Research Article: Artificial Antigen Presenting Cells: An Off the Shelf Approach for Generation of Desirable T-Cell Populations for Broad Application of Adoptive Immunotherapy

Date Published: February 22, 2016


Author(s): AN Hasan, A Selvakumar, RJ O’Reilly.



Adoptive transfer of antigen specific T-cells can lead to eradication of cancer and
viral infections. The broad application of this approach has further been hampered by the
limited availability of adequate numbers of T-cells for treatment in a timely manner. This has
led to efforts for the development of efficient methods to generate large numbers of T-cells
with specificity for tumor or viral antigens that can be harnessed for use in cancer therapy.
Recent studies have demonstrated that during encounter with tumor antigen, the signals
delivered to T-cells by professional antigen-presenting cells can affect T-cell programming and
their subsequent therapeutic efficacy. This has stimulated efforts to develop artificial
antigen-presenting cells that allow optimal control over the signals provided to T-cells. In
this review, we will discuss the cellular artificial antigen-presenting cell systems and their
use in T-cell adoptive immunotherapy for cancer and infections.

Partial Text

Targeted eradication of cancers and viral infections can be achieved with adoptive
immunotherapy involving the infusion of T-cells directed against viral or tumor antigens (Figure 1). Recent clinical trials have shown that adoptive
transfer of transplant donor derived virus specific T-cells have the capacity to provide
protection against, and successfully eradicate EBV and CMV infections developing in recipients
of hematopoietic stem cell transplants [1–5]. More recently, third party donor derived virus specific
T-cells have also demonstrated effective eradication of CMV, EBV and adenoviral infections in
recipients of hematopoietic stem cell transplants [6–8] and EBV infections in solid organ
transplants [6].

T-cells require several signals to become activated and perform their function. The
first signal imparted is when the T-cell receptor interacts with the corresponding MHC on an
APC. The next required signal is that of co-stimulation, provided upon binding of the

The limited availability of cells constitutes a serious obstacle to the use of DC for
vaccine therapies or for generating T-cells for adoptive immunotherapy. DC generated for
clinical use are derived from the peripheral blood monocytes of patients or transplant donors.
This requires a large amount of blood or leukapheresis to be collected, which is both expensive
and time-consuming. In tumor bearing patients, additional constraints with this approach are
presented due to the effects of chemotherapy leading to a decreased number of DCs in the
peripheral blood, as well as the suppressive cytokines released in the tumor mileu which impair
the function of the host DC [21]. The differentiation and
maturation of DC is inhibited by the soluble immunosuppressive factors secreted by the tumors
such as IL-10, TGFβ, PGE2, and VEGF. These immature DC have abnormally low expression of
MHC-II and low or undetectable levels of costimulatory molecules, rendering them incapable of
processing and presenting antigens, and therefore, unable to induce an effective immune response
against the tumor [22]. Certain tumors may further induce
the patients’ own APC to express other costimulatory molecules, like B7.H1, that
preferentially stimulate regulatory T-cells to suppress immune responses [23].

T-cells are broadly classified as naïve or antigen experienced based on their
encounter with antigen and differentiation status. Antigen specific T-cells are further
classified based on their differentiation status into central memory (TCM), effector memory
(TEM), and terminally differentiated effector cells (TE) [27]. Recent emerging data describes a population of T-cells with stem cell like
properties (TSCM) that would have the potential for prolonged persistence and further
replication in-vivo [28–30]. In earlier clinical trials, adoptively transferred
anti-tumor T-cells clones, even when infused in large numbers, demonstrated only limited
clinical efficacy, which was primarily attributed to the lack of persistence of the T-cells
infused [13,14].
Subsequent studies evaluated the potential of different T-cell subsets with respect to
in-vivo activity and persistence. In both animal models and humans, recent
studies have shown that adoptively transferred TCM phenotype T-cells, with high expression of
L-selectin (CD62L), CCR7 and CD44 provide durable immunity against infections such as CMV [31–33]. Berger
et al. [31] demonstrated that TCM derived T-cells when
adoptively transferred into macaques persisted for prolonged periods in-vivo
and re-acquired the phenotypic markers of TCM cells, and subsequently Wang et. al. [34] showed prolonged engraftment of TCM derived cells in an
immunodeficient mouse model. In TCR transgenic mouse models, Restifo et al. have demonstrated
that antigen specific naïve and TCM cells are more effective than TE cells in
eradicating large established tumors, and paradoxically, differentiated T-cells displaying high
functional activity in-vitro were less effective in eradicating tumors
in-vivo [35]. In recent clinical
trials, persistence of adoptively transferred T-cells has been correlated with regression of
disease [36]. Therefore, such TCM cells are a desirable
T- cell population for adoptive immunotherapy because they have the potential to provide durable
protection against disease by virtue of their lymphoid homing properties
in-vivo [27], lending to their
prolonged survival after infusion.

AAPC are a developing technology for use in adoptive immunotherapy. AAPC use the
kinetics known about antigen presentation, but adapt a platform in which an APC provides
specific signals delivered using a designed template to stimulate T-cell expansion. The use of
these artificial platforms allow for expression of specific molecules on these cells providing a
more controlled stimulation of T-cells, therefore permitting the propagation of T-cells with
specific phenotype and activity. AAPCs can be derived from cell lines using viral transduction
of genes encoding specific co-stimulatory molecules and/or HLA molecules, or from synthetic
materials such as polystyrene coated with specific cytokines and/or co-stimulatory molecules

Systems for non-specific expansion of T-cells were initiated using magnetic beads
coated with anti-CD3 and anti-CD28 antibodies. Initial studies with this artificial non-cell
based system demonstrated preferential long-term expansion of CD4+ T-cells [53], however, this AAPC system did not support the long-term
growth of purified CD8+ T- cells [54]. Maus et al.
attempted to overcome this limitation by engineering a cell based AAPC using additional
co-stimulation. The human erythroleukemia cell line K562 was used, which does not express HLA
class-I or class-II, but expresses adhesion molecules ICAM-1 and LFA-3. These K562 cells were
engineered to stably express the human low-affinity Fcγ receptor, CD32 (K32), and the
co-stimulatory molecule human (h) 4-1BB ligand (K32/4-1BBL). The K32/4-1BBL coated with anti-CD3
and anti-CD28 antibodies were then used as AAPCs [55].

The expansion and enrichment of antigen specific T-cells from a starting population
of polyclonal CD3+ T-cells containing minimal concentrations of the desired T-cells has remained
challenging. Two main cell based AAPC systems have thus far been developed and evaluated for
this purpose, and for potential application for adoptive immunotherapy. Latouche et al. first
described the generation of mouse fibroblast NIH 3T3 cell based AAPC transduced to express a
single human MHC class-I allele (HLA A0201) and critical T-cell co-stimulatory molecules as a
platform for in-vitro expansion of epitope specific T-cells restricted by a
single HLA allele [83]. In engineering these AAPC, the
choice of co-stimulatory molecules was further improvised in an effort to maximize the effects
of signal 1 and 2 for T-cell activation. This AAPC was accordingly transduced to express the
co-stimulatory molecule B7.1 and the adhesion molecules LFA-3 and ICAM-1. In addition, these 3T3
AAPC expressing HLA A0201 were also transduced to co-express peptide epitopes of influenza and
MART-1 proteins to stimulate the expansion of antigen specific T-cells responding to specific
peptide-MHC complexes. Successful generation of epitope specific CD8+ T-cells bearing an
effector memory phenotype was achieved using 3T3 AAPC that were directed against both viral and
tumor antigens, and were cytotoxic against tumor cell targets as well as peptide loaded targets
in-vitro. A higher efficiency of T-cell expansion was attained using 3T3 AAPC
compared to autologous peptide loaded DC; AAPC yielding 2 fold higher T-cell expansions with a
cytolytic activity that was 1.6 to 4-fold higher. Importantly, T-cells generated using these
AAPC did not demonstrate activity against targets lacking HLA A0201 or HLA A0201 expressing
targets lacking the appropriate antigen, thus establishing the ability of this AAPC system to
foster the generation of HLA restricted epitope specific T-cells.

In order to achieve durable T-cell immunity, CD4+ T-cell help is critical. Indeed, in
patients receiving adoptively transferred cytomegalovirus specific CD8+ T-cells, the infused
CD8+ T-cells were only shown to have long term in-vivo persistence in the
presence of CMV specific CD4+ T-cells [96]. Yee et al.
have subsequently demonstrated complete regression of metastatic melanoma upon infusion of
cloned CD4+ T-cells directed against NY-ESO-1, suggesting that CD4+ T-cells can potentially
mediate direct effector function in addition to providing help to effector CD8+ T-cells [15]. Therefore, approaches for the generation of CD4+ T-cells
are critical to enhance the success of adoptive immunotherapy. Nadler et al. first reported the
development of an AAPC system for the generation Th1 type CD4 + T-cells. In this report, the
previously described K562 cells expressing CD80 and CD83 [89] were used as the backbone, and were successively transduced to co-express HLA class
– II alleles DRB1 0101 and DRB1 0701 as well as CD64, the common Fc γ receptor,
the invariant chain (li) and the α and β chain of HLA DM. These cells were then
used for the generation of AAPC expressing HLA class-II alleles, DRB1 0101 and DRB1 0701 [97]. These studies demonstrated successful expansion of DR1
and DR7 specific T-cells responding to CMVpp65 as well as MART1, bearing a Th1 cytokine profile
in response to specific antigenic stimulation in-vitro.

Recent studies have led to the identification of T-cell subsets with the capacity for
longer in-vivo persistence and the cytokines regulating the propagation of such
T-cells. This knowledge has launched the development of a new generation of AAPC specifically
engineered to deliver cytokine cocktails facilitating the expansion of TCM and TSCM cells for
adoptive immunotherapy. Interleukin-15 is a γ chain cytokine that is critical for the
survival and homeostatic proliferation of NK cells and memory phenotype CD8 T-cells [99–101]; and
in the presence of antigen, it specifically induces the proliferation of TCM phenotype antigen
specific CD8+ T-cells [102–104]. IL-15 mediates its functional activity by binding with its unique high
affinity receptor subunit IL-15Rα forming an IL-15Rα /IL-15 complex
(15Rα/15) which then shuttles to the cell surface to bind with the β (CD122) and
common γ chain subunits to initiate signaling in receptive lymphocytes [105–107]. We
generated HLA A0201+ NIH 3T3 based AAPC that were also transduced to co-express IL15Rα
and IL-15 genes. T-cell stimulation using IL15Rα/IL-15 expressing AAPC fostered the
preferential expansion of antigen specific T-cells bearing a TCM phenotype [108]. We and others have shown that IL-15 can prolong the
in-vivo persistence of antigen specific T-cells [34], specifically when administered in complex with its high affinity receptor
IL-15Ra/IL-15 [109]. AAPC systems expressing and
secreting such IL-15Ra/IL-15 complexes may be a useful technique for the efficient generation of
TCM cells for clinical applications. IL-21 is another such cytokine that can be developed within
this approach. More efficient systems for the consistent expansion of TH1 type CD4+ T-cells need
to be developed for clinical use and to further study: (1) for defining epitopes of tumor and
viral antigens presented by class-II alleles that would enhance the effect of CD8+ T-cells (2)
the functional activity and CD8+ cell help afforded by co-infusion of CD4 and CD8 T-cells
in-vivo (Table 2).





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