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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5346_Библиотеки_им_академика_М_И_Перельмана
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peptides. This simultaneous engagement brings about the activation of the T cell
followed by the destruction of the cancer cell [41].
BiTEs secreting CAR-T cells are successful in preclinical models in overcoming antigen
expression and antigen escape [42].Choiet al. have shown that CAR-T cell secreting BiTEs
against EGFR (an antigen often overexpressed in glioblastoma) eliminated heterogenous
glioblastoma in mouse models. Furthermore, CAR-T cells secreting BiTE did not show
toxicity in in vivo xenograft models [43]. Promisingly, in July 2017 the CD19-targeted BiTE
blinatumomab was granted approval by the FDA for the treatment of relapsed or refractory
B-cell precursor acute lymphoblastic leukemia (ALL) in adults and children.
3.1.4 CAR-T cells secreting antibodies
Researchers have been exploring the possibility of engineering CAR-T cells that can
secrete antibodies. This ingenious solution has been used in the production of an
antiprogrammed death-ligand 1 (PD-L1) antibody. As a mechanism of the immune checkpoint pathway, PDL1 which is overexpressed on tumor cells interacts with PD1 expressed
by T cells and thereby negatively regulates immune response and avoids the immune
attack. Aiming at overcoming the immunosuppressive TME, thus great efforts have been
made to reduce the negative effects of PD-1 and PD-L1 that promote T cell dysfunction
[44,53].Liet al. have developed CAR-T cells secreting checkpoint inhibitors targeting
PD-1. This study had positive outcomes, and show in a xenograft mouse model of lung
cancer an efficient eradication of the tumor [45]. Suarez et al. engineered CAR-T cells
secreting anti-PD-L1 at the tumor site to prevent T cell exhaustion. This new technology
was tested in a humanized mice model of cell renal cell carcinoma and a five times reduction
in tumor growth was shown as well as a diminished tumor weight of 50%–80% [44].
13Engineering solutions to design CAR-T cells
3.1.5 CAR-T cells used as a vehicle for oncolytic viruses
CAR-T cells have also been employed for the deposition of oncolytic viruses directly to
the tumor site. Oncolytic viruses are a type of virus that have the capacity to infect and
induce lysis of cancer cells but not normal cells. This indirectly impacts the tumor growth
via vasculature shutdown. Oncolytic viruses exist in nature, but they can also be created
in the laboratory by altering other viruses [47,54]. Van Seggelen et al. have proven in vitro
on a murine breast tumor and on human lung tumor the increased efficacy of the combined therapy [47]. Also, CAR-T cells secreting soluble herpesvirus have been designed.
Herpesvirus entry mediator binds to the inhibitor receptor B lymphocyte and
T lymphocyte on B cell lymphoma cells, leading to tumor suppression [42].
3.2 CAR-T cells engineered with LOGIC gates
To further improve the overall effectiveness of the CAR-T cells, precision logic gates
have been explored. Logic gates, just like in the field of electronics, can be used to implement timely and logical activation or inactivation of signaling. This includes AND, OR,
and NOT circuits, as described [55] (Fig. 4).
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Fig. 4 Engineering solutions for CAR-T cells. (A) Tandem CAR, two different antigen binding domains
are expressed in the same CAR (B) Bicistronic CAR, two different antigen binding domains are
expressed on two different CARs (C) Tricistronic CAR, three different antigen binding domains are
expressed on three different CARs (D) CAR with OFF switch, inducible caspase 9 (iCASP9) induces apoptosis if a small dimerizing molecule is exogenously administered (E) NOT gate, CAR-T cell activates
only if the antigen A is present and antigen B is absent (F) Synthetic Notch (Syn-Notch) CAR, if antigen
A binds, then a synthetic transcription factor is released and it enters the nucleus where drives the
expression of a second receptor for antigen B. (Created with BioRender.com.)
3.2.1 CAR-T cells with OR gate
CAR-T cells engineered with an OR gate have two different antigen receptors and the
cells can be activated by either one of them [56]. The idea of the OR gate has been proposed to solve the problem of antigen loss that patients run into during the treatment with
CD19 CAR T-cells. In fact, cancers can lose irreversibly CD19 expression via a variety of
genetic resistance mechanisms and thus cause a relapse in patients [57]. Especially, in solid
tumors, this strategy has shown to be effective in preclinical models [58,59]. But also, it
was shown positive results in B-cell malignancies.
The three main types of OR gate that have been proposed are: tandem or bispecific
CAR, bicistronic or cotransduced CAR, and tricistronic CAR.
3.2.1.1 Tandem CAR
Tandem CAR is yet another exploratory idea in which the CAR contains two antigens
binding domains. Hegde et al. developed a tandem CAR-T cell capable of targeting both
HER2 and IL13Rα2 simultaneously. Their technology has shown enhanced antitumor
activity with a reduction of antigen escape in a glioblastoma murine model [58].
3.2.1.2 Bicistronic CAR
Unlike the Tandem CAR, the bicistronic CAR-T cells are engineered with two different CAR receptors expressed on the same T cell. Qin et al. engineered a CD19/CD22
bicistronic CAR-T cells and compared it with tandem CAR-T cell targeting also CD19/
CD22 in a preclinical study. Their results have shown that the bicistronic CAR-T cells
have been more effective in clearing leukemia and thereby prevented further relapse in
preclinical models [60].
15Engineering solutions to design CAR-T cells
3.2.1.3 Tricistronic CAR
Tricistronic CAR-T cells are CAR-T cells that can target three different antigens.
Bielamowicz et al. designed tricistronic CAR-T cells cotargeting HER2, IL13Rα2, and
EphA2 and tested it in glioblastoma model, and proved that the tricistronic CAR-T cells
could overcome the antigenic heterogeneity in glioblastoma and thus improve the therapy
outcomes [59].
The CAR-T cells strategy using dual-targeting is used in clinical trials for B-cell acute
lymphoblastic leukemia (B-ALL) and diffused large B-cell lymphoma (DLBCL)
(clinicaltrials.gov identifier: NCT03241940, NCT03448393).
3.2.2 CAR-T cells with AND gate
An AND gate can be created in CAR-T cells by splitting the primary signal (CD3ζ) and
the costimulatory signal (CD28 or 4-1BB) on two different receptors each of them
targeting a different antigen. Thus, when the first T cell receptor binds to its
corresponding antigen, the primary signal is initiated but this alone is insufficient for full
T cell activation. The secondary signal from the costimulatory receptor is acquired when
the T cell is fully activated [55,61]. A more robust AND gate can be construct with the
synthetic Notch (synNotch) receptor. SynNotch receptor detects a target antigen via
extracellular recognition domain, this triggers a proteolytic cleavage that releases the
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intracellular domain of the receptor. The intracellular domain is a synthetic transcription
factor that once released, enters into the nucleus and links to user-specified transgenes,
and drives the expression of a second receptor that if constitutively expressed, would be
toxic [55]. In another study, Srivastava et al. reported an engineered synNotch receptor
specific for expressing receptor tyrosine kinase-like orphan receptor 1 (ROR1) receptor
only when in proximity with tumor cells, resulting in a better safety profile [62].
3.2.3 CAR-T cells with NOT gate
NOT gate was designed to prevent toxicities in healthy tissues.In fact, NOT gatesare T cells
engineered with A-AND-NOT-B circuits, where antigen A activate T cells, but antigen
B stronglyinhibitsthem [55]. Furtherstudiesare requiredto consolidate this concept.In fact,
thereis onlyone publishedstudyby Fedorovet al. in whichCAR-T cells are presentedwith a
receptor that activates the CAR-T cell when it binds to the specific antigenbut can also contain a receptor that temporarily deactivate CAR-T cell functions [63].
3.2.4 CAR-T cells with OFF switches
To prevent toxicity derived from CAR-T cells, numerous ways to switch them off have
been designed. One approach is the use of a suicide gene, for instance, inducible caspase 9
(iCASP9) that induces apoptosis if a small dimerizing molecule is exogenously administered [64]. Further suicide genes explored are Herpes simplex virus thymidine kinase
(HSV-TK), which renders T cells vulnerable to ganciclovir which deactivates DNA
polymerase resulting in cell death [65].
3.3 Engineering the chimeric antigen receptor
The signaling endodomain in CAR construct plays an important role not only in the
signaling activation of the CAR-T cell but also in the survival of the cell [66]. Recently,
the immunoreceptor tyrosine-based activation motifs (ITAMs) of the CD3z
endodomain was reduced from three functional ITAMs to only one functional ITAMs
and it was shown to have a greater therapeutic profile [67].
Due to the high impact of the costimulatory domains on the functions of CAR-T
cells, they have been explored in an attempt to enhance their activity and persistence.
Several combinations have been tested and even new costimulatory domains have been
introduced. For example, inducible T cell costimulator (ICOS) is a costimulatory
molecule from the CD28 family and it has been shown that the presence of ICOS
can dramatically enhance the persistence of T cells [68].
Alternate costimulatory molecules are actively being explored to enhance the beneficial effect of CAR-T cells. Some examples of these efforts include the herpesvirus entry
mediator (HVEM), addition of this costimulatory domain can enhance the development
of CD8 positive memory T cell [69]; tool-like receptor 2 (TLR2) which when added to
the end of m28z CAR enhances the cytotoxic capacity the tumor-specific T cell [70];
DNAX-activating protein 10 (DAP10) incorporated at the end of the CAR construct
improves the T cells antitumor property [71].
These modifications on the CAR construct alter their functionality to display desirable
characters, for example, CD28 and 4-1BB costimulatory domains. Here, the CD28
domain enhances the glycolysis and influences the effector memory, while 4-1BB increases
the respiratory capacity, fatty acid oxidation, and mitochondrial biogenesis [72].
Ligtenberg et al. engineered CAR-T cells to coexpress catalase. This turned the cells more
resistant to reactive oxygen species (ROS), which are highly abundant in the TME and
impair the T cell antitumor efficacy. They have shown high antitumor activity in ovarian
carcinoma in vitro despite the presence of high amounts of ROS in the tumor
surrounding [73].
Also, combinations of costimulatory domains have been designed. Guedan et al.
designed a third-generation CAR-T cell combining ICOS and 4-1BB to display greater
antitumor activity in xenograft tumor models of lung cancer, ovarian cystadenocarcinoma,
and pancreatic adenocarcinoma [68].
4. Gene delivery to engineer CAR-T cells
4.1 Viral vectors to engineer CAR-T cells
Both retroviral and lentiviral vectors need three basic genes: gag, pol, and env for survival
and function. The gag encodes the structural proteins, the pol encodes enzymes required
for reverse transcription and integration into the host cell genome, and env encodes the
viral envelope glycoprotein [74]. The genome size of retroviral and lentiviral vectors is
7–10 kb and 9.7 kb, respectively [75,76].
Viral vectors are efficient in integrating their genome into the host genome. This
essentially results in the viral vector being non-site-specific leading to undesirable effects
such as rendering the functional genes ineffective or even increase the off-target gene
editing [77,78]. Retroviral vector on the other hand tends to integrate with the
LMO2 gene in the hematopoietic cells. This integration could result in dysregulation
and possibly lead to induced leukemia [79]. Though theoretically possible, in reality,
long-term follow up did not show integration-mediated oncogenesis in patients treated
with genetically modified T cells [77]. The use of Lentiviral vectors has been controversial as they are derived from the human immunodeficiency virus. So far, they have shown
great potential in delivering a gene to produce CAR-T cells and often has shown better
specificity and target profile [74]. Bobisse et al. have shown that lentiviral vector delivery
systems have a higher transfer efficiency and transgene expression compared with
gamma-retroviral vectors [80].
Though strained with ethical challenges, Lentiviral vectors have gained more attention than retroviral vectors for their ability to efficiently transduce nonproliferating or
slowly proliferating cells [74].
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This mode of approach is never free from inherent risks. More studies are needed to
understand ways to mitigate known risks before presenting them to patients in the clinical
setting. One curious case of viral integration was reported in the clinical trial
(NCT01029366) in the Abramson Cancer Center of the University of Pennsylvania.
The engineered CAR-T cells were infused into a patient with chronic lymphocytic leukemia. A lentiviral vector was used to engineer the CAR-T cells targeting the CD19
protein. The lentiviral vector integrated CAR gene into the TET2 gene, thus causing
TET2 gene deletion. The patient underwent complete remission and it was seen that
94% of CAR-T cells present in the patient originated from the single cell in which
the TET2 gene was deleted. It was also noted that the T cell population was from a single
clone that integrated the gene [81].
The use of both, lentiviral and retroviral vectors have shown a positive response in
clinical practice. In clinical trials, the complete remission rate for B-cell acute lymphoblastic leukemia has been higher than 60% in patients with refractory disease [82].
A complete remission has been observed in 40%–70% of non-Hodgkin lymphoma [83].
The large-scale production of lentiviral vectors is initiated with the culture of cell line
derivates of the HEK293T cell line. After few days of expansion, cells are transiently
transfected with plasmid DNA encoding the necessary proteins for lentiviral vector production. Thus, the plasmid transfected contains the genes for the envelope protein the
gag, and the pol. In the following days, the cell line produces functional viral vectors that
are filtrated, purified, and sterilized. The purification of the viral vector is a critical step in
the production since impurities could lead to inflammatory responses both in vitro and
in vivo [74].
In summary, viral vectors are an effective and reliable machinery to generate CAR-T
cells and often express the CAR for a prolonged amount of time [84]. Currently, there
are several challenges that limit their clinical use. Notably, the high cost of production
and the safety concerns such as the mutagenicity, generation of infectious viruses, variegated transgene expression, and transcriptional silencing [85,86]. Like the other strategies,
viral vectors to hold a promising yet challenging road ahead.
4.2 Transposon systems to engineer CAR-T cells
Class II DNA transposons or sometimes referred to as jumping genes have been used in
nonviral engineering applications [87]. Simplistically, they are a piece of DNA sequences
capable of changing their location within the genome via a “cut and paste” mechanism
[88]. These transposons construct contains the gene of interest and also a gene coding for
transposase. Transposase can insert this piece of DNA into the genome. Some labs have
been able to synthesize these transposons with varied success. This preparation step is
essentially composed of two parts. First, the transposon carries the gene of interest and
is inserted into the desired location in the genome. Second, the transposase excises
the DNA sequence and reintegrates it into another region of the chromosome, thus catalyzes the “cut-and-paste” process [87,88]. Compared with viral vectors, transposon sys-
tems are simple and cost-effective to manufacture. Furthermore, their capacity to carry
the cargo is much greater. However, the higher the genetic payload the lower is the transposition efficiency [89,90]. Though promising for large-scale manufacture, the main disadvantage over the viral vectors is the reduced transduction efficiency. This impediment
is so pronounced that achieving higher efficiency of gene delivery and a stable and
prelonged gene expression at the same time has turned out to be a challenge [87].
Another disadvantage of the transposon system is its ability to randomly insert the gene
into an unintended location in the genome. This could lead to potential safety issues and
diminished efficacy. Furthermore, by nature, transposons are naturally inclined to change
their location in the genome. This could result in unexpected mutagenesis [91]. Even if
the electroporation of transposons results in a faster production process of CAR T-cells
when compared with viral vectors, this strategy, currently as it stands, often results in
lower cell viability, and importantly the expression level of the transgene is often lower
when compared with the viral vector [92].
Though these disadvantages seem discouraging, the advantage it holds for manufacturing scale-up is hard to ignore. Labs across the globe have progressed their research in
developing improved strategies to tackle the existing challenges. Notably, there are two
principal types of transposons systems that are currently being explored in generating
CAR-T cells: Sleeping Beauty system (SB) and the piggyBac. The transposon SB has been
discovered by Dr. Hackett and his colleagues. They identified in the fish salmon family an
ancestral transposon gene that was working ten million years ago. They spent almost three
years to reconstruct the transposon piece-by-piece, and then they successfully applied the
SB system to gene therapy, to transport new genes into genetically-damaged cells [93]. The
SB system is more specific, by its ability to insert the gene of interest in a location that is
highly abundant in thymine and adenosine (TA) sequences [94]. As a result, the SB system
has shown a much safer genotoxicity profile when compared with viral vectors [87].In
2008, Cooper et al. demonstrated that it was possible to adopt the SB system to develop
CAR-T cells expressing CD-19 and that these cells could be effectively used in the treatment of refractory leukemia and lymphoma [90,95]. The SB system is noticeably superior
over conventional viral vectors owing to their simple and cost-effective process of
manufacturing. The efficiency of gene transfer is higher than the conventional DNAmediated random integration. Also, T cell activation before transfection, which is needed
with retroviral vectors, is not required by the SB system [90].
The SB system makes use of Minicircles (MCs) plasmids. MCs are supercoiled circular
DNA vectors used as a valid alternative to plasmids in delivering the SB transposon system. Thanks to their small size they allow a cost-effective and rapid approach in the preparation of the CAR-T cells compared with the conventional plasmid. Furthermore, the
CAR expression in cells transduced with MCs was higher than the one in cells transduced
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with viral vectors [96,97]. Recently, Barabas et al. produced a highly soluble SB capable
of spontaneously penetrating cells and they used it to generate CAR-T cells. This
decreased the overall genotoxicity [98]. SB is being tested in a phase I trial. SB is being
adopted to generate CD19+ CAR-T cells from autologous (NCT00968760) or allogeneic (NCT01497184) human stem cells and show an overall survival of 100% and 63%,
respectively.
PiggyBac system, on the other hand, is the other transposon system largely adopted in
genetic manipulation. The piggyBac transposase introduces transposons in TTAA
sequences [99]. It has been employed to produce a novel type of CAR-T cells that target
B cell maturation Antigen. In addition to benefiting from the reduced cost of production,
T stem cells shown a higher percentage of T cell memory phenotype. Furthermore,
piggyBac, having a higher gene payload capacity, allows the incorporation of multiple
genes in addition to CAR. A phase 1 trial has already shown promising results [100]. Early
clinical trials are underway and a more accurate safety profile of these transposon systems
needs to be further assessed.
4.3 Genome editing technology in CAR-T cells
Transcription activator-like effector nucleases (TALENs) and zinc-finger nucleases (ZFN)
are two chimeric, customizable restriction enzymes. They can be engineered to target specific loci into the genome and induce DNA double-strand breaks that stimulate errorprone nonhomologous end joining or homology-directed repair, enabling knockouts
and addition of genes [101–103]. ZFN links two different zinc proteins to a cleavage
domain, Fok-1 endonuclease. The created technology can cut the DNA in a predetermined site [104]. The technology of TALEN has the DNA-binding protein domain
derived from TALE proteins, this domain is bound to Fok-1 that mediates the break of the
double-strand DNA [103,105]. The main advantage over viral vectors and transposons
gene delivery systems is the ability to target site-specific loci in the genome. However,
the disadvantage lies in the high cost of manufacturing. This is because of the need for
the proteins to be designed individually for each targeting locus [102,106].
Current research in CAR-T cell therapy is focused on using TALEN and ZFN technologies to knockout genes gene of interest. ZFN has been applied to permanently eliminate the expression of the TCR α or β chains in CD19-specific CAR-T cells (generated
with Sleeping Beauty technology) and it has been shown that the generated cells were not
responding to TCR stimuli (TCR receptor responsible for graft-versus-host-disease
GVHD) [107]. In another study mRNA coding for an engineered ZFN has been
electro-transferred in CD19-specific T cells to disrupt HLA expression [108].
An early clinical trial using TALEN technology to knock out the T cell receptor alpha
chain (TRAC) has shown promises. In a clinical trial done by Qasim et al. at the Great
Ormond Street Hospital two infants with relapsed refractory CD19+ B cell, acute
lymphoblastic leukemia were treated with a CAR-T cell produced using this technology.
Impressively, both infants reached complete remission [109].
4.4 CRISPR-Cas9 to engineer CAR-T cells
Another popular and exciting technology that has got researchers excited across the globe
is that of the CRISPR-Cas 9 (clustered regularly interspaced short palindromic repeatsCRISPR associated protein 9) system. Understandably, CRISPR-Cas9 is currently
widely studied because of the potential it holds. CRISPR was first identified as RNA
mediated adaptive antiviral immune system in bacteria [110].
Briefly, CRISPR-Cas9 needs three elements to function: a CRISPR RNA
(crRNA), a transactivating CRISPR RNA (tracrRNA), and a Cas9 nuclease protein.
The crRNA is homologous to the target DNA sequence and interacts with tracrRNA
to form a complex that helps the protein Cas9 to bind to target the DNA sequence [78].
For gene editing purposes the system is simplified by bringing together the crRNA and
the tracrRNA into a single RNA transcript [78]. Since CRISPR-Cas9 is based on the
interaction RNA-DNA of few base pairs, the CRISPR-Cas9 system tends to have more
off-target effects when compared with ZFNs or TALENs technologies. By contrast,
ZFNs or TALENs rely on highly specific protein-DNA interaction.
Eyquem et al. have introduced CD19 CAR gene via CRISPR-Cas9 technology into
CAR-T cells targeting T cell receptor α constant (TRAC) locus. In their study, they have
shown that the CAR expression was uniformly distributed on the T cells. These T-cells
also showed enhanced potency in the mouse model [111].
The potential of CRISPR-Cas9 technology was discovered when CRISPR-Cas9
was used to knock out PD-1 gene ex vivo in a hepatocellular carcinoma mice model.
The resulting PD-1-deficient CAR-T cells shown enhanced antitumor activity [112].
CRISPR-Cas9 was also used to experimentally knock-out the CD7 gene and prevent
CAR-T cells from fratricide [113]. Currently, albeit very early, the CRISPR technology
is being investigated in clinical trials both for the knock-out of PD-1 and the endogenous
TCR (NCT03545815).
Interestingly, a combination of CRISPR-Cas9 and TALEN is been explored and is
termed Cas-CLOVER. This combined technology has been employed to successfully
delete αβTCR and β2M [42].
21Engineering solutions to design CAR-T cells
4.5 Other technologies to engineer CAR T-cells
MegaTAL, a fusion protein of meganuclease to TAL repeats has been used to disrupt
T cell receptor expression in primary human T cells. Early studies using this method have
been proven to be more efficient, alongside the advantage of showing low levels of toxicity and higher specificity by reduced off-target cleavage [114]. Further studies are
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ongoing to better understand the role that nucleases can play in future patient treatments
using gene editing.
In the recent past, alternative approaches to genome editing techniques have been
proposed. A notable example is the application of a protein expression blocker
(PEBL) that is the result of the combination of an antibody-derived single-chain variable
fragment specific for CD3 ε with 21 different amino acid sequences. After the transduction in T cells, PEBLs have been found colocalized intracellularly blocking the surface
expression of CD3 and TCR αβ. Interestingly, the combined expression of CAR and
PEBL can be accomplished in a single-step procedure [115].
Short hairpin RNA (shRNA) has been adopted to knockout adenosine 2A receptors
(responsible for potent inhibition of antitumor T cell responses) to augment CAR-T cell
functions. To assist with the transfer of genes to the target cells encapsulation using
shRNS using retroviral vector has shown early success [116].
MacLeod et al. engineered CAR-T cells with an adeno-associated virus 6 (AAV6)
encoding for an anti-CD19 CAR transgene and targeting TRAC locus with a sitespecific I-CreI homing endonuclease, thus knocking-out the native TCR gene [117].
It is important to recognize that, even with modern methods at hand, none of the
gene-editing techniques can claim cent percent efficiency in transferring the gene of
interest. Therefore, CAR-T cells need further purification procedures [118].
4.6 Nanotechnologies to engineer CAR-T cells
Nanoparticles made of cationic polymers have been implemented to transfect RNA and
DNA with an efficiency respectively of 25% and 18%. Higher transfection efficiency, up
to 50%, has been achieved in Jurkat human T cell lines with minimal toxicity [119].
Wayteck et al. explored another technique to introduce small interfering RNA
(siRNA) into cytotoxic T lymphocytes. The technique is based on photoporation and
consists of attaching gold nanoparticles to the cell surface and illuminate it by a pulsed
laser. Thus, membrane pores are transiently generated and allow the delivery of siRNA
into the cell that subsequently silences the target gene. This method is less toxic when
compared with other comparable methods [120].
One more approach to knock-out selected genes in T cells is the one described by
Moffett et al. in which nanocarriers deliver mRNA encoding megaTAL nuclease which
is designed to target the TRAC region of the TCR alpha gene [121].
The in vivo reprogramming of T cells has also been explored. Smith et al. designed
polymer nanoparticles that contain the DNA that could effectively enter into the
T cells and reprogram the cellular machinery to express CD19-specific CAR. This could
potentially be used in targeting leukemia. The engineered nanoparticle, made of biodegradable poly(βamino ester), was functionalized on the surface with T-cell-targeting
anti-CD3e f(ab’)2 to target T cells. This nanoparticle is loaded with piggyBac
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