Expert Review of Vaccines

ISSN: 1476-0584 (Print) 1744-8395 (Online) Journal homepage: http://www.tandfonline.com/loi/ierv20

In situ production of therapeutic monoclonal

antibodies

Todd J Suscovich & Galit Alter

To cite this article: Todd J Suscovich & Galit Alter (2015) In situ production of
therapeutic monoclonal antibodies, Expert Review of Vaccines, 14:2, 205-219, DOI:
10.1586/14760584.2015.1001375

To link to this article: http://dx.doi.org/10.1586/14760584.2015.1001375

Published online: 12 Jan 2015.

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 SPECIAL FOCUS y RNA Vaccines Review

In situ production of
therapeutic monoclonal
antibodies
Expert Rev. Vaccines 14(2), 205–219 (2015)

Todd J Suscovich* and The use of antibodies as a treatment for disease has it origins in experiments performed in
the 1890s, and since these initial experiments, monoclonal antibodies (mAbs) have become
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Galit Alter
Ragon Institute of MGH, MIT, and
Harvard, Cambridge, MA 02139, USA
*Author for correspondence:
TJSuscovich@mgh.harvard.edu
one of the fastest growing therapeutic classes for the treatment of cancer, autoimmune
disease, and infectious diseases. However, treatment with therapeutic mAbs often requires
high doses given via long infusions or multiple injections, which, coupled with the
prohibitively high cost associated with the production of clinical-grade proteins and the
transient serum half-lives that necessitate multiple administrations to gain therapeutic
benefits, makes large-scale treatment of patients, especially patients in the developing world,
difficult. Due to their low-cost and rapid scalability, nucleic acid-based approaches to deliver
antibody gene sequences for in situ mAb production have gained substantial traction. In this
review, we discuss new approaches to produce therapeutic mAbs in situ to overcome the
need for the passive infusion of purified protein.

KEYWORDS: adeno-associated virus . adenovirus . ex vivo gene transfer . gene therapy . in situ production
. monoclonal antibody . non-vectored gene delivery

However, technological advances made over

Antibodies as therapeutic proteins
While monoclonal antibody (mAb)-based ther-
apeutics account for over one-third of all bio-
logics currently in development [1], the use of
antibodies as a therapeutic was first explored
in the 1890s. In 1890, von Behring and Kita-
sato demonstrated that immunity to diphtheria
and tetanus could be transferred from animals
given small doses of diphtheria or tetanus toxin
to naı̈ve animals via the transfer of serum [2].
These early experiments provided the founda-
tion for the use of polyclonal antisera as a ther-
apeutic, enabling the development of the
11 US FDA-approved polyclonal antisera cur-
rently licensed for use, largely as post-exposure
prophylaxis in high-risk, immunocompromised
patients [3]. These antibodies are purified and
pooled from vaccinated individuals (e.g., teta-
nus) or animals (e.g., botulism) with high titers
of antibodies [4]. However, the identification of
immune individuals and the collection of plas-
maphereses to generate these polyclonal sera
are time consuming and expensive, strongly
limiting the widespread use of antisera as either
a pre-exposure prophylactic or a therapeutic.
the last 40 years in areas including mAb dis-
covery, recombinant DNA technology and
large-scale cell culture have fully unlocked the
therapeutic potential of antibodies.
Antibody discovery techniques
In 1975, Köhler and Milstein reported the
generation of a cell line producing an antibody
of a single specificity, that is, a mAb [5]. This
cell line was generated by fusing splenocytes
from a vaccinated mouse with a mouse mye-
loma cell line, and the resulting hybridoma
continuously produced antibodies against the
antigen used for vaccination. Suddenly, this
method made the production of single anti-
bodies a reality, thereby enabling the screening,
selection and optimization of the most potent
antibodies. Furthermore, using this technology,
it now became possible to develop antibodies
targeting a specific protein or antigen, thus
allowing the targeted development of therapeu-
tic antibodies.
As a direct result of this technology, in
1986, the first therapeutic mAb was approved
for use in humans (muromonab-CD3) [6].

informahealthcare.com 10.1586/14760584.2015.1001375
2015 Informa UK Ltd ISSN 1476-0584 205

 Review Suscovich & Alter
Mouse antibody Chimeric antibody
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Humanized antibody Human antibody
Figure 1. Humanization of therapeutic monoclonal anti-
bodies. Mouse antibody sequences, indicated in red, can induce
an immune response in humans, decreasing the therapeutic effi-
cacy of the monoclonal antibody. To prevent the induction of
this immune response, chimeric and humanized antibodies have
been developed. To generate a chimeric antibody, the entire vari-
able domain of the mouse heavy and light chains are grafted
onto a human antibody backbone, indicated in blue. To generate
a humanized antibody, the specific antigen recognition domains
(complementarity determining regions) are grafted onto a human
antibody backbone. As the humanized antibody has a smaller
proportion of mouse antibody sequences, they are significantly
less immunogenic than chimeric antibodies.
Muromonab-CD3 is a mouse mAb isolated using hybridoma
technology that targets CD3". As CD3" is present on the
surface of T cells, this antibody was used as an immuno-
suppressant for the treatment of acute transplant rejection.
While effective, patients treated with muromonab-CD3 gener-
ated anti-mouse antibodies, as it is a fully mouse mAb [7].
These newly generated antibodies against muromonab-CD3
resulted in the rapid clearance of the mAb from the circulation,
thereby reducing its clinical potency with repeat administration.
To circumvent the generation of an immune response
against the infused antibody, subsequent therapeutic mAbs are
often chimeric, humanized or fully human (FIGURE 1). Specifically,
with rapid advances in recombinant DNA technology over the
same timeframe, it became possible to isolate the genes encod-
ing the mouse mAb and manipulate them to make the result-
ing antibody less immunogenic while retaining its function. To
accomplish this, either the entire antigen-binding domain (i.e.,
the variable domains) or the individual sequences responsible
for antigen recognition (i.e., the complementarity-determining
regions) from the mouse antibody are grafted onto a human
antibody backbone, generating chimeric or humanized antibod-
ies, respectively. As the majority of the protein sequence from
206
chimeric and humanized mAbs is human, these second-
generation therapeutics are significantly less immunogenic than
their mouse counterparts. Furthermore, using current mAb
discovery techniques, it is now possible to rapidly clone and
produce high-affinity, fully human antibodies. Importantly,
even fully human antibodies can be immunogenic due to the
recognition of complementarity-determining region-derived
epitopes by CD4 T cells; however, using antibody engineering,
it may be possible to even further reduce the immunogenicity
of fully human antibodies [8]. Since the approval of muromo-
nab-CD3, 36 additional mAbs have been approved for use in
either the EU or the USA (TABLE 1), with over 300 additionally
in the pipeline [1]. Of these new mAbs therapeutics, only a
small subset is fully mouse antibodies without any degree of
humanization.
While the advent of hybridoma technology rapidly expanded
the therapeutic potential of mAbs, recent advances in antibody
discovery techniques, including antibody display (e.g., phage
display [9], yeast display [10] and ribosome display [11]), vaccina-
tion of humanized mice and single-cell RNA sequencing will
expand this potential even further. Antibody display utilizes a
library of carriers (e.g., bacteriophages, yeast or ribosomes)
engineered to display human antibody fragments. These anti-
body fragment-expressing carriers are then exposed repeatedly
to antigens of interest, enabling the selection/propagation (also
referred to as panning) of carriers displaying antibodies capable
of binding to the target antigen with remarkably high affinity.
Importantly, the library of antibodies displayed can be derived
from any source, including vaccinated or immune individuals.
Therefore, by varying the antibody library, antibody display
allows for the identification of human antibodies targeting vir-
tually any antigen. Of the 12 fully human mAbs currently
approved for use, four were identified using phage display (ada-
limumab, belimumab, raxibacumab and ramucirumab), with
adalimumab, the first fully human, antibody phage display-
isolated mAb approved for use in 2002 [12].
While antibody phage display allows for the in vitro selection
of human mAbs, humanized transgenic mice, such as the Xen-
omouse [13] or the Kymouse [14], allow for the directed in vivo
induction and selection of human antibodies. In these trans-
genic mice, the endogenous mouse antibody variable regions are
deleted using gene targeting, and the human variable regions are
inserted using artificial chromosomes. Normal B-cell develop-
ment and IGH and IGK/IGL loci recombination occurs in these
mice, and following immunization, normal antibody maturation
occurs, except that the antibodies produced contain fully human
variable regions. Hybridomas can then be generated or B-cell
clonal repertoires can be sequenced and expressed (as described
below) from the immunized mice to generate fully human
mAbs. Transgenic humanized mice have proven to be extremely
useful in the identification of human mAbs as eight of 12 human
mAb therapeutics currently approved for use were generated in
humanized transgenic mice (panitumumab, golimumab, canaki-
numab, ustekinumab, ofatumumab, denosumab, ipilimumab
and nivolumab), and the first human mAb generated from
Expert Rev. Vaccines 14(2), (2015)
 Downloaded by [Cornell University Library] at 12:47 02 November 2017

Table 1. Therapeutic monoclonal antibodies approved for use in the USA and/or EU.
International Trade name Target Type Approval Indication
name
US EU
Muromonab- Orthoclone CD3 Mouse IgG2a 1986 1986 Acute organ transplant rejection
CD3 Okt3

informahealthcare.com
Abciximab Reopro GPIIb/IIIa Chimeric IgG1 Fab 1994 1995 Prevention of cardiac ischemia
(integrin aIIbb3)
Rituximab MabThera, CD20 Chimeric IgG1 1997 1998 Non-Hodgkin’s lymphoma
Rituxan
Daclizumab Zenapax IL-2R Humanized IgG1 1997 1999 Acute organ transplant rejection
Basiliximab Simulect IL-2R Chimeric IgG1 1998 1998 Acute organ transplant rejection
Palivizumab Synagis RSV F protein Humanized IgG1 1998 1999 Respiratory syncytial virus infection
Infliximab Remicade TNF-a Chimeric IgG1 1998 1999 Crohn’s disease, ulcerative colitis, rheumatoid
arthritis, ankylosing spondylitis, psoriatic
arthritis, plaque psoriasis
Trastuzumab Herceptin HER2 Humanized IgG1 1998 2000 Breast cancer
†
Gemtuzumab Mylotarg CD33 Antibody drug conjugate 2000 NA Acute myelogenous leukemia
ozogamicin (gemtuzumab [Humanized
IgG4]: calicheamicin)
Alemtuzumab MabCampath, CD52 Humanized IgG1 2001 2001 Chronic lymphocytic leukemia, cutaneous
Campath-1H T-cell lymphoma, and T-cell lymphoma
Brentuximab Adcetris CD30 Antibody drug conjugate 2001 2012 Hodgkin’s lymphoma, systemic anaplastic
vedotin (brentuximab [chimeric IgG1]: large cell lymphoma
monomethyl auristatin E)
Adalimumab Humira TNF-a Human IgG1 2002 2003 Rheumatoid arthritis, juvenile idiopathic
arthritis, psoriatic arthritis, ankylosing
spondylitis, Crohn’s disease, and plaque
psoriasis
Ibritumomab Zevalin CD20 Radiolabeled mouse IgG1 2002 2004 Non-Hodgkin’s lymphoma
tiuxetan
Efalizumab Raptiva CD11a Humanized IgG1 2003 2004 Plaque psoriasis
Omalizumab Xolair IgE Humanized IgG1 2003 2005 Asthma
In situ production of therapeutic mAbs

Tositumomab- Bexxar CD20 Radiolabeled mouse IgG2a 2003‡ NA Non-Hodgkin’s lymphoma

I131
†
Withdrawn from market in 2010.
‡
Withdrawn from market in 2014.
§
Approved for use in Japan.
Review

NA: Not approved.

207
 Downloaded by [Cornell University Library] at 12:47 02 November 2017

208
Table 1. Therapeutic monoclonal antibodies approved for use in the USA and/or EU (cont.).
International Trade name Target Type Approval Indication
name
Review

US EU
Cetuximab Erbitux EGFR Chimeric IgG1 2004 2004 Colorectal cancer, non-small-cell lung cancer,
squamous cell carcinoma of the head and
neck
Bevacizumab Avastin VEGF Humanized IgG1 2004 2005 Colorectal, lung, breast (outside the USA),
glioblastoma (USA only), kidney and ovarian
cancer
Suscovich & Alter

Natalizumab Tysabri Integrin a4 Humanized IgG4 2004 2006 Multiple sclerosis and Crohn’s disease
Ranibizumab Lucentis VEGF Humanized IgG1 2006 2007 Macular degeneration
Panitumumab Vectibix EGFR Human IgG2 2006 2007 Colorectal cancer
Eculizumab Soliris C5 Humanized IgG2/4 2007 2007 Paroxysmal nocturnal hemoglobinuria
Certolizumab Cimzia TNF-a Pegylated humanized Fab 2008 2009 Crohn’s disease and rheumatoid arthritis
pegol
Catumaxomab Removab EPCAM Rat/mouse bispecific NA 2009 Malignant ascites
CD3
Golimumab Simponi TNF-a Human IgG1 2009 2009 Rheumatoid arthritis, psoriatic arthritis,
ankylosing spondylitis and ulcerative colitis
Canakinumab Ilaris IL-1b Human IgG1 2009 2009 Cryopyrin-associated periodic syndrome
Ustekinumab Stelara IL-12 Human IgG1 2009 2009 Plaque psoriasis and psoriatic arthritis
IL-23
Ofatumumab Arzerra CD20 Human IgG1 2009 2010 Chronic lymphocytic leukemia
Tocilizumab RoActemra, IL-6R Humanized IgG1 2010 2009 Rheumatoid arthritis
Actemra
Denosumab Prolia RANK-L Human IgG2 2010 2010 Bone loss
Belimumab Benlysta BAFF Human IgG1 2011 2011 Systemic lupus erythematosus
Ipilimumab Yervoy CTLA-4 Human IgG1 2011 2011 Metastatic melanoma
Pertuzumab Perjeta HER2 Humanized IgG1 2012 2013 Breast cancer
Raxibacumab Abthrax Protective Human IgG1 2012 NA Inhaled anthrax
antigen of
anthrax toxin
†
Withdrawn from market in 2010.
‡
Withdrawn from market in 2014.
§
Approved for use in Japan.
NA: Not approved.

Expert Rev. Vaccines 14(2), (2015)

 In situ production of therapeutic mAbs Review

transgenic humanized mice was approved for use in humans in

2006 (panitumumab) [12].

Uveitis, rheumatoid arthritis and psoriasis

The newest technique that has been adapted to isolate

Ulcerative colitis and Crohn’s disease

human mAb sequences is the high-throughput sequencing of

Multicentric Castleman’s disease

the antibody repertoire. With this technology, DNA or RNA is
Chronic lymphocytic leukemia isolated from bulk B-cell populations and sequenced using
next-generation sequencing. The matching heavy- and light-
chain sequences can be bioinformatically paired, and the anti-

Metastatic melanoma
body produced in vitro and screened for function. Importantly,
recent advances in single-cell sequencing of DNA/RNA has rev-

Neuroblastoma
Gastric cancer
Breast cancer

olutionized this application dramatically, as paired heavy- and

Indication

Melanoma
light-chain sequences can be directly identified and screened [15].
While no therapeutic human mAbs have been produced using
the technology, this technology is only in its infancy and its
therapeutic potential likely remains untapped and will explode.
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One area in which high-throughput sequencing of antibody

In review

In review
In review

repertoires will be particularly useful is in the treatment/

2013

2014

2014

2014
2014

prevention of infectious diseases, where immune repertoires can

NA§
EU
Approval

be sequenced in real time at the start of an epidemic/pandemic

Table 1. Therapeutic monoclonal antibodies approved for use in the USA and/or EU (cont.).

in immune individuals to rapidly develop protective monoclo-

nal therapeutics.
In review
In review
2013

2013

2014
2014
2014
2014

2014
US

Production of therapeutic mAbs

Even though advances in antibody discovery technologies made
it possible to produce antibodies targeting specific antigens,
therapeutic mAbs often require relatively high doses to be effec-
Glycoengineered humanized

tive for the treatment of solid tumors (up to hundreds of milli-

Antibody drug conjugate

grams per kilogram), largely due to their poor penetrance into

(Herceptin:mertansine)

tumors [16]. Initially, therapeutic mAbs were purified from the

Humanized IgG1
Humanized IgG4

ascitic fluid of mice injected with hybridomas. While the ascitic

Chimeric IgG1

Chimeric IgG1
Human IgG1

Human IgG1

Human IgG4

fluid contained relatively high concentrations of antibodies (on

the order of 1 g/l), large-scale production of therapeutic mAbs
would require an excessively large number of mice. Further-
Type

IgG1

more, ascites induction in mice causes a significant amount of

pain and distress to the animal and is currently considered
unethical. Therefore, therapeutic mAb production was quickly
moved into mammalian cell culture expression systems, and
a4b7 integrin

virtually all therapeutic mAbs approved to date are produced in

mammalian cell culture systems. While the initial cell lines pro-
VEGFR2
Target

IL-17a

ducing therapeutic mAbs produced antibodies on the order of

CD20
HER2

PD-1

PD-1
GD2
IL-6

100–500 mg of antibody per liter of tissue culture [17], advan-

ces in antibody production technology, including production
cell line generation and optimization, large-scale bioreactor-
based tissue culture and fed-batch production, have resulted in
Trade name

significant increases in antibody production [18]. As a result of

(Pending)
Keytruda

these advances, therapeutic mAbs are often produced on the

Cyramza

Unituxin
Kadcyla

Gazyva

Entyvio

Opdivo
Sylvant

Withdrawn from market in 2010.

Withdrawn from market in 2014.

order of 5 g or more per liter of tissue culture in 15,000–

20,000 l bioreactors.
Approved for use in Japan.

Although these advances in antibody production technology

have reduced the cost of producing therapeutic mAbs from
NA: Not approved.
Pembrolizumab
International

Obinutuzumab

US$ 300/g to approximately US$ 100/g these costs, the down-

Ramucirumab

Secukinumab
Vedolizumab
Trastuzumab

Dinutuximab

stream quality control requirements and the large dose of anti-

Nivolumab
emtansine

Siltuximab

bodies required for efficacy still make the production of

name

therapeutic mAbs extremely costly [19,20]. This expense signifi-

cantly limits the use of therapeutic mAbs in the developing
†
‡
§

informahealthcare.com 209
 Review Suscovich & Alter

Heavy chain be replaced with a single or regularly

Vectored cDNA Ex vivo spaced injections resulting in the continu-
delivery Light chain modification ous and sustained production of thera-
cDNA peutically efficacious doses of mAbs. To
Non-vectored date, three main approaches have been
delivery used to ectopically produce mAbs in situ
(FIGURE 2): ex vivo genetic modification of
cells, viral vector-mediated gene transfer
and non-vectored gene transfer.

Ex vivo genetic manipulation of cells

In vivo, antibodies are produced by tran-
sient plasmablasts and long-lived plasma
cells, which are capable of secreting over
3000 antibody molecules a second [23].
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While this naturally high secretion rate

and long persistence in vivo [24] make
plasma cells ideal candidates for ex vivo
manipulation and engineering, these cells
are difficult to access (bone marrow or
Tumor tissue localized). Furthermore, these cells
already produce antibodies, making pro-
Pathogen Cytokines duction of additional antibodies in these
cells difficult, but not impossible [25].
However, a wide array of non-B cells has
Figure 2. Strategies to produce therapeutic monoclonal antibodies in situ. Three
been demonstrably modified to secrete
approaches have been used to produce therapeutic monoclonal antibodies in situ:
ex vivo modification of primary cells, vectored gene delivery and non-vectored gene therapeutic mAbs in vivo.
delivery. During ex vivo modification of primary cells, cells are harvested from the sub- The ideal target cell type for ex vivo
ject, transduced ex vivo with antibody-encoding vectors or transfected with antibody- modification should be easily accessible,
encoding nucleic acids, expanded and re-implanted into the subject. Alternatively, the manipulatable and engraftable. Early work
modified cells can be used to seed a bioscaffold and implanted as a pseudo-organ.
in this field demonstrated that many cell
During vectored gene delivery, the antibody-encoding genes are cloned into a viral vec-
tor (e.g., adenovirus or adeno-associated virus), which is then directly injected into the types can be modified to secrete mAbs
subject. By contrast, during non-vectored gene delivery, the antibody-encoding genes in vitro. Noël et al. demonstrated that
are directly injected into the subject as either plasmid DNA or RNA. DNA/RNA uptake myoblast, hepatocyte, keratinocyte and
can be increased using electroporation or packaging into lipid nanoparticles or polymers. fibroblast cell lines could all produce
mAbs at levels similar to or greater than
world where they could have the greatest impact. In particular, hybridomas in vitro following transduction with retroviral con-
one area in which mAbs are currently in development is as a structs and that similar result could be seen using primary
prophylactic against HIV infection [21]. However, to gain wide- cells [26,27]. Importantly, these cells retained their ability to pro-
spread utility as a prophylactic, it has been estimated that the duce mAbs following implantation in vivo, with consistent
cost of production of a mAb would have to be reduced to the secretion seen for over a year [28].
level of dollars per gram [19]. As these reductions are likely While the clinical efficacy of these antibodies was not tested
beyond what can be accomplished via advances in cell culture in these models, therapeutic effects following the ex vivo engi-
technology, novel approaches to produce therapeutic mAbs are neering of cells to secrete mAbs have been observed. Using a
imminently needed. The remainder of this review focuses on severe combined immunodeficiency mouse model of cancer,
one promising approach – in situ production of therapeutic human peripheral blood mononuclear cells were transduced
mAbs – and the strategies being used to accomplish this. with an antibody-encoding lentivirus and injected into mice [29].
Mice receiving cells transduced with the antibody-encoding len-
In situ production of therapeutic mAbs tivirus demonstrated a significant reduction in tumor growth
Given the high cost associated with the production of therapeu- compared to the control mice. Additionally, fibroblasts modified
tic mAbs, in situ mAb production has been a long-term goal of to secrete an HIV-specific mAb demonstrated antiviral activity
the gene therapy field. Using gene therapy-based approaches to following implantation into humanized mice [30], resulting in a
turn cells of the body into ‘in situ bioreactors’ producing thera- 100,000-fold lower viral load following HIV infection.
peutic mAbs, the high doses, long infusion times and side Intriguingly, while some early studies examining ectopic,
effects typically associated with mAb-based treatment [22] could in situ antibody production simply injected modified cells into

210 Expert Rev. Vaccines 14(2), (2015)


the mouse, the HIV-specific antibody-secreting fibroblasts were
injected not as a suspension of cells, but were rather implanted
as a pseudo-organ generated in vitro using bioscaffolds [30]. In
multiple studies examining the ectopic expression of therapeutic
proteins other than mAbs, implantation of transduced cells in a
bioscaffold resulted in increased protein expression and a pro-
longed therapeutic effect when compared with transduced cells
injected alone, strongly suggesting that implantation of mAb-
secreting pseudo-organs derived from a patient’s own cells has
therapeutic potential.
As a proof of concept for the engineering of therapeutic
mAb-secreting tissues, Compte et al. generated antibody-
secreting engineered blood vessels using primary human endo-
thelial cells and implanted these tissues into nude mice [31].
Stable antibody production was observed for at least 7 weeks.
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Importantly, a clear therapeutic effect was observed in the
mice receiving the antibody-secreting blood vessels, with a sig-
nificantly reduced tumor growth rate and a significantly
increased survival time. As additional advancements are made
in bioscaffold development and tissue engineering, the potential
of antibody-secreting engineered pseudo-organs may become
fully realized.
While many of the early studies reporting the ex vivo engi-
neering of cells to secrete mAbs focused on manipulating
terminally differentiated cell types ex vivo, terminally differenti-
ated cells often have a finite lifespan, potentially limiting their
use for the persistent, long-term production of mAbs in situ.
By contrast, the self-renewal capacity of stem cells affords them
a significantly longer lifespan, making them strong candidates
for modification ex vivo. To date, adult mesenchymal [32,33],
hematopoietic [34] and neural [35] stem cells have all been suc-
cessfully engineered in vitro to secrete mAbs in vivo. Hemato-
poietic stem cells engineered to secrete an HIV-specific antibody
readily differentiated into B cells that secreted the transduced
antibody in vivo [34], resulting in an 80-fold reduction in viral
load and a 200-fold reduction in the number of infected cells
following challenge with HIV. Similarly, mice injected with
mesenchymal stem cell-like stem cells engineered to secrete a
trastuzumab-like antibody demonstrated a significantly increased
survival rate compared with the control animals [32].
In addition to the increased lifespan of stem cells making
them an attractive candidate for ex vivo engineering, both mes-
enchymal stem cells and neural stem cells can home to and
seed tumors in vivo following injection [36,37]. As therapeutic
mAbs penetrate into solid tumors poorly [16], the homing and
seeding of the tumor with cells engineered to secrete antibodies
targeting the tumor allows for more effective eradication of the
malignant cells. In fact, neural stem cells engineered to secrete
trastuzumab readily homed to the tumor and secreted anti-
body [35]. Importantly, the intratumoral concentration of trastu-
zumab was significantly increased in mice treated with the
antibody-secreting neural stem cells compared with mice
directly treated with trastuzumab [35].
It should be noted that while the tumor-homing potential of
stem cells can be beneficial, stem cells have also been shown to
informahealthcare.com
In situ production of therapeutic mAbs Review
have pro-angiogenic [38] and immunomodulatory effects [39].
These effects can increase tumor growth and metastasis [40],
potentially limiting the effectiveness of ex vivo-engineered stem
cells as a therapeutic. To bypass this limitation, ex vivo-
engineered stem cells could be implanted in association with a
bioscaffold, limiting their dissemination throughout the body.
In support of this, stem cells engineered to secrete an antibody
targeting a tumor antigen expressed on the cell surface and
implanted in a bioscaffold at a site distal to the tumor still
demonstrated a therapeutic effect: tumor growth in mice receiv-
ing the antibody-secreting stem cells was significantly reduced
in comparison to the mice receiving the control stem cells [32].
Vectored production of therapeutic mAbs
Although the ex vivo engineering of autologous cells to secrete
therapeutic mAbs has shown potential, the cost and labor
required to collect, engineer and implant the modified cells will
likely limit the scalability of this approach. Therefore, addi-
tional strategies to produce antibodies in situ are needed. Vec-
tored gene therapy, that is, the delivery of foreign genes using
viral vectors, has a long history of being used to express thera-
peutic proteins in vivo, including antibodies. Even though strat-
egies employing other viruses [41–43] and bacteria [44] have been
used, the vectors typically used in vectored gene therapy are
adenovirus (AdV), adeno-associated virus (AAV) and retrovi-
ruses. While retroviruses have been used to ectopically express
mAbs in vivo with therapeutic effects [45,46], because of the
inherent risk of insertional mutagenesis due to integration of
the retroviral genome, most of the research harnessing viral vec-
tors has used AdVs or AAVs.
Adenoviral-mediated gene delivery
AdVs are non-enveloped, double-stranded DNA viruses that
infect a large variety of cell types. AdVs are one of the most
frequently used and best-studied viral vectors for the expression
of therapeutic proteins in vivo, and multiple studies have dem-
onstrated in vivo production of mAbs using antibody-encoding
AdVs [47–58]. Furthermore, in many of these studies, the
expressed mAbs had a demonstrable therapeutic effect, includ-
ing inhibition of tumor growth [50–53]; protection from infec-
tion with Yersinia pestis [54,55], respiratory syncytial virus [57]
and West Nile virus [58]; and the suppression of pulmonary
edema [56].
While therapeutic effects of AdV-encoded mAbs have been
observed, the expression of mAbs in vivo following infection
with AdVs is highly variable. Following systemic administration
of antibody-encoding AdVs, peak concentrations greater than
1 mg/ml have been reported early (Day 3) after AdV deliv-
ery [54]. However, by Day 5, the antibody titer had begun to
decline, and long-term concentrations ranging from 20 ng/ml
to 40 mg/ml have been reported [48,51], illustrating the transient
nature of AdV-mediated gene delivery. Importantly, in the
majority of the studies in which a therapeutic or protective
effect was observed, the challenge was carried out near the peak
antibody titer. Moreover, because many AdVs are highly
211
 Review Suscovich & Alter
prevalent, pre-existing immunity can greatly affect the clinical
efficacy of AdV-mediated antibody gene transfer [59,60]. Thus,
the prevalence of pre-existing anti-AdV immunity coupled with
the transient nature of AdV antibody expression significantly
limits enthusiasm for using AdVs as a delivery vehicle for the
in situ expression of mAbs [61].
AAV-mediated gene delivery
In contrast to the limited durability of AdV-mediated ectopic
protein expression, expression of proteins from AAVs can per-
sist for years [62]. AAVs are small, single-stranded DNA viruses
that are capable of infecting both dividing and non-dividing
cells [63]. Furthermore, because AAVs are not associated with
any known disease and AAV-based vectors (which lack the viral
rep and cap genes required for integration) do not integrate
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into the genome, AAVs have become the most attractive viral
vector for gene therapy [64–67], and the first AAV-based gene
therapy product was recently approved for use in Europe
(Glybera; [68]).
In 2002, Lewis et al. reported the first instance of a mAb
being produced from an AAV-based vector [69]. Although
only low-level antibody production was observed in vivo
(<10 mg/ml), this low antibody titer persisted for at least
6 months. While low antibody levels were observed in this ini-
tial experiment, follow-up experiments using optimized anti-
body expression cassettes significantly enhanced antibody
levels [70]. Further refinements to the antibody-encoding cas-
sette, including the utilization of a self-cleaving picornavirus 2A
peptide, resulted in a peak serum antibody level greater than
8 mg/ml, with sustained production of 1 mg/ml up to 4 months
post-AAV delivery [71]. Not surprisingly, the expression of a
therapeutic mAb (anti-mouse VEGFR2) at this level had signifi-
cant therapeutic effects: tumor dormancy was observed in over
half of the treated mice for 3 months, with complete regression
of even large tumors observed in a subset of treated mice.
Since these initial descriptions of AAV-mediated in situ
expression of mAbs, a large number of additional studies have
demonstrated the therapeutic effectiveness of AAV-expressed
mAbs. AAV-mediated expression of mAbs has now been shown
to suppress tumor growth in mouse models of cutaneous T-cell
lymphoma [72], colon cancer [73], liver cancer [73,74] and lung
cancer [74–76]. Furthermore, AAV-mediated antibody expression
has been effective against tumors, both prophylactically and
therapeutically [77]. Beyond tumor models, AAV-mediated
expression of mAbs has also been shown to provide protection
from infection with HIV [70,78,79], respiratory syncytial virus [57],
influenza [80], prion disease [81], anthrax [82] and malaria [83] and
the first clinical trials utilizing AAV-mediated expression of a
mAb (two different HIV-specific mAbs) are currently under-
way [84,85]. Intriguingly, AAV-mediated expression of mAbs has
demonstrated therapeutic potential in areas in which mAbs have
only recently begun to be exploited, including addiction [86,87],
Alzheimer’s disease [88] and macular degeneration [89].
While AAV has become the de facto vector of choice for the
expression of therapeutic antibodies in vivo, it is not without
212
its limitations. First, unlike AdV-mediated antibody expression,
which occurs rapidly, the expression of mAbs using AAV can
take weeks to reach therapeutically efficacious or protective
concentrations. De et al. treated two groups of mice with either
an AdV expressing a mAb against Bacillus anthracis lethal toxin
or an AAV expressing the same mAb [82]. The mice were then
challenged with B. anthracis lethal toxin at different time points
(1, 3, 7, 14, 28, 64 and 182 days) post-AdV/AAV treatment.
In contrast to the AdV-treated mice, which were protected
from the toxin when challenged 1 day post-treatment, AAV-
treated mice were only protected beginning 14 days post-treat-
ment. However, the protection in the AdV-treated mice waned
between Week 8 and Week 26; the AAV-treated mice were
completely protected from the toxin even at Week 26. These
results suggest that in instances where immediate, but not long-
term, antibody expression is required, such as during an infec-
tious pandemic or bioterrorism event, AdV-mediated antibody
expression may be superior to AAV-mediated antibody expres-
sion. By contrast, AAV may be the vector of choice when
slower, but persistent, antibody expression is required, such as
for cancer immunotherapy. When both rapid and persistent
antibody expression is required, co-delivery of antibody-
encoding AdVs and AAVs will likely prove effective, as demon-
strated by De et al. [82].
Non-vectored production of therapeutic mAbs
Vector-mediated expression of mAbs has shown success in ani-
mal models and has begun Phase I clinical trials; however,
vector-mediated delivery has several disadvantages that are
shared among vectors, including the development of or pre-
existing anti-vector immunity, a limited coding capacity and
difficulties in producing clinical-grade products, which may
limit its broad use in clinical settings, especially for the treat-
ment of otherwise healthy individuals. Unlike vectored delivery,
non-vectored gene delivery (i.e., direct delivery of DNA or RNA)
is not confined by many of these limitations as an immune
response does not develop against transferred DNA/RNA, there
are no restrictions on the size of the construct, it can be produced
easily and cheaply in a cell-free manner and it has an excellent
safety profile.
DNA as a vehicle
However, in spite of these apparent advantages, in situ expres-
sion of mAbs via direct delivery of DNA has gained limited
traction largely due to the poor expression observed following
injection. Tjelle et al. were the first to report the in situ pro-
duction of mAbs by the direct transfection of plasmid DNA in
2004 [90]. Following a single injection of DNA and in vivo
electroporation, low levels of a human mAb (50–200 ng/ml)
could be detected in the mouse. While the antibody titers
in vivo declined after 7–14 days, this decline was associated
with the development of an immune response against the
human mAb, and the plasmid DNA-mediated production of a
fully mouse version of the same antibody persisted in the
serum for over 7 months. Furthermore, when injected into
Expert Rev. Vaccines 14(2), (2015)

severe combined immunodeficiency mice, which lack T or
B cells, increased antibody titers were observed, with no decrease
in antibody expression observed after 28 days [91].
Similarly, Perez et al. also reported in situ mouse mAb
expression following the delivery of DNA via in vivo electropo-
ration [92]. A peak level of 1.5 mg/ml of antibody was observed;
however, this concentration decreased over time, with average
levels of approximately 200 ng/ml observed 5 months post-
plasmid delivery. Interestingly, by incorporating tetracycline-
responsive elements into the promoter controlling the expression
of the antibody, they demonstrated that it was possible to regu-
late the expression of the antibody in vivo via treatment with the
antibiotic doxycycline. Similar regulated in vivo antibody expres-
sion following AAV-mediated gene delivery has also recently
been reported [93]. Overall, these studies demonstrate that injec-
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tion of mAb-encoding DNA can result in durable mAb expres-
sion, albeit at low levels.
Importantly, despite the low levels of mAbs produced
in vivo, protection from infection has been observed following
plasmid DNA-mediated antibody delivery [94]. Following injec-
tion of a plasmid encoding an influenza-specific mAb, serum
levels of 4–20 mg/ml and pulmonary levels of 50–300 ng/ml
were observed 20 days after injection. Despite these relatively
low antibody titers, following challenge with a lethal dose of
influenza on Day 20, all of the plasmid-treated mice demon-
strated a significant reduction in lung viral load and weight
loss, with a corresponding increase in survival. Furthermore,
the protection afforded by these antibodies persisted even
though the circulating and tissue titers declined over time. One
hundred twenty days after plasmid injection, the antibody levels
in the blood and lung decreased to 600 ng–4 mg/ml and 4 ng–
60 ng/ml, respectively. However, even with these low antibody
titers, a significant reduction in lung viral load was observed
following challenge on Day 120. Combined, these data demon-
strate that plasmid DNA can be used as a delivery modality to
produce persistent levels of functional mAbs in vivo.
While only relatively low levels of mAb expression following
delivery of plasmid DNA in vivo have been reported, the con-
centration of antibody required for a therapeutic effect differs
drastically depending on the disease being targeted. While ther-
apeutic mAbs used for the treatment of cancer often require
high doses, this is largely due to the poor penetrance of mAbs
into solid tumors [16]. By contrast, mAbs used for the treatment
of autoimmune disease, such as ustekinumab (anti-IL-12/IL-23)
and adalimumab (anti-TNF-a), require significantly lower
serum concentrations to be effective (6 mg/ml for adalimumab
and 0.31 mg/ml for ustekinumab) [12]. Therefore, while the
serum concentrations currently attainable following delivery of
antibody-encoding plasmid DNA may not be sufficient for the
treatment of cancer, these levels are above or near the concen-
trations associated with a therapeutic effect in the treatment of
autoimmune disease.
Importantly, injections at multiple sites can boost in vivo
antibody titers. While delivery into one site resulted in peak
expression levels of approximately 10 mg/ml, declining to
informahealthcare.com
In situ production of therapeutic mAbs Review
approximately 5 mg/ml after 70 days, delivery into three differ-
ent sites resulted in peak expression levels of approximately
30 mg/ml. Furthermore, following injection into three sites,
the titer of the antibody remained over 10 mg/ml over the
course of 70 days [94]. These results suggest that with further
refinement of the delivery strategy, it may be possible to
increase in situ antibody production even higher.
RNA as a vehicle
While several studies have demonstrated the production of
therapeutic mAbs in situ following delivery of plasmid DNA,
to date, there have been no studies demonstrating production
of mAbs directly from RNA. Importantly, RNA has several
advantages over DNA as a non-vectored delivery strategy,
including reduced immunogenicity and no risk of genomic
integration. Furthermore, unlike DNA, RNA does not require
entry into the nucleus to be effective, potentially eliminating a
significant limitation of DNA [95].
Although there have been no studies demonstrating the
in situ production of mAbs using RNA, RNA has been success-
fully used to produce secreted proteins in situ [96–98]. Using a
chemically modified mRNA encoding erythropoietin,
Kormann et al. demonstrated that a single intramuscular injec-
tion of 5 mg of mRNA resulted in a fourfold increase in the
serum erythropoietin level 14 days post-delivery, with the
erythropoietin level remaining significantly increased 28 days
post-injection [98]. This increased erythropoietin level conse-
quently resulted in a significantly increased hematocrit level in
the mice both 14 and 28 days following delivery of the
mRNA. Similarly, Kariko et al. demonstrated that intraperito-
neal injection of 100 ng of HPLC-purified, chemically modi-
fied, erythropoietin-encoding mRNA resulted in a significant
increase in the serum erythropoietin level that persisted for
4 days post-injection, with corresponding increases in hemato-
crit persisting for 20 days post-injection [96]. Importantly, injec-
tion of recombinant erythropoietin protein resulted in only a
transient (6 h) increase in the serum erythropoietin level, dem-
onstrating that the persistent increase in erythropoietin levels
following delivery of mRNA was due to the continued produc-
tion of erythropoietin in situ. Furthermore, as little as 1 ng of
this mRNA was shown to have a therapeutic effect, illustrating
the potency of mRNA as a delivery modality [96].
Beyond erythropoietin, both surfactant protein B (SFTPB) [98]
and VEGF [97] have been produced in situ using mRNA, with
significant therapeutic effects. Following delivery of a SFTPB-
encoding mRNA directly to the lung of mice with a condi-
tional deficiency of SFTPB using an endotracheally inserted
microsprayer, near wildtype levels of SFTPB were detected [98].
This level of SFTPB resulted in a significant increase in the
survival of the conditionally deficient mice compared to the
control mice. Furthermore, repeated delivery of the SFTPB-
expressing mRNA (twice a week) protected the mice from
pulmonary failure until the end of the study, further demon-
strating the therapeutic effects of the delivery of mRNAs
in vivo. In a different study, Zangi et al. demonstrated the
213
 Review Suscovich & Alter
therapeutic effectiveness of mRNA-delivered VEGF in a model
of myocardial infarction [97]. The intramyocardial injection of
the VEGF-encoding mRNA at the time of infarct induction
resulted in a significant increase in both the short-term and
long-term survival of the mRNA-treated mice compared to the
untreated controls.
While there have only been a handful of studies expressing
therapeutic proteins in situ using RNA, and no studies demon-
strating in situ production of mAbs, the therapeutic potential
of RNA is likely to be greater than that of DNA, especially
when transient expression is needed. For example, prolonged
exposure to VEGF can have detrimental effects, including
increased vascular permeability [99]. Therefore, while transient
expression of VEGF can be beneficial, persistent expression
may be harmful. In support of this, while mRNA-mediated
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expression of VEGF resulted in prolonged survival of mice fol-
lowing myocardial infarct induction, plasmid DNA-mediated
expression of VEGF actually reduced survival, highlighting the
benefits of transient expression from RNA [97].
However, while the transient nature of RNA can be an
advantage, it can also significantly limit broader therapeutic
potential. To circumvent this, self-replicating RNAs (or repli-
cons) based on single-stranded RNA viruses (e.g., flaviviruses
and alphaviruses) have been developed [100–103]. These replicons
encode all of the viral proteins required for viral replication,
but none of the viral structural proteins, which have been
replaced with one or more genes of interest. Following delivery
into a cell, the viral non-structural proteins are produced,
allowing for the replication of the replicon and the expression
of the gene of interest. Importantly, the gene of interest is
expressed at an extremely high level, accounting for 10–20% of
the total protein of the cell [104]. Furthermore, due to the self-
propagation of the replicon, the gene of interest continues to
be expressed in cells irrespective of cell division, and long-term
(>2 months) replicon-mediated gene expression has been
observed [101,102]. Therefore, via the use of replicons, it will
likely be possible to durably express high levels of therapeutic
proteins, including mAbs, in situ.
Conclusions
Since the first serum transfer experiments were performed in
the 1890s, antibody-based therapeutics have evolved rapidly.
While therapeutic antibody infusion and ex vivo cell manipula-
tion have shown clinical efficacy in humans and animal models,
respectively, these techniques are costly and slow. Therefore, for
indications in which mAbs must be rapidly delivered, such as
during a pandemic, this approach cannot be used. To date, vec-
tored gene delivery has demonstrated the most success in the
in situ production of mAbs, and approaches utilizing this tech-
nique have advanced to clinical trials. Depending on the vector
used, either rapid, short-term antibody production (e.g., AdV
vectors) or delayed, long-term antibody production (e.g., AAV
vectors) can be observed in situ. However, viral vectors are lim-
ited by pre-existing immunity against the vector, the relatively
low coding capacity of the commonly used vectors and
214
manufacturing difficulties. Conversely, synthetic approaches,
including the delivery of non-vectored nucleic acids, offer
remarkable flexibility and the greatest speed of production, have
shown promise in animal models and, while still in their
infancy, are currently being vetted in clinical trials. However,
each of the approaches discussed in this review has their advan-
tages and disadvantages. Importantly, as the range of indications
for which therapeutic mAbs can be used is diverse and the dos-
ing requirements vary considerably based on the indication, it is
likely that no single strategy will be ideal, but rather, distinct
strategies for antibody gene transfer will be of utility for spe-
cific clinical indications.
Expert commentary
Despite their promise, key technological advances are still
required to make either vectored or non-vectored antibody gene
delivery approaches a reality. To gain widespread acceptance
for vectored gene delivery, particularly with AAV, advances
must be made in limiting/subverting anti-vector immunity,
high-throughput production methodologies and strategies to
turn off the vector. For non-vectored gene delivery, delivery
approaches must improve.
Vectored delivery of mAb-encoding genes has proven to be
quite effective as a prophylactic against a number of infectious
diseases, including HIV and influenza [79,80]. However, while
AAV-based vectors are largely considered safe, with the first
AAV vector-based gene therapy approved for use in Europe,
treatment with AAV induces both a humoral [105] and T-cell
[106] response against the vector itself. Furthermore, while mul-
tiple AAV subtypes have been identified, a significant portion
of the population has already been exposed to these viruses and
thus harbors pre-existing immunity against many of these sub-
types [107]. Because even low levels of anti-AAV antibodies can
limit transduction and gene expression [108], the presence of
pre-existing antibodies targeting AAV can significantly decrease
the efficacy of AAV-based mAb expression strategies, greatly
limiting the utility of AAV in particular geographic areas or for
applications requiring repeated administration. While several
promising approaches to subvert this immunity have been
developed, including chemical [109] and genetic [110] modifica-
tion of the viral capsid, in order to enable the wide use of
AAV-based gene therapy, additional strategies to evade the pre-
existing anti-vector immunity and prevent the induction of
de novo anti-vector immune responses must be developed.
Similar to the mAb therapeutics currently in use, vectored
gene delivery approaches are produced using eukaryotic cell cul-
ture, which can make manufacturing costly and compli-
cated [111,112]. Currently, it is estimated that 104–105 viral
particles can be produced from a singly transfected cell [113].
However, one of the ongoing AAV-mediated, in situ antibody
production clinical trials will deliver 4  1012–1.2  1014 viral
particles per subject. Therefore, to gain widespread use, large
amounts of virus must be generated [85]. As approximately 2 
1014 viral particles can be grown per 1 liter culture [114], large-
scale batch cultures must be generated and quality controlled
Expert Rev. Vaccines 14(2), (2015)
 In situ production of therapeutic mAbs Review

to produce sufficient clinical-grade product. While significant
advances have been made in producing recombinant viral vec-
tors, including the use of HSV-complemented cultures [115] and
insect production cell lines [114], further advances will be
required to make production of viral vectors economical.
While vectored gene delivery has been exploited more widely
than non-vectored gene delivery, non-vectored gene delivery
promises to provide a flexible, non-immunogenic, high-
throughput, cost-effective solution for vectored-antibody deliv-
ery approaches. Currently, the greatest limitation associated
with non-vectored gene delivery is the poor titers obtained fol-
lowing plasmid DNA delivery. However, all of the initial stud-
ies have been carried out using suboptimal electroporation
conditions/equipment. Yet, delivery strategies are rapidly evolv-
ing, already showing great promise in the expression of a mAb
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field; a goal that has not been met. However, with the recent
advancements in both vectored and non-vectored gene delivery
approaches, we are closer than ever in attaining this goal. The
first clinical trials for the in situ production of mAbs are cur-
rently underway, scheduled for completion by January
2015 [85]. These trials make the critical step from animal mod-
els to humans and will provide the pivotal proof-of-concept
studies that in situ production of therapeutic mAbs is feasible.
Over the coming 5 years, the expected advancements in both
vector design/manufacturing and vehicles for the delivery of
non-vectored DNA/RNA, coupled with advancements in anti-
body discovery techniques and antibody engineering, including
the use of recombinant antibody fragments (e.g., single-chain
Fv fragments, minibodies, camelid-based nanobodies and
immunoadhesins) [21], will allow the potential of the in situ

fragment at levels greater than 3 mg/ml [116]. Additionally,
non-electroporation-based strategies have improved dramati-
cally over the past several years [117], with lipid-based delivery
vehicles showing great promise for RNA delivery. Thus, with
further technical improvements in electroporation and advan-
ces in material sciences, novel delivery strategies will likely
increase the in situ expression of non-vectored antibody gene
delivery strategies further.
Five-year view
The in situ production of therapeutic proteins, including thera-
peutic mAbs, has been a long-term goal of the gene therapy
production of mAbs to become fully realized. Furthermore, as
the indications for the administration of mAbs broaden
beyond cancer, autoimmune disease and infection, in situ pro-
duction of mAbs will gain increased widespread use.
Financial & competing interests disclosure
The authors have no relevant affiliations or financial involvement with
any organization or entity with a financial interest in or financial conflict
with the subject matter or materials discussed in the manuscript. This
includes employment, consultancies, honoraria, stock ownership or options,
expert testimony, grants or patents received or pending, or royalties.
No writing assistance was utilized in the production of this manuscript.

Key issues
. Monoclonal antibody (mAb)-based therapeutics are one of the fastest growing classes of therapeutics, with over 300 currently in
various stages of clinical development. However, the large doses required, coupled with the high cost of production and long-
administration times, limits the use of mAb-based therapeutics as a widespread prophylactic or therapeutic treatment in resource-poor
settings.
. Three main approaches have been used for the in situ production of therapeutic mAbs: ex vivo cell manipulation, vectored gene
delivery and non-vectored gene delivery.
. Multiple cell types have been successfully modified ex vivo to produce mAbs with demonstrable therapeutic effects, and advancements
in tissue engineering will likely increase the titer of antibodies obtainable. However, the therapeutic usefulness of this approach is limited
by the cost and labor required to collect, engineer and implant the modified cells as well as the mutagenic risk associated with ex vivo
manipulation of cells.
. Vectored gene delivery has demonstrated the greatest success for in situ mAb production due to its ability to generate high titers of
mAbs. These antibodies have shown significant therapeutic benefits, particularly as a prophylaxis against infectious diseases, and the first
clinical trials using this approach have begun. However, the therapeutic usefulness of this approach is limited by the development of or
pre-existing immunity against the vector, the relatively low coding capacity of the commonly used vectors and
manufacturing difficulties.
. Non-vectored gene delivery utilizing plasmid DNA has shown limited success for the in situ production of mAbs, while RNA
has been successfully used to express therapeutic secreted proteins in situ, but not antibodies. While this approach still struggles
to achieve clinically effective titers, recent advances in in vivo nucleic acid delivery approaches may drastically improve this
therapeutic approach.

informahealthcare.com 215
 Review Suscovich & Alter
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