Advanced Drug Delivery Reviews 56 (2004) 1219 – 1231

www.elsevier.com/locate/addr

Folate-mediated targeting of T cells to tumors

Edward J. Roy a, Ute Gawlick a, Brent A. Orr a, David M. Kranz b,*
a
Neuroscience Program, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
b
Department of Biochemistry, University of Illinois at Urbana-Champaign, 600 S. Mathews Avenue, Urbana, IL 61801, USA
Received 30 August 2003; accepted 5 January 2004

Abstract

Recently various strategies have been developed to exploit in a clinical setting the well established finding that T cells can
specifically recognize and destroy tumor cells. Several independent approaches to the targeting of T cells against cancer have
been explored, including the use of bispecific antibodies (anti-T cell/anti-tumor cell) to redirect T cells, vaccines to induce
tumor-reactive T cells, and adoptive transfer of ex vivo activated, tumor-reactive T cells. In this review, we focus on studies in
which high-affinity folate receptors (FRs) on tumor cells have served as targets for redirecting or enhancing the effectiveness of
activated T cells. Bispecific antibody conjugates of folate and antibodies to the T cell receptor (TCR) complex can generate
tumor-reactive T cell responses. The development of folate/antibody conjugates specific for the T cell co-stimulatory molecule
CD28 could yield activated T cells that recognize endogenous peptide – major histocompatibility complex (MHC) antigens on
tumor cells. Finally, we discuss a less investigated area in which high-affinity FRs on macrophages, or other antigen presenting
cells (APCs), may provide opportunities in the design of tumor-antigen-specific vaccines.
D 2004 Elsevier B.V. All rights reserved.

Keywords: Immunotherapy; T cell responses; Folate/antibody conjugates; Antigen presenting cells; Macrophages

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1220
2. T Cell immunotherapy of cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1220
2.1. Bispecific antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1221
2.2. T cell vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1222
2.3. Adoptive T cell therapies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1222
3. Folate-mediated targeting of T cells to cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1222
3.1. Tumor models. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1223
3.2. Bispecific folate/antibody conjugates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1223
3.3. Optimal T cell targeting: cytokine co-treatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1225
4. Potential strategies for eliciting effector and memory T cells against tumors . . . . . . . . . . . . . . . . . . . . . . . . 1225

* Corresponding author. Tel.: +1-217-244-2821; fax: +1-217-244-5858.

E-mail address: d-kranz@uiuc.edu (D.M. Kranz).

0169-409X/$ - see front matter D 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.addr.2004.01.006
1220 E.J. Roy et al. / Advanced Drug Delivery Reviews 56 (2004) 1219–1231

4.1. Co-stimulatory molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1225

4.2. Antigen presenting cells: folate-mediated uptake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1226
5. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1228
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1228
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1228

1. Introduction cells against cancers. While it is beyond the scope of

Early studies of Gorer showed that mice could
reject very large tumor burdens that are not histocom-
patible [1], although the nature of the immune re-
sponse had not yet been uncovered. In hindsight, these
findings revealed the tremendous potential of T cells
in the elimination of cancers. The molecular basis of
the T cell recognition process began to be elucidated
by many investigators, notably with the discoveries by
Zinkernagel and Doherty [2] that T cells recognize a
foreign antigen only when it is associated with a self
protein encoded by the major histocompatibility com-
plex (MHC). Subsequent work by Boon and others
showed that specific tumor-antigen peptides, bound to
a product of the MHC, could be isolated and shown to
stimulate cytotoxic T lymphocyte (CTL) activity
[3,4]. There are now many peptides that have been
associated with different types of cancers, and that
have formed the basis for specific tumor vaccines in
pre-clinical and clinical trials [5].
The cell surface molecules involved in T cell
immunity have been the topics of study by many
laboratories [6], yielding valuable information about
the basic mechanisms of T cell biology and guiding
the development of many strategies for eliciting T
cell activity against tumors. Among the key T cell
molecules are the ah T cell receptor (TCR) that
recognizes the peptide/MHC antigen and that asso-
ciates with subunits of the signaling complex called
CD3 (Fig. 1). Co-stimulatory T cell molecules such
as CD28 are essential in driving T cells through the
proliferative and differentiation pathways required
for effective targeting. The ligands for the co-stim-
ulatory molecules, and the peptide/MHC antigens
themselves, are normally presented to the T cells
by ‘‘professional’’ antigen presenting cells (APCs)
[7]. APCs include B cells, macrophages, and, in
particular, dendritic cells.
The field of T cell-based therapies has expanded
over the past 20 years to include a variety of strategies
aimed at stimulating and redirecting the activity of T
the present review to cover these in detail, an over-
view of the general approaches is presented in Section
2. In Section 3, we summarize how the high-affinity
folate receptors (FRs) found on some tumor cells have
been used to specifically focus the activity of T cells
against the tumors. These studies include the genera-
tion of folate/antibody conjugates against epitopes of
the TCR/CD3 complex. Tumor models that have been
developed to study these effects include syngeneic
tumors in immunocompetent mice, FR + human
tumors transplanted into immunodeficient mice, and
FR+ choroid plexus tumors that arise endogenously in
SV40 transgenic mice. As with many clinical
approaches to generating T cell anti-tumor responses,
the use of cytokines, especially IL-2 and IL-12, to
optimize T cell activity have been tested. Finally, in
Section 4 we describe two strategies that are aimed at
eliciting longer term immune responses against can-
cer, both involving folate-mediated induction of ef-
fector and/or memory T cells that are specific for
tumor antigens.
2. T Cell immunotherapy of cancer
The normal interactions between a naı̈ve T cell
and an APC that lead to T cell activation are shown
in Fig. 1A APCs take up foreign proteins or tumor
cells and display processed peptides bound to a
protein of the MHC. The heterodimeric ah TCRs
on a small fraction of T cells are capable of binding
to these specific peptide/MHC products. Ligation of
the TCR/CD3 complex in addition to ligation of co-
stimulatory molecules such as CD28 with its ligand
B7 leads to T cell activation and proliferation [8]. T
helper cells that express the class II MHC-specific
accessory molecule CD4 and CTLs that express the
class I MHC-specific accessory molecule CD8 are
activated by the corresponding peptide/MHC com-
plexes. Our discussion here will focus largely on
CTLs, as most folate-mediated studies have been
 E.J. Roy et al. / Advanced Drug Delivery Reviews 56 (2004) 1219–1231 1221

Fig. 1. Activation and effector phases of CTL function. (A) A naı̈ve T cell is activated by two signals; one through the TCR/CD3 complex and
one through the co-stimulatory molecule CD28. The naı̈ve T cell expresses an ah TCR that, in association with the accessory molecule CD8,
has sufficient affinity for a specific peptide MHC complex (pMHC2) that is expressed by an APC. CD28 binds to its ligands, members of the B7
family, on the APC. (B) The activated CTL from panel A is capable of recognizing and lysing a tumor cell that expresses pMHC2, without
participation of CD28.

conducted with CTLs. Once activated, CTLs can
migrate to the site of a tumor where they recognize
and lyse tumor cells that express the antigenic
peptide/MHC (Fig. 1B). Recognition of peptide/
MHC by CTLs is facilitated by the action of CD8
that enhances binding to the class I product and
recruits other signaling molecules such as the kinase
p56lck [9,10]. Three general approaches to stimulate
and/or redirect the activity of T cells are discussed
below.
2.1. Bispecific antibodies
Initial studies showed that covalent attachment of
an anti-TCR antibody to the surface of a tumor cell
that lacked the appropriate pep/MHC allowed the
tumor cell to be lysed [11]. These studies soon led to
the development of bispecific anti-TCR/anti-tumor
cell antibodies that also redirected the activity of
activated CTLs against the tumor [12,13]. In the
decade following, many different tumor antigens
1222 E.J. Roy et al. / Advanced Drug Delivery Reviews 56 (2004) 1219–1231
were examined in studies with bispecific antibodies
directed against the ah TCR or the CD3 subunits
that form the TCR/CD3 complex [14,15]. The mech-
anisms for activating T cells typically involved either
the use of activating anti-CD3 antibodies, or alter-
natively, the use of bacterial superantigens such as
Staphyloccocal enterotoxin B that stimulate large
fractions of T cells [16]. Antibodies to CD3 allowed
recruitment of all T cells, whereas antibodies specific
for particular variable regions of the TCR would
recruit only subpopulations of T cells that bear that
variable region.
In principle, activating agents must in some way
allow the engagement of the co-stimulatory molecule
CD28 by its ligands of the B7 family (e.g. through
binding of the Fc region of anti-CD3 antibodies to Fc
receptors (FcR) on APCs or binding of superantigens
to class II MHC on APCs). Efforts to mimic these
interactions led to the characterization of bispecific
antibody conjugates that contain an anti-CD28 anti-
body and an anti-tumor cell antibody [17,18]. This
approach seeks to convert a tumor cell into an APC
thereby allowing the tumor to act as both the initiator
and the target of T cell responses.
2.2. T cell vaccines
There are currently many clinical trials that involve
the use of antigenic peptides, tumor cells, or tumor
mRNA to stimulate T cell responses [19 – 21]. These
vaccines are designed to elicit internalization and
subsequent presentation of the administered tumor
antigens by professional APCs (especially dendritic
cells, the most potent APCs in the immune system)
and thereby to generate effector and memory T cells
against tumors. Many studies have begun to elucidate
the numerous factors involved in T cell vaccines, as
reviewed most recently by Finn [21]. Attempts to
exploit folate-mediated T cell activity will be depen-
dent on the knowledge that is gained in these areas.
For example, some efforts now include the ex vivo
isolation and activation of dendritic cells that can
process and present tumor peptides along with co-
stimulatory ligands for optimal T cell activation.
Nussenzweig, Steinman and colleagues have explored
strategies to enhance the uptake of antigen using
antigen/antibody conjugates that bind to dendritic cell
surface molecules such as DEC205 [22]. The complex
set of factors that are involved in the potential success
of T cell vaccine approaches include the existence of
an adequate T cell repertoire in a patient (e.g. immu-
nosuppression could exist due to previous therapies,
factors released by the tumor, or the age of the
patient), the nature of the antigen (e.g. defined pep-
tides, proteins, whole tumor cell preparations, or
mRNA), the optimal state of dendritic cell maturation
(where dendritic cells are used ex vivo), the route of
administration, and delivery schedule. Considerable
pre-clinical and clinical work will be needed to begin
to resolve these issues.
2.3. Adoptive T cell therapies
Two recent reports on the treatment of melanoma
patients with ex vivo activated T cells have encour-
aged additional efforts towards adoptive T cell thera-
pies [23,24]. A disadvantage of adoptive therapy is
the tedious and time-consuming need for culturing of
each patient’s T cells. A further issue is whether the T
cell repertoire of a patient has been ‘tolerized’ against
tumor antigens to the extent that T cells with adequate
TCR avidity and activity can not be expanded in vitro
[25 –27]. A possible strategy under these circumstan-
ces is to combine adoptive T cell therapy with gene
therapy using TCRs isolated and/or evolved in vitro
for specificity or higher affinity against a peptide
antigen [10,28 – 30]. This approach has been applied
with some success in animal models, using TCR
genes isolated from viral or tumor-specific T cells
[31,32].
An alternative approach to the use of TCR genes
has been to express a chimeric gene fusion that
encodes an antibody-binding region (VH – VL) and a
T cell signaling region such as the CD3 ~ subunit
[33 –35]. While such T cells (called ‘‘T-bodies’’) have
been engineered with a number of different antibodies
to demonstrate redirected activity of T cells, it remains
to be seen if the approach yields various other T cell
functions in vivo (e.g. T cell migration, persistence,
and memory) that are important in tumor targeting.
3. Folate-mediated targeting of T cells to cancer
The purpose of this review is to focus on those
studies that have involved targeting of T cells against
 E.J. Roy et al. / Advanced Drug Delivery Reviews 56 (2004) 1219–1231
tumors that express the high-affinity FR. The expres-
sion of FR on tissues and tumors [36 – 39] is the topic
of other articles in this issue, and will thus not be
discussed further here.
3.1. Tumor models
Several different tumor models have been used to
test the efficacy of T cell targeting to FR+ tumors.
Animal models of bispecific antibody targeting
against human tumors have typically relied on the
transplantation of the human tumor lines into immu-
nodeficient SCID or nude mice [40,41]. In these
cases, in vitro activated human T cells are also
adoptively transferred to provide a source of effector
cells. We have described a murine model that allows
activation of an endogenous T cell population and
transplantation of human tumors, an approach that is
desirable in translation of therapies to human cancer.
In our model, the human FR+ tumor line KB was
transplanted into 2C TCR/RAG / mice that bear a
single monoclonal population of T cells [42]. 2C
TCR/RAG / mice express TCR transgenes for a
and h chains from a mouse CTL clone called 2C and
knockout of the recombination-activation gene-1 that
is required for gene rearrangement in B and T cells
[43]. The CD8+ monoclonal T cell population can be
activated in vivo with the agonist peptide recognized
by CTL 2C called SIYR, but their TCR does not
recognize human tumor cells. However, the 2C TCR/
RAG / model is limited by the absence of a number
of cell types (B cells, CD4+ helper T cells, and CD4+
regulatory T cells) that are known to affect the
immune response against tumors.
A syngeneic model of FR+ tumors has involved the
BALB/c tumor M109, a spontaneous lung carcinoma
[44]. In this tumor model, an indirect approach was
used to target tumor cells, involving the induction of
an anti-hapten antibody response in BALB/c mice,
followed by transplantation of M109, and administra-
tion of folate/hapten conjugates. While the rejection of
M109 required the presence of anti-hapten antibodies,
it is not clear if T cells were required as a component
of the effector response. Nevertheless, this syngeneic
tumor model could be used to evaluate the various
folate-mediated targeting strategies that require im-
munocompetent mice, as described below. Other
BALB/c tumor lines that express various levels of
1223
the FR have been described and could in principle
also be used as syngeneic models of T cell targeting
[45,46].
We have used an endogenously arising FR+ tumor
model that involves the SV40 large T antigen trans-
genic line of mice called SV11 [47,48]. These mice
develop endogenous brain tumors and ultimately
succumb to the tumor around 100 days of life. Tumors
derived from the choroid plexus express very high
levels of the FR [49], similar to human choroid plexus
carcinomas and ependymomas. MRI can be used to
monitor the status of tumors, thus providing a non-
invasive method for comparing various targeting
regimens. Because tumors arise endogenously, SV11
mice represent the scenario encountered clinically, in
which an individual’s T cells are tolerant of the tumor
[50]. Studies of adoptively transferred T cells, derived
from syngeneic C57Bl/6 mice that are not transgenic
for SV40 large T antigen, showed the potential of T
cell therapies, as the lifespan of treated SV11 mice
could be more than doubled [51]. SV11 mice are
tolerized against the SV40 large T antigen, and hence
they may lack the T cell repertoire capable of eliciting
strong anti-SV40 responses. However, SV11 mice
may contain T cells with TCRs that have lower
affinities for particular SV40 peptide epitopes and
these T cells may be activated against the tumor upon
sufficient stimulation [51].
3.2. Bispecific folate/antibody conjugates
Among the first tumor antigens targeted with the
bispecific antibody approach was an ovarian carcino-
ma antigen recognized by two monoclonal antibodies
called Mov18 and Mov19 [36,40,52,53]. The tumor
antigen recognized by these antibodies was a folate
binding protein now known to bind folate with
relatively high-affinity (KD f 1 nM). Mezzanzanica
et al. showed that Mov18 specific antigen was stable
on the cell surface and was present for a sufficient
time to allow for redirected lysis of ovarian carcino-
mas by T cells [40]. Several clinical trials for patients
with ovarian carcinoma were only marginally success-
ful, limited at least in part by the induction of human
anti-mouse antibody (HAMA) responses [54]. More
recent pre-clinical studies have used chimeric versions
of the bispecific antibody in an attempt to avoid
HAMA responses [55].
1224 E.J. Roy et al. / Advanced Drug Delivery Reviews 56 (2004) 1219–1231

As an alternative to the bispecific antibody ap-
proach, we explored the use of bispecific folate con-
jugates in which folate was coupled to antibodies
specific for TCRs. Folate/antibody conjugates offer
the advantages of having a smaller size compared to
traditional bispecific antibodies, a factor that has been
correlated with increased tumor penetration [56]. The
folate/antibody conjugate also should exhibit a de-
creased probability of generating anti-Ig immune
responses. Conjugates of folate and anti-TCR or anti-
CD3 antibodies were shown to retarget the lysis of FR+
tumor cell lines (Fig. 2A) [46]. Furthermore, these
conjugates were capable of modest improvements in
survival when SV11 mice were treated with the super-
antigen followed by folate/anti-CD3 IgG [57].
Subsequent studies showed that the smallest bis-
pecific antibody yet produced, folate-coupled to a
single-chain Fv antibody ( f 25 kDa), was also very
efficient in mediating T cell lysis of FR+ tumor cells
[58]. Folate/scFv and folate/Fab conjugates were

Fig. 2. Folate-mediated activities of CTLs. (A) A conjugate of folate and an anti-TCR/CD3 antibody can mediate the lysis of FR+ tumor cells by
an activated CTL. (B) In principle, a conjugate of folate and an anti-CD28 antibody could convert a FR+ tumor cell into an ‘APC’. In this
scenario, the CTL is activated by engagement of the TCR/CD3 complex with a specific peptide-MHC tumor antigen and by engagement of the
CD28 with the folate/anti-CD28 antibody bound to FR on the tumor cell surface.
 E.J. Roy et al. / Advanced Drug Delivery Reviews 56 (2004) 1219–1231
equally effective in promoting tumor rejection in the
KB tumor model in TCR/RAG mice [42]. This study
also showed the importance of pre-activating the
endogenous T cell population prior to treatment with
folate conjugates. The advantage of smaller conju-
gates stems from their ability to penetrate into solid
tumors, but their most notable disadvantage is a very
short serum lifetime (minutes). While intact IgG anti-
bodies have considerably longer serum lifetimes
(days), the Fc region can invoke FcR mediated effects
involving excessive cytokine release, as was original-
ly observed with the anti-CD3 antibody OKT3 in
humans. These side-effects can be minimized by using
a mutated Fc region that has reduced binding to the
FcR [59].
The issues that remain to be resolved for this
approach hold true for all bispecific antibody
approaches, including those that have been tested
clinically. Most importantly, the success will depend
on the degree to which a sustained and robust T cell
response can be achieved (see below). Their poten-
tial will also depend on the level of FR expressed
on tumors and the extent to which FR-negative
tumor cells can thrive and thereby escape redirected
T cell activity. An early report suggested that the FR
may enhance tumor growth and therefore may be
associated with selective pressure to maintain the
target [60].
3.3. Optimal T cell targeting: cytokine co-treatments
In the past decade it has become clear that various
cytokines play key roles in the induction and mainte-
nance of T cells. It is still unclear how these cytokines
can best be used clinically to generate a powerful T
cell response against tumors. Perhaps the most rele-
vant cytokines for purposes of a CD8+ T cell response
are IL-2, IL-12, and IL-15. IL-2 has long been known
to be critical factor for T cell proliferation and it has
been used clinically in many immunotherapeutic regi-
mens [61,62]. Early studies showed that IL-2 at high
doses caused significant side-effects but the most
recent trials with adoptive transferred T cells have
used IL-2 at lower doses to help alleviate the adverse
responses [62]. It has been suggested that IL-2 may
not be optimal in that it promotes activation-induced
cell death of self-reactive CD8+ T cells, whereas the
cytokine IL-15 can prevent apoptosis of CD8+ T cells
1225
and therefore may allow more sustained T cell activity
[63,64]. It is too early to tell whether a standardized
and perhaps alternating regimen of IL2 and/or IL-15
could improve T cell therapeutic approaches. IL-2 and
a-IFN have been used in combination with folate
conjugated haptens to promote anti-tumor immune
responses mediated by antibody-dependent cellular
cytotoxicity (ADCC) [44]. To the best of our knowl-
edge, IL-15 has not been tested with folate-mediated
approaches.
IL-12 has also received considerable attention as a
possible adjuvant for tumor vaccines since it acts as a
T cell potentiating agent [65,66]. IL-12 is secreted by
APCs and TH cells and it acts on CD8+ T cells to
generate longer term effector cells and possibly,
memory T cells [65,67]. Studies with SV11 mice have
shown that IL-12 alone, without folate/antibody con-
jugates, induces extensive T cell infiltration into
choroids plexus tumors [68]. Unfortunately, attempts
to combine IL-12 treatments with bispecific folate/
anti-TCR antibody to boost the strength of the T cell
response have led to lethal effects (see below). IL-12
treatment of cancer patients in a phase II trial unex-
pectedly had severe toxicity [69], but it is not clear if
the lethality that we observed in mice is related
mechanistically [70]. Nevertheless, studies described
below indicate that IL-12 could be a very important
component of T cell therapy that uses FR-targeted co-
stimulation, with minimal toxicity.
4. Potential strategies for eliciting effector and
memory T cells against tumors
4.1. Co-stimulatory molecules
The approaches with bispecific antibodies de-
scribed above rely on the FR as the target for cross-
linking the TCR/CD3 complex on the T cell. In order
to induce long-term immunity against a tumor, it
would be useful to generate T cells that recognize
tumor-associated pepMHC complexes. Self-reactive,
low-avidity T cells that have escaped clonal deletion
during thymic education can become CTL effector
cells upon sufficient stimulation [71], and peripherally
anergized CTLs can be recovered by repetitive in vitro
stimulation [72]. While the activation of self-reactive
CTLs has been demonstrated, it appears that T cell
1226 E.J. Roy et al. / Advanced Drug Delivery Reviews 56 (2004) 1219–1231
activation is often transient and anergy prevails upon
subsequent stimulation [73]. As indicated, the activa-
tion of naı̈ve T cells is highly dependent on signaling
through the CD28/B7 co-stimulatory pathway in ad-
dition to the TCR/pepMHC interaction. Because tu-
mor cells typically lack B7 molecules, optimal
stimulation requires processing of tumor antigens
through APCs. Stimulation of the TCR without
CD28 can in some cases lead to peripheral T cell
anergy against the peptide-MHC antigen (i.e. on the
tumor).
The crucial role of CD28 in the activation of low-
avidity potentially tumor-reactive T cells has made it a
target for eliciting an anti-tumor T cell response. In
this context, various strategies have emerged: (1) the
development of bispecific antibody conjugates which
simultaneously crosslink CD28 on T cells to tumor
antigens (Fig. 2B), (2) transfection of tumor cells with
the B7 gene [74], and (3) transfection of T cells with a
secreted bispecific protein composed of B7 and an
anti-tumor single-chain antibody [75]. The advantage
of the first approach is that it does not require ex vivo
manipulation the patient’s tumor or T cells. The
objective of these strategies is to convert tumor cells
into competent APCs that are capable of activating
anti-tumor T cell responses against tumor pepMHC
antigens. Under the optimal conditions, the approach
might also promote the generation of memory T cells
that could target re-occurring cancers.
Human clinical trials of ovarian cancer using
bispecific antibody to CD3 and FR showed an in-
crease in tumor redirected lysis when combined with a
bispecific antibody to CD28 and FR [76]. There is
some evidence that long-term immunity is influenced
by the strength of the initial T cell response as induced
by bispecific agents [77]. The induction of optimal
memory cells may require both co-stimulatory signals
and IL-12 or other cytokines. Under normal condi-
tions, T cell activation and memory induction appear
to involve the action of IL-12 secreted by activated
APCs upon CD40/CD40L interaction, the so-called
third signal [78]. Our lab has investigated the role of
CD28 co-stimulation against FR+ SV11 tumors by
testing various folate/anti-CD28 antibody conjugates
alone and in combination with IL-12 (manuscript in
preparation). Again, the advantage of using folate as
the targeting agent is that it can be readily coupled to
antibodies and tested in vitro and in vivo.
4.2. Antigen presenting cells: folate-mediated uptake
As indicated above, during the course of our
studies with IL-12 and folate/anti-TCR antibody con-
jugates we observed that animals treated with a
combination of these agents were highly susceptible
to lethal reactions that occurred within 48 h of treat-
ment. The toxicity was observed with both SV11 and
normal C57Bl/6 mice, indicating that the reaction was
independent of FR+ tumors. Although the basis of this
toxicity has not been further resolved, we suspect that
a toxic-shock like syndrome may be responsible. In
this scenario, the folate/anti-TCR conjugates might
bind to FR+ macrophages that have been activated
through an IL-12-dependent mechanism (e.g. IL-12
induces g-IFN, which is known to induce FRs on
macrophages [79]). IL-12 may also act through the
synergistic activation of T cells. The observed effect
may be analogous to that of bacterial superantigens,
which induce toxic-shock by binding to class II MHC
expressed on APCs such as macrophages and to Vh
regions of the TCR on T cells. This cross-linking leads
to the massive release of T cell cytokines and subse-
quent organ failure.
Several observations suggest that the high-affinity
FRs on activated macrophages (or dendritic cells, if
they are expressed on these cells also) could provide
a useful mechanism for the delivery of T cell-specific
antigens or whole tumor cells (Fig. 3): (1) Low and
his colleagues have previously shown that the high-
affinity FR could be exploited as a target for the
endocytosis of folate-coupled macromolecules, in
order to facilitate their uptake [80 –85]; (2) macro-
phages are known to express the ligands (B7.1,
B7.2) for co-stimulation of T cells and Nussenzweig,
Steinman and colleagues have shown that receptor-
mediated uptake of protein antigens by dendritic
cells may provide a strategy for creating optimized
vaccines [22]; (3) activated macrophages express
FRs [79] at levels that are even diagnostic for
inflammatory reactions [86], and hence the FR may
be an ideal candidate for facilitating the endocytosis
of T cell antigens by APCs; and (4) T cells reside in
regions where these cells might be able to present
antigen.
As pointed out by Lu and Low, it is possible that
antibody-mediated uptake of tumor cells by activated
macrophages may also have been involved in the
 E.J. Roy et al. / Advanced Drug Delivery Reviews 56 (2004) 1219–1231 1227

Fig. 3. Possible strategy for generating vaccines using FR-mediated uptake of antigens. As macrophages express the high-affinity FR, it may be
possible to facilitate their uptake and processing of specific antigens to T cells by conjugation of known antigens or whole tumor cells to folate.
The enhanced levels of the specific peptide/MHC (pMHC2) could activate T cells that attack tumor cells expressing pMHC2.

effects observed in their study, using folate/hapten
conjugate [44,87]. In principle, antibody-mediated
uptake of tumor cells by APCs could be more effec-
tive than folate-mediated uptake of antigens, as the
former relies on well-known binding and internaliza-
tion properties of FcR on dendritic cells and macro-
phages. In contrast, it is unclear if folate-mediated
uptake of antigens by macrophages will be as efficient
as FcRs and it is not known if dendritic cells bear
high-affinity FRs.
1228 E.J. Roy et al. / Advanced Drug Delivery Reviews 56 (2004) 1219–1231
5. Concluding remarks
The destruction of tumor cells by T cells is now
regarded as a desirable goal in the design of cancer
immunotherapeutics. There are several distinct
approaches toward T cell immunotherapies that are
in various stages of clinical and pre-clinical studies.
At this time it is not known what the optimal approach
will be and in fact it is quite possible that different
approaches will be required, depending on the type of
cancer and the patient. While it is still unclear whether
T cell therapies that involve folate-mediated targeting
will provide measurable advantages, the principles of
high-affinity interactions with FRs and the use of
small molecule (folate) as conjugates facilitates the
exploration of their use in this platform. Accordingly,
folate conjugated to an anti-T cell antibody redirects
the activity of a T cell against a FR positive tumor.
Whereas antibodies against the TCR complex can
directly mediate lysis of the tumor by an activated
cytotoxic T cell, antibodies against co-stimulatory
molecules provide an opportunity to induce T cells
with tumor specificity (i.e. tumor-specific pepMHC
that are recognized by the TCR). T cells stimulated in
this manner, in combination with the appropriate
cytokine regimen, could develop into longer-term
memory cells that provide greater protection against
the cancer.
In another approach, FRs expressed on activated
macrophages could be targeted with folate/antigen
complexes. Activated macrophages serve as APCs,
with appropriate co-stimulatory ligands for the acti-
vation of resting T cells. Hence, it may be possible to
enhance an inflammatory T cell reaction against tumor
antigens, thereby stimulating a potent anti-tumor re-
sponse. An advantage of this approach is that it does
not rely on the expression of high-affinity FRs by a
tumor. While both of these approaches will require
significant additional pre-clinical work, the ability to
readily conjugate folate to proteins of interest will
facilitate studies to explore their potential in the area
of T cell therapeutics.
Acknowledgements
We thank other members of the lab for their efforts
on projects involving folate-mediated targeting of T
cells to tumors. The work was supported by grants
from the NIH.
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