical engineering at Auburn Univ.
(Email: rjp0029@auburn.edu). His research
group focuses on combining computational and
experimental methods to design and identify
therapeutic proteins, with an emphasis on
rapidly designing high-affinity binding proteins
and comprehensively characterizing epitope
repertoires. He received his BS and PhD in
chemical engineering at Pennsylvania State
Univ. under the supervision of Costas Maranas,
researching computational protein engineer-
ing. He completed his postdoc at the Univ. of
California, Santa Barbara, researching autoimmune disease biomarker
discovery under the supervision of Patrick Daugherty. After completing
his postdoc, Pantazes was a scientist at Serimmune, Inc.
Copyright © 2017 American Institute of Chemical Engineers (AIChE)
ing has improved significantly. In addition to many protein
structures being reliably predicted, protein complexes can
be designed to meet exacting geometric criteria, thera­
peutic proteins with abilities comparable to those of natural
proteins can be computationally developed, and trans-
membrane proteins with transport functions are becoming
possible. Computational protein engineering has reached
a threshold where it can reliably be of use in a growing
number of applications.
Areas requiring improvement exist, most significantly
in designing catalytic proteins (i.e., enzymes) with catalytic
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computers,” SoftwareX, 1–2, pp. 19–25 (Sept. 2015).
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of Protein Structures,” Trends in Biochemical Sciences, 14 (7),
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8. Kuhlman, B., “Design of a Novel Globular Protein Fold with
Atomic-Level Accuracy,” Science, 302 (5649), pp. 1364–1368
(Nov. 2003).
9. Moult, J., et al., “Critical Assessment of Methods of Protein
Structure Prediction: Progress and New Directions in Round XI,”
Proteins: Structure, Function, and Bioinformantics, 1 (4–14)
(Sept. 2016).
10. Sali, A., and T. L. Blundell, “Comparative Protein Modelling by
Satisfaction of Spatial Restraints,” Journal of Molecular Biology,
234 (3), pp. 779–815 (Dec. 1993).
11. Schwede, T., et al., “SWISS-MODEL: An Automated Protein
Homology-Modeling Server,” Nucleic Acids Research, 31 (13),
pp. 3381–3385 (July 2003).
12. Zhang, Y., “I-TASSER Server for Protein 3D Structure Prediction,”
BMC Bioinformatics, 9, p. 40 (Jan. 2008).
13. Krivov, G. G., et al., “Improved Prediction of Protein Side-chain
Conformations with SCWRL4,” Proteins: Structure, Function, and
Bioinformantics, 77 (4), pp. 778–795 (Dec. 2009).
14. Hsia, Y., et al., “Corrigendum: Design of a Hyperstable 60-Subunit
Protein Icosahedron,” Nature, 540, p. 150 (Dec. 2016).
15. Mou, Y., et al., “Computational Design and Experimental Verifica-
tion of a Symmetric Protein Homodimer,” Proceedings of the
National Academy of Sciences, 112 (34), pp. 10714–10719
(Aug. 2015).
Copyright © 2017 American Institute of Chemical Engineers (AIChE)
properties comparable to those of natural enzymes. Promis-
ing research is underway to design enzymes and make
significant algorithmic improvements, such as multistate
design (i.e., designing a protein for more than a single
requirement) and backbone flexibility.
Given the pace of developments, computational protein
engineering could be a mature and widespread field in the
next decade. It is already possible to create proteins for
many purposes, and in the near future, it should be feasible
to design a protein from scratch for most catalytic, mate-
rial, and medical applications. CEP
16. King, N. P., et al., “Accurate Design of Co-Assembling Multi-­
component Protein Nanomaterials,” Nature, 510, pp. 103–108
(June 2014).
17. Ecker, D. M., et al., “The Therapeutic Monoclonal Antibody
Market,” MAbs, 7 (1), pp. 9–14 (2015).
18. Nam, D. H., et al., “Generation of Inhibitory Monoclonal Anti­
bodies Targeting Matrix Metalloproteinase-14 by Motif Grafting
and CDR Optimization,” Protein Engineering, Design and Selecion,
30 (2), pp. 113–118 (2016).
19. Liu, X., et al., “Computational Design of an Epitope-Specific Keap1
Binding Antibody Using Hotspot Residues Grafting and CDR Loop
Swapping,” Scientific Reports, 7, p. 41306 (2017).
20. Sun, F., et al., “Engineering a High-Affinity Humanized Anti-CD24
Antibody to Target Hepatocellular Carcinoma by a Novel CDR
Grafting Design,” Oncotarget, doi: http://dx.doi.org/10.18632/onco-
target.17228 (2017).
21. Pantazes, R. J., and C. D. Maranas, “OptCDR: A General Com-
putational Method for the Design of Antibody Complementarity
Determining Regions for Targeted Epitope Binding,” Protein Engi-
neering, Design and Selection, 23 (11), pp. 849–858 (Nov. 2010).
22. Entzminger, K. C., et al., “De Novo Design of Antibody Comple-
mentarity Determining Regions Binding a FLAG Tetra-Peptide,”
Science Reports, 7, p. 10295 (2017).
23. Li, T., et al., “OptMAVEn — A New Framework for the De Novo
Design of Antibody Variable Region Models Targeting Specific
Antigen Epitopes,” PLoS One, 9 (8) (Aug. 2014).
24. Pantazes, R. J., and C. D. Maranas, “MAPs: A Database of
Modular Antibody Parts for Predicting Tertiary Structures and
Designing Affinity Matured Antibodies,” BMC Bioinformatics,
14, p. 168 (May 2013).
25. Poosarla, V. G., et al., “Computational De Novo Design of Anti­
bodies Binding to a Peptide with High Affinity,” Biotechnology and
Bioengineering, 114 (6), pp. 1331–1342 (June 2017).
26. Rask-Andersen, M., et al., “Trends in the Exploitation of Novel
Drug Targets,” Nature Reviews Drug Discovery, 10, pp. 579–590
(Aug. 2011).
27. Joh, N. H., et al., “De Novo Design of a Transmembrane
Zn2+-transporting Four-Helix Bundle,” Science, 346 (6216),
pp. 1520–1524 (Dec. 2014).
28. Goparaju, G., et al., “First Principles Design of a Core Bio­
energetic Transmembrane Electron-transfer Protein,” Biochimica
et Biophysica Acta (BBA) — Bioenergetics, 1857 (5), pp. 503–512
(May 2016).
CEP  October 2017  www.aiche.org/cep  57
SBE SPECIAL SECTION:
BIOMOLECULAR ENGINEERING
Plant Metabolic Engineering
for Chemicals, Fuels,
and Precursors
Timothy A. Whitehead Narrowing the metabolic engineering divide
Michigan State Univ.
Sean Cutler
between plants and microorganisms
Ian Wheeldon will require faster design-build-test cycles
Univ. of California, Riverside
and better genome engineering tools.
P
lants are the ultimate source of foods, fibers, pharma-
ceuticals, and fuels. This article discusses the meta-
bolic engineering of plants to produce fuels and value-
added chemicals, with a focus on the general concepts and
challenges that differentiate plant and microbial engineering.
Two examples of successful metabolic engineering of plant
traits are highlighted, and emerging techniques to prevent
transgene release into the wild and to advance the speed of
the design-build-test cycle are discussed.
Conversion schemes for commodity
chemicals and fuels from plant biomass
Plant biomass is an output of carbon dioxide, sunlight,
water, and soil-derived nutrients. This biomass can be used
directly or indirectly for fuels and value-added chemicals
(Figure 1).
For thousands of years, pharmacologically active mol-
ecules have been extracted from plants, including opioids
from poppies and antimalarial medicines such as artemisinin
from Chinese wormwood. Today, chiral building blocks are
sourced from plants for a variety of pharmaceuticals. For
example, shikimic acid is produced in anise seeds for the
synthesis of the antiviral oseltamivir (marketed as Tamiflu)
(1) and scopolamine is extracted from corkwood leaves for
synthesis of various bronchodilators (most notably tio­
tropium, which is marketed as Spiriva) (2).
Plant biomass can be converted to biofuel using physico-
chemical and/or enzymatic treatments to break it down into
sugars, followed by upgrading via fermentation or chemical
catalysis. Diesel and jet fuel can be prepared from seed oils
from palm trees or plants like Camelina sativa. Finally, fast-
58  www.aiche.org/cep  October 2017  CEP
growing plants like tobacco can be genetically engineered to
accumulate value-added chemicals (3) and drugs (4). Thus,
there is a wide diversity of feedstocks and speciality chemi-
cals that originate in plants.
Comparing products made by
engineered plants and microbes
Not every chemical can be produced by plants. One
major issue is volatility: many plant-produced volatile
compounds leak into the atmosphere, making collection dif-
ficult. Take, for example, the C5 monomer isoprene, which
is used in rubber. Although trees and other plants emit over
500 million tons of isoprene annually, little is left in plant
biomass (5). It is much easier to recover volatiles for down-
stream purification from off-gases in closed fermentation
vessels. To that end, Danisco, in collaboration with Good-
year, has developed a bioprocess for production of isoprene
using metabolically engineered E. coli (6). Researchers
have implemented several strategies to mitigate the escape
of plant-produced volatiles, including adding a protecting
group or handle to keep the molecule inside of the plant (7)
and redirecting biosynthesis to plant subcompartments.
Another issue is compound toxicity that can limit plant
yields. This is the case for classical fermentation products
— such as citric acid and ethanol — that can be produced
by fermentation at near-quantitative yields and high product
titers. Even though many plants naturally produce many of
these compounds (e.g., citric acid in citrus fruits), the pro-
cess of extracting and purifying these products from diffuse
point sources is economically less favorable than fermenta-
tion using inexpensive sugar sources like beet molasses.
Copyright © 2017 American Institute of Chemical Engineers (AIChE)

Fatty Acids (for Biodiesel)
Metabolic
Etoposide Structural
Engineering
Biology
Polyhydroxybutyrate
Genome
Editing
Non-Natively
Produced
Protein
Engineering
and Design
Scopolamine Shikimic Acid
Chiral Building Blocks
However, in many cases it is more desirable to pro-
duce biofuels or bioproducts directly in the host plant.
Biodiesel is one such example. For example, the non­food
oilseed crop Camelina can yield two metric tons of bio-
diesel with approximately 40% by weight lipids per hectare.
Seed lipids are trapped in oil bodies that can be recovered
and hydrotreated to yield a renewable diesel that is inter­
changeable with petroleum-derived diesel. Alternatively,
the recovered lipids can be subjected to catalytic cracking
to alter the distribution of chain lengths to the appropriate
range for jet fuel and gasoline. In general, products accumu-
late at high abundance if they are part of the reproductive
strategy of the organism — oil accumulation in the seeds,
sucrose in beets and sugarcane, and so forth.
A large engineering gap exists
between plants and microbes
The plant metabolic engineer has many potential targets.
Classical approaches to improve yield of a desired prod-
uct include overexpressing key biosynthetic genes and/or
deleting genes that produce a competing metabolite. More
fundamentally, increasing overall biomass yields — which
can result from changes in the efficiency of photosynthesis,
Copyright © 2017 American Institute of Chemical Engineers (AIChE)
t Figure 1. Many different
chemicals and fuels can
be produced from plants;
the photos in the center
are several representative
species. The outer boxes
show various chemicals
that can be produced from
Sucrose
plants, including shikimic
acid and morphine. The
inner ring shows different
Precursors
enabling technologies that
facilitate production routes
for these and other fuels
and chemicals.
Mathematical
Modeling
Artemisinin
Morphine
Pharmaceuticals
pathogen tolerance, water use, and fixed nitrogen use —
can improve the economic suitability of any given process.
Encoding degradation enzymes that are sequestered or inac-
tive until deconstruction conditions are present can enhance
the digestibility of biomass. Alternatively, a large research
push over the past half-decade aims to increase the trans-
formation of lignin to value-added products (8), including
altering biosynthesis of lignin precursors.
Despite the scope and range of these approaches, there
are comparatively fewer studies on plants than microbes by
metabolic engineers (and, specifically, by those housed in
engineering departments). As an example, in 2016, the jour-
nal Metabolic Engineering published 100 research articles
on microbes and three on plants or algae.
There are several reasons for this difference. First,
traditional plant breeding techniques have been used to
produce desired plants successfully over the past 10,000
years. Many plants contain multiple copies of each chromo-
some and have many wild relatives, and thus thousands of
progeny with diverse genetic backgrounds can be screened
fairly easily in modern breeding programs. Second, for many
desirable traits, the link between genotype and phenotype is
not well understood — in fact, for many medicinally active
CEP  October 2017  www.aiche.org/cep  59
SBE SPECIAL SECTION:
BIOMOLECULAR ENGINEERING
compounds, neither the biosynthetic nor regulatory genes are
completely known.
The largest stumbling block may be the engineering
gap between microorganisms and plants. Within micro­
organisms, gene-editing technologies that harness transcrip-
tion activator-like effector nucleases (TALEN) and clustered
regularly interspaced short palindromic repeats (CRISPR)
can be used to knock out genes at defined locations. How-
ever, gene replacement or repair at defined loci within plants
is still an unsolved problem.
In plants, standardized biological “parts” like promoters,
terminators, cis- or trans-activators, etc., have been shown to
respond with very high variance between biological repli-
cates, even under ideal laboratory conditions (9), and are thus
not as useful as their microbial counterparts. In fact, plant
biologists who transform genes at random locations must
then go through expansive and exhaustive screening of seeds
to identify constructs with appropriate transgene(s) expres-
sion levels.
Finally, the time required for a design-build-test cycle
is daunting even for well-studied plants. These cycles can
range from several months for model species to up to a
year for commercially relevant softwoods such as poplar.
In contrast, a design-build-test cycle on the baker’s yeast
Saccharomyces cerevisiae takes one week — two to three
days to generate a construct and the remainder of the time to
perform fermentation and analysis. Because of the difference
in cycle times for iterating design concepts, forward design
in plants has lagged far behind that in microbes.
Overcoming the disadvantages of plant metabolic
engineering will require rethinking current strategies. One
way forward for plant metabolic engineers is to create better
Low
High
Light
Light
Leaf transitions Leaf remains
from sun to shade shaded
NPQ remains activated NPQ begins to relax
CO2
Fixation
CO2
Fixation
PsbS NPQ PsbS NPQ PsbS NPQ Fixation
zeaxanthin zeaxanthin
zeaxanthin
VDE ZEP VDE ZEP VDE ZEP
violaxanthin
violaxanthin violaxanthin
ZEP speeds up NPQ relaxation
VDE balances ZEP activity during NPQ induction
PsbS adjusts NPQ level to maintain WT amplitude
p Figure 2. Researchers found that overexpression of the PsbS, VDE, and
ZEP genes helped increase photosynthesis faster in periods when the leaf
transitions from high light to low light. Source: Adapted from (10). Reprinted
with permission from AAAS.
60  www.aiche.org/cep  October 2017  CEP
initial models. The example discussed in the next section
shows impressive productivity gains produced by careful
upstream modeling. Other improvements can be anticipated
as the engineering gap between microorganisms and plants
is narrowed. The second example showcases a potential
solution to optimize biosynthetic pathways in plants without
extensive, sequential genetic modifications.
Example 1: Improving efficiency of photosynthesis
Recent research has shown that simple genetic perturba-
tions identified by mechanistic modeling can substantially
improve plant biomass yields under real-world condi-
tions. An elegant example of this modeling approach was
published recently (10). The study demonstrated that crop
leaves in full sunlight dissipate damaging excess absorbed
light energy as heat (Figure 2). When sunlit leaves become
shaded, dissipation continues for many minutes and reduces
photosynthesis. The transition from high dissipation to low
dissipation is longer than the reverse process, and the high-
to-low time delay reduces the efficiency of photosynthesis
by up to 30% under real-world conditions. The researchers
postulated that minimizing the time delay could improve
photosynthetic efficiency.
The team built a regulatory model that explains how
the time delay was controlled. They suggested that over­
expression of three genes — Photo System II subunit S
(PsbS), violaxanthin de-epoxidase (VDE), and zeaxanthin
epoxidase (ZEP) — could minimize the time delay. They
developed three independent tobacco lines overexpressing
these three genes, and found that the time delay in the modi-
fied plants was shorter than the time delay in the control
line. More importantly, under field conditions, plant biomass
yields improved by 15% relative to the control line.
Low Notably, simultaneous overexpression of all three genes
Light
was necessary, as previous literature showed that over­
expression of a single gene did not improve biomass yields.
This groundbreaking work is important because it shows that
careful modeling of the extensive regulation-controlling plant
CO2 traits can identify comparatively simple solutions to improve
plant yields, abiotic stress tolerance, and product formation.
Example 2: Chemical control of plant water use
One emerging goal for researchers is tunable chemical
control over plants. Achieving this goal has the potential
to avoid many of the technical challenges inherent in plant
metabolic engineering. For example, synthetic biologists
routinely use combinatorial construct assembly methods to
manipulate enzyme expression levels and optimize microbial
pathways. Such refactoring approaches are underutilized in
plant synthetic biology due to the cost and time required to
make and characterize transgenic plants.
In contrast, refactoring multigene expression can be
Copyright © 2017 American Institute of Chemical Engineers (AIChE)
accomplished from a single or a few transgenic plants if
researchers could apply chemicals to the plant exterior to
activate regulatory modules that could turn beneficial traits
on and off. These regulatory modules can be multiplexed to
facilitate the engineering of complex, field-ready inducible,
and repressible traits.
In a demonstration of the power of this emerging
technology, one author (Cutler) and his colleagues re-­
engineered a natural signaling pathway controlling water
use to respond to a commonly used agrochemical (Figure 3)
(11). His research team had previously worked on funda-
mental control over water use with abscisic acid (ABA).
ABA interacts with a hormone receptor, Pyrabactin Resis-
tance 1 (PYR1), which then undergoes a conformational
change to bind another protein that triggers gene expression
differences. The team redesigned the ligand-binding pocket
of PYR1 to no longer recognize ABA and to bind with
potency to the U.S. Food and Drug Administration (FDA)-
approved agrochemical mandipropamid. Transgenic plants
containing this designed hormone receptor could regulate
water use if mandipropamid was applied.
Further development of this technology could allow
biologists to control gene expression of unrelated transgenes,
to further repurpose the binding pocket of PYR1 or related
hormone receptors to respond to new agrochemicals, and to
develop predictive models of response thresholds as a func-
tion of agrochemical concentration. These breakthroughs,
in turn, will minimize the engineering gap between plants
and microbes for the plug-and-play design of enhanced plant
traits in non-model organisms.
Biocontainment strategies for field-grown plants
Restricting the flow of transgenes out of genetically
engineered organisms into wild relatives or nontransgenic
domesticated plants remains a challenge for plant synthetic
biologists. A recent report concluded that although gene
flow has occurred, no examples have demonstrated an
adverse environmental effect of gene flow from a crop to
a wild, related plant species (12). Nonetheless, given the
unfounded yet widespread societal concerns, it is essential
that plant synthetic biologists attempt to prevent and/or limit
the effects of transgene flow. Scientists must therefore build
standardized parts that can mediate biocontainment.
For microbial systems, researchers have approached this
problem by developing a modified genetic code that limits
an engineered gene’s effects to a specialized host organism
(13). Employing this approach in plants will take exten-
sive development and likely be costly because it relies on
non-natural amino acids. Conventional proposals for plant
bio­containment exploit transgenes containing site-specific
recombination sites, which can be recombined to delete
engineered sequences using pollen­and or seed-specific

(a) t Figure 3. Scientists suc-

cessfully engineered a natural
ABA Signaling Module Engineered Agrochemically Controlled signaling pathway that controls
Receptor Draught Tolerance
water use to respond to a com-
mon agrochemical. (a) The plant
hormone abscisic acid (ABA) con-
trols transpiration and modulates
drought tolerance. Its biochemical
signaling functions via a chemi-
cally induced dimerization (CID)
module that stabilizes a complex
WT PYR1MANDI of its receptor (PYR1) and down-
PYR1MANDI stream effector (PP2C) (left).
Engineering of the PYR1 binding
pocket to bind the agro­chemical
(b) mandipropamid (PYR1MANDI)
Transcriptional Activation Transcriptional Repression enabled the induction of drought
“off-on” “on-off” tolerance by a low-cost molecule
suitable for field deployment
(middle). Arabidopsis thaliana
plants containing the redesigned
PP2C* PP2C*
PYR1 binding pocket could
PP2C* PP2C* regulate water use depending on
madipropamid application (right).
Agchem Agchem (b) The PYR1-PP2C CID module
can be repurposed for transgene
activation (left) and repression
(right). Source: Adapted from (11).
Reprinted with permission from
Nature Publishing Group.

Copyright © 2017 American Institute of Chemical Engineers (AIChE) CEP  October 2017  www.aiche.org/cep  61
SBE SUPPLEMENT:
BIOMOLECULAR ENGINEERING
expression of recombinases. These recombinase enzymes
would recognize the transgene recombination sites and
remove this DNA from progeny.
While relatively simple, these strategies have limitations.
For example, they prevent the sexual propagation of homo-
zygous transgenic materials; when using pollen-specific
versions of the technology, hybrids between wild pollen
and transgenic eggs are possible, and seeds can still trans-
mit transgenes to their progeny and impact fitness outside
managed agro-ecosystems. Thus, new methods to ensure
biocontainment are urgently needed.
Chemically regulated systems — like those built by
Cutler and his colleagues — have great potential to moder-
TIMOTHY A. WHITEHEAD, PhD, is the Johansen Crosby
Endowed Chair Associate Professor in the Dept.
of Chemical Engineering and Materials Science
and the Dept. of Biosystems and Agricultural
Engineering at Michigan State Univ. He received
his BE in chemical engineering from Vanderbilt
Univ. and his PhD in chemical engineering from
the Univ. of California, Berkeley, and he held
a postdoctoral appointment in the Dept. of
Bio­chemistry at the Univ. of Washington. His
research group develops new technologies for biomolecular engineer-
ing, with a focus on protein engineering and design. He has published
35 peer-reviewed journal papers and is the recipient of numerous
awards, including the CAREER award by the National Science Founda-
tion and the Young Scientist Keynote at the 2017 Protein Engineering
Global Summit meeting. He serves as an Executive Committee member
for an NIH T32 Biotechnology Training Grant on Plant Biotechnology
for Health and Sustainability and is a member of AIChE, the Society for
Biological Engineering, and the American Chemical Society.
SEAN CUTLER, PhD, is a professor of plant cell biology
at the Univ. of California, Riverside. He received
his BA and MSc from the Univ. of Toronto and
PhD from Stanford Univ. (2001). He started his
own research group as an assistant professor at
the Univ. of Toronto in 2002 and subsequently
moved to the Univ. of California, Riverside, in
2007. His work combines genetics, chemistry, and
synthetic biology to understand and manipulate
plant physiology. His current research focuses
on understanding how plants respond to drought stress and using
this information to improve drought tolerance in crops, focusing on
the stress hormone abscisic acid (ABA). Discoveries from his lab have
included ABA receptors, new agrochemical lead compounds, and engi-
neered proteins that can be used to improve crop yields during drought.
He has received the American Society of Plant Biology’s Charles Albert
Shull Award (2011), the Valent Biosciences’ Young Scientist Award
(2010), and the International Plant Growth Substances Association’s
Distinguished Researcher Award (2016). He serves on the editorial
board of The Plant Cell.
IAN WHEELDON, PhD, is an associate professor in the
Dept. of Chemical and Environmental Engineering
at the Univ. of California, Riverside. He received
his Bachelor’s of Applied Science from Queen’s
Univ., Canada, and a Master’s of Applied Science
from the Royal Military College of Canada. He
completed his PhD at Columbia Univ. and trained
as a postdoctoral fellow at Harvard Medical
School and the Wyss Institute for Biologically
Inspired Engineering. His research group focuses
on developing synthetic biology tools for applications in biocatalysis.
He is the recipient of several awards, including the Air Force Office of
Scientific Research Young Investigator Award.
62  www.aiche.org/cep  October 2017  CEP
ate the consequences of transgene flow. Both “on-off” and
“off-on” chemically regulated modules could be constructed
to contain the transgene to the desired plant (Figure 3b).
Nevertheless, such designs are currently speculative.
Closing thoughts
Although plants are the ultimate source of many of our
chemicals and fuels, the large engineering gap between
microbes and plants currently limits the power of much of
synthetic biology. Better genome engineering and predictive
tools must be built if the promise of direct chemical and fuel
production in plants is to be met. CEP
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