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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5608_Библиотеки_им_академика_М_И_Перельмана.pdf
Chapter 9
CAR-T Bioprocessing
AdebolaAdeniran, SalinaHandy, andAbdulrahmanBaki
Abstract Over the past 30 years, immunotherapy has grown from bench experi-
ments to a viable clinical option in the battle against cancer with nearly 1000 currently registered clinical trials Ivica NA, Young CM. Tracking the CAR-T
Revolution: Analysis of Clinical Trials of CAR-T and TCR-T Therapies for the
Treatment of Cancer (1997-2020). Healthcare (Basel) 19;9(8):1062.
In chimeric antigen receptor T-cell (CAR-T) immunotherapy, a patient’s own
immune cells are engineered to express a CAR which recognizes cancer cells,
expanded exvivo and then introduced back into the patient, where the engineered
cells will mount an immune response against the targeted cancer cells.
Immunotherapy manufacturing requires careful consideration of the collection of
immune cells from the patient, introduction of receptor into the cells, method of cell
expansion, formulation for cryopreservation, and the unique supply chain that must
connect clinics, hospitals, and manufacturers to supply individual patients. In this
chapter, we provide a brief overview of the current clinical landscape, discuss current practices and key challenges at each manufacturing step using clinical examples where available, and highlight emerging technologies that may accelerate the
progress of CAR-T therapies.
Keywords CAR-T · T-cell · Immunotherapy · Bioprocessing · Apheresis · Vector ·
Activation · Transduction · Expansion · Automation · Formulation ·
A. Adeniran (*)
Cell Therapy Process Development, The Center for Breakthrough Medicine,
King of Prussia, PA, USA
e-mail: badeniran@cfbm.com
S. Handy
Manufacturing Science and Technology, Lonza, Houston, TX, USA
A. Baki
Cell and Gene Therapy, Medicinal Science and Technology, GlaxoSmithKline,
Stevenage, UK
221© The Author(s), under exclusive license to Springer Nature Switzerland AG 2024
K. Gadamasetti, S. A. Kolodziej (eds.), Bioprocessing, Bioengineering
and Process Chemistry in the Biopharmaceutical Industry,
https://doi.org/10.1007/978-3-031-62007-2_9
222
Cryopreservation · Supply chain · Yescarta · Kymriah · Tecartus · Breyanzi ·
Abecma · Carvykti
A. Adeniran et al.
9.1 Introduction
9.1.1 Generation andFunction
Chimeric antigen receptor T-cells, also known as CAR-T cells, represent an exciting, growing class of anticancer immunotherapies. In CAR-T immunotherapy, a
patient’s own immune cells are engineered to express a chimeric antigen receptor
(CAR) which recognizes cancer cells, expanded exvivo and then introduced back
into the patient, where the engineered cells will mount an immune response against
the targeted cancer cells. CAR-Ts have proven themselves to be a promising therapy, with ongoing remissions reported to last as long as a decade in a small subset
of leukemia patients [1]. However, it should be noted that success with CAR-T cells
is generally limited to hematological malignancies and longer survival rates are
observed in patients with lower disease burdens [2].
CARs consist of an ectodomain and intracellular signaling stimulatory domains
[3]. The ectodomain contains an antigen-binding domain, which is typically a
single- chain fragment variant (scFv) from an antibody, a signal peptide to ensure
proper trafcking to the cell membrane, and a hinge region between the signal peptide and recognition domains [4]. The transmembrane domain, a hydrophobic alpha
helix, connects the ectodomain and the intracellular signaling domain. The intracellular signaling domain is responsible for initiating the intracellular signaling cascade to produce an anticancer immune response (Fig.9.1).
To date, CAR-T therapies such as Yescarta, Kymriah, Tecartus, Breyanzi,
Abecma, and Carvykti have been approved for a variety of leukemias and lymphomas across different patient populations (Table9.1). In this chapter, we review the
key steps of CAR-T manufacturing, highlighting many available technologies, challenges to the eld, and provide relevant examples from clinical trial manufacturing
where available.
9.2 Transgene andVector Bioprocessing
9.2.1 Introduction
The intrinsic ability of viruses to incorporate genetic material into a cell for the
purpose of altering cell function for a therapeutic benet has made viral vectors an
extremely useful tool in gene therapy, being considered key raw material required
for CAR-T cell manufacturing in the United States [5]. As the amount of success in
9 CAR-T Bioprocessing
223
Fig. 9.1 CAR-T cell. Depiction of a CAR-T cell interacting with the target antigen on a cancer
cell. The CAR is composed of a target binding domain, a hinge region to connect the target binding
domain to the transmembrane domain, the co-stimulatory domain, and the signaling domain
clinical trials and potentially approved CAR-T drug products continue to increase
and rapidly advance to mainstream manufacturing, large-scale production and purication of high-titer viral vectors are becoming key [6] to meeting demand and
potential global distribution.
Notably three viral vector technologies, adenovirus, adeno-associated viruses
(AAV), and lentiviruses, have demonstrated much success in both clinical and preclinical studies [7] attracting global attention and focus into the development of
viral vector technology. In particular, lentiviral vectors (LVVs), derived from lentiviruses, have become increasingly popular especially for CAR-T drug product
development, as the rst lentiviral vector-based CAR-T immunotherapy,
Kymriah®(Novartis), was FDA approved in 2017. Due to their increased safety proles, broad tropism, and ability to integrate transgenes of interest into host cell
genomes, LVVs have been the major vector of choice for treatments which require
introduction of genetic material into host cells. For the purpose of this section and
the following overview, the bioprocessing of LVVs will be reviewed as well as key
challenges, current methods, and emerging technologies.
224
Table 9.1 Approved CAR T therapies to date (Wang etal. [20]; United States Food and Drug
Administration 2021)
CAR T therapy Indications Company
Yescarta
(Axicabtagene
Ciloleucel)
Kymriah
(tisagenlecleucel)
Tecartus
(brexucabtagene
autoleucel)
Breyanzi
(Lisocabtagene
maraleuce)
Abecma
(idecabtagene
vicleucel)
Carvykti
(ciltacabtagene
autoleucel; ciltacel)
Abbreviation: USFDA United States Food and Drug Administration
Adult relapsed or refractory large
B-cell Lymphoma
Adult relapsed or refractory large
B-cell Lymphoma
Pediatric and young adult relapsed
or refractory acute lymphoblastic
leukemia (ALL)
Adult relapsed or refractory mantle
cell lymphoma (MCL)
Adult relapsed or refractory B-cell
precursor acute lymphoblastic
leukemia (ALL)
Adult relapsed or refractory large
B-cell lymphoma
Adult relapsed or refractory
multiple myeloma
Adult relapsed or refractory
multiple myeloma
Kite Pharma 2017
Novartis 2017
Kite Pharma 2020
Juno Therapeutics 2021
Celgene 2021
Janssen Biotech,
Legend Biotech
A. Adeniran et al.
First
approval
year
(USFDA)
(USFDA)
(USFDA)
(USFDA)
(USFDA)
2022
(USFDA)
9.2.2 Lentiviral Vector Design
Lentiviral vectors (LVVs) are derived from the human immunodeciency virus type
1 (HIV-1) and have conventionally been produced using a multi-plasmid DNA construct system where separate plasmid constructs are carry essential genomic information required for viral vector production. With time, as our viral vector knowledge
and experience have expanded, the LVV construct design has undergone multiple
rounds of modications and enhancements with a particular focus in increasing
safety. As a result, three generations of LVVs have been developed over time each
with an increasing safety prole, and in addition, genetic modications suggested to
increase gene expression, transduction efciency, and vector stability during bioprocessing steps. For clinical applications, the third-generation construct design,
thought to be the modern packaging system [8], is the vector design of choice and
most widely used, due to its increased safety prole which avoids the potential for
replicative competent virus to be generated.
To provide some context, the third-generation construct design, modied from
earlier generations of LVV construct designs, has undergone the complete removal
of regulatory genes, such as the tat gene, from packaging plasmid constructs, while
9 CAR-T Bioprocessing
225
the rev gene, originally within the packaging plasmid, was placed on a separate
fourth plasmid construct of its own. In addition, deletions were introduced into the
3’LTR region of the transfer plasmid to reduce the risk of generating replication
competent LVVs (RCL), which thus created the third-generation self-inactivating
lentiviral vector (SIN) [7]. Reports of use of earlier generations of LVVs indicated
their cause of the development of lymphomas in primate models due to the generation of replicative competent virus [9] which had raised a safety concern around the
use of these LVVs. Generation of RCLs can occur as a result of recombination
events that reconstitute a pathogenic virus [10] which can lead to adverse events.
However, by removing unessential genes and splitting and modifying viral genetic
sequences within the construct design used to produce LVVs, the development of
the third-generation SIN LVVs has greatly reduced the potential risk of generating
RCL rendering it an unlikely event. Nonetheless, to ensure safety to patients, the
Food and Drug Administration (FDA), the European Medicines Agency, and most
regulatory agencies have required that all cell products transduced with retroviral
vectors be tested for the presence of RCL prior to infusion of the cell drug product
into patients for treatment. In addition, the FDA suggests that every 3 months
patients be tested for the presence of recombinant virus during the rst year of having received the gene therapy [3, 9].
The ability of retroviral-based vectors, such as LVVs, to stably integrate genetic
information into the host genome is a key advantage with the potential to increase
cell drug product efcacy in patients by allowing long-term sustained transgene
expression and has been utilized as a powerful technique allowing for gene expression manipulation and engineering capabilities [10]. However, the integration
potential of LVVs as retroviral-based vectors has raised additional safety concerns
around their use, and their ability to cause insertional mutagenesis. The gammaretroviral vector, a retroviral-based vector, has demonstrated an oncogenic and transformative potential in transduced cells as a result of insertional mutagenesis. Several
accounts of patients experiencing adverse events leading to the development of leukemia have been reported [11] with the use of gammaretroviral vectors. Insertional
mutagenesis is a phenomenon that occurs when the viral vector integrates genetic
material into an area of the host genome that is essential for cellular function causing a genetic mutation or disruption of cell function which has a potential to become
cancerous.
The process of integration for most retroviruses is not random, and each class of
retrovirus has its characteristics preferential site of integration. In comparison to
other retroviruses, lentiviruses have been shown to prefer integration of genetic
material within transcriptional units, in addition they also have the unique ability to
translocate across the nuclear pore of intact nuclear envelopes which may increase
their integration and oncogenic potential [3]. To address this concern, it is imperative to design efcient and safe viral vectors by achieving tissue or cell-type specicity [7].
A potential alternative, which some groups have investigated to address some of
the concerns around the use of integrating LVVs, is the development of non-integrating lentiviral vectors (NILVs). Some reports have demonstrated that integration
226
A. Adeniran et al.
into the host cell genome may not be required for transgene expression in target
cells both invitro and invivo [11]. NILVs have been developed by inducing genetic
mutations into the genes responsible for the integration of viral DNA into the host
genome. These modications result in a vector episomal DNA species that is nonintegrative and remains within the nucleus as extrachromosomal molecules. Though
there have been reports of many applications for this technology, there has also been
low levels of expression associated with NILVs in comparison to their integrating
LVVs counterparts. For other clinical applications that require high expression of
the transgene further investigation into techniques increasing transgene expression
will need to be explored [11], but results using these types of vectors prove promising and there may be therapeutic advantage to look forward to as progression
continues.
Some of the notable advantages of using LVs for clinical applications are their
ability to transduce both dividing and non-dividing cells in comparison to other
viral vectors of the Retroviridae family, such as gammaretroviral vectors, which are
only able to transduce actively dividing cells. This characteristic provides a major
benet allowing for a variety of cell types such as neurons, hematopoietic stem
cells, and immune system cells, like T-cells, to be transduced and receive therapeutic genetic material [8]. LVVs also exhibit reduced immunogenicity, ideal for use in
patients, reducing the likelihood of adverse events and severe immune reactions to
occur. LVVs also have the ability to express multiple genes from a single vector
which could be benecial for certain diseases that require high-level expression of
multiple genes [7].
An added benet has been the ability to pseudotype the viral envelope, which has
broadened the range of tropism, where the HIV-1 envelope can be exchanged for an
envelope from another virus. The more commonly used and widely considered gold
standard envelope protein is the vesicular stomatitis virus Indiana glycoprotein
envelope (VSV-G) [8]. The VSG-G glycoprotein provides not only a broad tropism
for a variety of cell types but increased vector stability which is extremely important
for bioprocessing steps, though the VSV-G envelope has displayed cytotoxic effects
in host cells when not tightly regulated or when overexpressed which can limit its
application to the transient transfection platforms [8]. Other viral envelope proteins
have been investigated that exhibit decreased toxicity, comparable tropism and stability, for instance the glycoproteins, Cocal-G, PIRY, Chandipura, and VSV-New
Jersey which all derive from the same vesiculovirus family as VSV and the RD114
viral envelope deriving from the feline endogenous retrovirus [8] could all be potential alternatives to circumvent potential toxicity due to the viral envelope.
As we continue to expand our knowledge and acquire more information
about these efcient genetic delivery systems, it is important to keep in mind, for the
purpose of CAR-T drug product manufacturing, that optimization of the viral
vectors must also be conducive to facilitating a scalable production process that
will enable robustness, reproducibility, consistency, and cost-effectiveness in
producing LVVs.
9 CAR-T Bioprocessing
227
9.2.3 Cell Culture Technology fortheProduction ofLVVs
Each cell essentially is a miniature LVV production factory, producing a volumetric
product yield of LVVs. In theory the more dense the culture is, the higher the productivity or titer of LVV produced. It is important to point out that there are other
types of appropriate cells lines available for the production of viruses. The type of
cell line used will be dependent on the type of virus being produced, hence the type
of cell culturing will then be dependent on the type of cell line used. Specically,
LVVs typically are produced in an appropriate producer cell line or derivative, such
as the human embryonic kidney 293T (HEK293T) cell line. Traditionally, this cell
line is adherent-dependent requiring that culturing take place in a single monolayer
vessel that provides a surface for attachment and growth. For small-scale production, T-ask vessels can provide the appropriate surface area required allowing for
cell attachment and growth, they can come treated or non- treated depending on
culture needs. For large-scale production of viral vectors, this vessel type proves
unsuitable as the surface area provided does not support the high cell density growth
required for high titer viral vector production. Multiple surface cultivation systems,
like roller bottles, multi-layer asks, and cell factories, can alternatively be used to
increase the total surface area needed to support high cell density growth but can
still be considered low cell density culture systems as they have been reported to
result in moderate increases of viral vector titers [12]. Instead, these systems would
rather need to be “scaled-out” opposed to “scaled-up” where the number of culture
units are increased in parallel requiring large enough facility footprint and personnel
to accommodate and operate the numerous culture units which can prove to be both
labor and cost intensive.
Alternatively, other types of cell culture systems such as the hollow ber bioreactor technology, xed-bed bioreactors, and microcarriers can provide larger surface
areas in comparison to the aforementioned systems, supporting high cell density
growth and product yield than what has been seen to be possible in asks and roller
bottles. Hollow ber bioreactors are a closed system that utilizes a hollow ber
bioreactor cartridge that provides a porous support for cells to attach and grow, the
cartridge consist of two separate spaces: the extra-capillary and intracapillary space.
The cells are cultivated in the extra-capillary space and receive nutrients and metabolites via an exchange from the medium transported through the intracapillary space
[12]. Fixed-bed bioreactors or packed-bed bioreactors provide a xed-bed of microber carriers or disks for cell growth where cells receive nutrient and metabolites
through a perfusion mode. Microcarriers are small sphere-like beads which provide
high surface area for cells to attach and grow but interestingly enough can also be
grown in a three-dimensional suspension system, such as stirred tank bioreactors, as
opposed to the conventional two-dimensional planar culture systems [12]. Based on
clinical trial size, these cell culture technologies can prove sufcient providing a
solution to generating increased LVV titers; however, as demand may increase these
approaches with the exception of microcarriers still require a scale-out approach,
again requiring large enough incubator space and facility footprint.
228
A. Adeniran et al.
Recently there has been a shift away from anchorage-dependent adherent cell
culture technology to the development of suspension cell cultures to produce LVVs
as this has been shown within a limited workspace to be much more amendable to
scaling-up. Suspension cell cultures are derived from the adaptation of adherent
HEK293T cells to suspension culture conditions and can grow directly in shake
asks or bioreactors as they do not require a surface for attachment allowing for
high cell density growth to occur. In addition, these cells do not require serumcontaining media. Reports have described that suspension adapted cells in serumfree containing medium are able to retain their cell virus packaging abilities with
nearly a ve-fold improvement in comparison to original adherent 293T cells and
proved suitable for cultivation in a bioreactor setting yielding comparable LV titers
to shake ask induced titers [6]. The serum that has been traditionally used is a
derived animal component which is complex and unpredictable in nature and the
composition of the serum used in culture media can lead to batch variability resulting in inconsistencies within the process [6] as well as posing a risk of introducing
animal components and contaminants into the nal clinical grade vector stocks [12]
which can complicate further downstream processing steps. The ability to remove
the use of the highly variable animal derivative serum in culture media has been
highly favorable easing the GMP manufacturing process and satisfying safety compliance regulations.
9.2.4 Mode ofProduction forLVVs
Predominately the transient transfection method is used for the production of LVVs
where plasmid DNA constructs carrying the viral genes are introduced into the
appropriate cell line of choice. This is mostly attributed to time as this method can
quickly produce the LVVs. Transfection can be carried out using a variety of transfection reagents dependent on the needs of the process. Once such method which
has been heavily used is the static transfection process called the calcium phosphate
(CaPO4) co-precipitation method. Cell cultures are required to remain static as precipitates formed via CaPO4 co-precipitation settle on the cell monolayer for a period
of time to allow for endocytosis, uptake of the DNA, to occur [12]. A media
exchange typically occurs 24h post-transfection (hpt), to reduce cytotoxic effects
induced by CaPO4, and multiple harvests are carried out to increase the nal titer
produced. Though considered a cost-effective transfection reagent, CaPO4 is cytotoxic to cells requiring serum or albumin in culture media to reduce cytotoxic
effects, which as mentioned can pose a safety and compliance issue.
Other methods of transfection utilize reagents such as polyethyleneimine (PEI)
or lipofectamine for the production of LVVs. However, for large-scale production,
lipofectamine proves costlier than PEI.PEI is a linear polyethylenimine high-charge
cationic polymer that will bind to the anionic plasmid DNA creating a positively
charged transfection complex that will enter into cells by endocytosis. PEI can
9 CAR-T Bioprocessing
229
increase consistency across batches, is non-toxic to cells thus eliminating the need
for serum, and simpler to use as there is an unnecessary requirement for a tight regulation of transfection conditions as opposed to the CaPO4 method which requires pH
to be regulated at a certain value to increase transfection efciency. Though it is
important to keep in mind that many other factors can also affect the efciency of
transfection, theoretically all vector produced would be of similar quality, but due to
per cell scale factors such as plasmid ratio, reagent: DNA ratio, incubation and mixing time, and host cell viability the vector produced is typically heterologous and
further optimization would be required [8] for scale-up purposes to increase transfection efciency.
The development of a packaging cell line which constitutively expresses the vector components and viral envelope has the potential to streamline the production
process by replacing the need for costly GMP grade plasmid DNA and eliminating
the need for DNA preparation steps reducing the potential for source contamination.
However, cytotoxicity effects have been reported with constitutive expression of
some of the viral vector components. To mitigate those toxic effects, a potential
alternative is the tetracycline-inducible system to control expression or use of packaging cell lines that can express a number of less toxic envelope proteins [7, 8].
Stable cell lines in comparison to transient transfection can reduce cost, increase
reproducibility, and enable scalability ensuring manufacturability [7, 8].
Recently developed, a suspension adapted producer cell line derived from 293T
has been stably transfected with a single bacteria articial chromosome (BAC)
DNA construct that expresses all of the lentiviral vector components. This stable
cell line technology utilizes an inducible system where the addition of doxycycline
to culture media initiates transcription and production of LVVs yielding high titers
comparable to what has been achieved with transient transfection. This cell line
technology is suggested to be readily scalable in single-use stirred-tank bioreactors
maintaining genetic and functional stability [13].
For the purpose of producing high titer clinical grade LVVs at scales to meet
commercial demand, the use of suspension cell cultures has been the go-to choice
circumventing much of the challenges associated with the limited scalability that
previously mentioned cell culture technologies have not yet been able to overcome.
9.2.5 Upstream Bioprocessing
As the industry moves toward the use of suspension cell cultures for large-scale
production of LVVs, the typical vessels of choice to support the growth of cell cultures are bioreactors. Bioreactors provide an operator-controlled cell culture environment ideal for production processes that require larger and more consistent
batches. Cell cultures and viral production take place during what is called the
upstream bioprocessing portion of the process. The purpose of the upstream bioprocessing steps is to generate a sufcient amount of LVV bulk for the downstream