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*
Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037; and
Department of Anesthesiology, University of California, San Francisco CA 94110
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Some of the initial attempts in cancer immunotherapy were based on the ability of IL-2 to activate and expand T cells as well as other types of lymphokine activated killer (LAK)3cells (12). Patients were infused with high concentrations of IL-2 alone, with large numbers of LAK cells or with tumor infiltrating lymphocytes that had been expanded in vitro in the presence of IL-2 (13). Because high systemic concentrations of IL-2 proved toxic, other approaches were used to deliver the cytokine specifically to the tumor microenviroment to augment its immunogenicity. One such strategy was the construction of recombinant fusion proteins consisting of tumor-specific mAbs linked to IL-2 (14, 15). When administered in vivo, this molecule could promote eradication of tumors and their metastasis by CD8+ T cells and could lead to the development of a long-lived and transferable tumor immunity (16, 17, 18). It has been assumed that the mechanism of activity of the fusion protein is similar to that of soluble IL-2, because both molecules augment lytic activity of activated-effector cells.
Using transgenic mice that express HLA-A2.1/Kb molecules, we have recently identified several peptides derived from the Her-2/neu tumor-associated protein that are processed and presented in association with HLA-A*0201 (A2) molecules expressed by human tumors that express both A2 and Her-2/neu (19). CTL obtained from these mice were able to lyse a wide variety of human tumors. As reported by others, experiments to determine whether these CTL could prevent growth of human tumors in vivo in SCID mice required very high concentrations of exogenously provided IL-2 to optimize the activity of the adoptively transferred CTL (20). In an effort to avoid the use of such high concentrations of cytokine, we generated a fusion protein consisting of a single chain Her-2/neu-specific mAb linked to IL-2 (neu-Ab-IL-2). In the course of in vitro characterization of the fusion protein, we observed that it could mediate the formation of stable heteroconjugates between effector T cells expressing IL-2R and tumors expressing Her-2/neu. This resulted in lysis of any Her-2/neu-expressing cell by either CD8+ or Th1 CD4+ T cells in a non-MHC restricted manner. This cytolytic mechanism is mediated by Fas-Fas ligand (Fas L) rather than perforin and represents a novel form of redirected lysis that does not engage TCR. In addition, when delivered to SCID mice harboring human tumor cells, the fusion protein was able to direct the migration of non-tumor-specific T cells to the tumor site and this resulted in the elimination of tumor in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The Clone-4 TCR transgenic murine line has been previously described (21). BALB/c, C57BL/6, and C.B.-17 scid/scid mice were obtained from the rodent breeding facility at The Scripps Research Institute. All animals were housed under specific pathogen-free conditions.
Cell lines
p773 is an A2-restricted CTL line derived from the (A2/Kb x huCD8)F1 transgenic mice and specific for the Her-2/neu peptide spanning residues 773–782 (19). p773 CTL was maintained in vitro by weekly restimulation in 2 ml cultures by incubating with 0.2 x 106 irradiated-Jurkat-A2 cells (20,000 rads) preincubated with the p773 peptide (20 µM) and 5 x 105 irradiated C57BL/6 spleen cells (3000 rads) as fillers in complete RPMI media (RPMI 1640 medium supplemented with 10% FCS, 2 mM glutamine, 5 x 10-5 2-ME, and 50 µg/ml gentamicin) supplemented with 2% (v/v) T cell growth factor supernatant from Con A-stimulated rat spleen cells. Clone-4 TCR CTLs were obtained form the Clone-4-TCR transgenic mice that recognize an influenza hemagglutinin (HA) Kd-restricted peptide (21, 22). Spleen cells (2 x 106) from Clone-4 TCR mice were stimulated with 6 x 106 irradiated BALB/c splenocytes previously pulsed with 20 µM of the KdHA peptide in 2 ml of complete RPMI. Tumor cell lines used in these studies were as follows: NCI-H1355 (A2+/Her-2/neu+) provided by A.F. Gazdar (The University of Texas, Southwestern Medical Center, Dallas, TX), OV5 (A2-/Her-2/neu+) was provided by P.S. Goedegebuure (Washington University School of Medicine, St. Louis, MO) and LG-2 (A2+/Her-2/neu-) was purchased from the American Type Culture Collection (Manassas, VA) and maintained in complete RPMI at 37°C in a 5% CO2 environment. CTLL-2 cells, an IL-2-dependent T cell line, were maintained in complete RPMI supplemented with 5% TCGF. The Th1-cloned line, K3-Th1, was kindly provided by Dr. Susan Webb (The Scripps Research Institute).
Plasmid construction
The single chain neu-Ab-IL-2 fusion protein was constructed by PCR amplification of the Fv portion of the single chain (scFv) anti-Her-2/neu Ab, C6.5 (23). Synthetic oligonucleotides were produced, introducing a BamHI restriction site at the 5' end and a EcoRI site at the 3' end of the Fv to allow insertion into the pCDNA3 expression vector (Invitrogen, San Diego, CA). The human constant lambda (CL) chain was obtained by RT-PCR from mRNA of human bone marrow. An EcoRI site was added at the 5' end of the CL primer and an EcoRV site was added at the 3' end. The amplified CL chain was ligated into the pCDNA3 vector containing the anti-Her-2/neu scFv. Finally the murine IL-2 gene (obtained from R. Levy, Stanford University) was amplified by PCR adding an EcoRV site at the 5'end and a NotI site at the 3' end and ligating into the pCNDA3-scFv-CL to obtain the neu-scFv-CL-IL-2 fusion protein. We designated this construct neu-Ab-IL-2. For control purposes we constructed in a similar fashion the scFv linked to CL (neu-scFv-CL) designated neu-Ab. All constructs were assembled and inserted into the pCDNA3 expression vector as a single-reading frame and confirmed by restriction analysis and DNA sequencing. The specific genes encoding the VH and VL domains of the 37–38 anti-HA mAb (24) were cloned by rapid amplication of cDNA ends (RACE)-PCR utilizing the marathon cDNA amplification kit (Clonotech). Total RNA was isolated from the hybridoma cells and first and second strand cDNA was synthesized. An adaptor was ligated to the 5' double stranded cDNA to allow the amplification of the variable regions by PCR. Amplification was performed using an adaptor oligonuclotide and the antisense VH and VL primers. The amplified products were subcloned into the PCR2.1 vector (Invitrogen) and subjected to sequence analysis. The 37–38 anti-HA scFv-CL-IL-2 (37-38-Ab-IL-2) was constructed in the same fashion as the neu-scFv-CL-IL-2.
Expression and purification of the fusion protein
The neu-Ab-IL-2, neu-Ab, and 37-38-Ab-IL-2 plasmids were transfected into P3x63Ag8.658 (nonsecreting B cell myeloma) and cells were clonally selected for growth in the presence of 1 mg/ml of G418 (Life Technologies, Grand Island, NY). Supernatants from surviving clones of the neu-Ab-IL-2 and neu-Ab transfectants were screened for the ability to bind Her-2/neu+ cell line by FACS analysis. FACS analysis was performed by incubating 50 µl of the supernatants with tumor cells and then with 1 µg/sample of mouse-anti-human-CL (PharMingen, San Diego, CA); and next, with 1 µg/sample of goat-anti-mouse-FITC (Jackson ImmunoResearch, West Grove, PA). Positive clones from the neu-Ab-IL-2 transfectants that recognize Her-2/neu were tested further for their ability to induce the proliferation of the IL-2-dependent cell line, CTLL-2. One positive clone that transfected with each construct was selected. The neu-Ab-IL-2 and neu-Ab fusion proteins were purified on an affinity column of anti-human CL (PharMingen) covalently linked to protein G matrix (Pharmacia, Piscataway, NJ). Supernatants from neu-Ab-IL-2 and neu-Ab fusion proteins were passed over the column and eluted with 0.1 M glycine (pH 2.7). The eluate material was dialyzed against PBS. One liter of supernantant produced approximately 1 mg of purified fusion protein. All the experiments presented here were performed with affinity column purified-fusion proteins. The 37-38-Ab-IL-2 fusion protein has the ability to bind HA-positive cells and also has IL-2 activity (data not shown). The 37–38-Ab-IL-2 protein was purified in the same manner as neu-Ab-IL-2.
Conjugate formation
To assess T cell-target cell conjugate formation, 1 x 106/ml CTLL-2 cells were incubated for 1 h in a solution containing 0.1 µg/ml of the green fluorochrome, calcein AM (Molecular Probes, Eugene, OR). Tumor cells (OV5) were incubated with 50 µg/ml of the red fluorochrome, dihydroethidium (Molecular Probes). Both cells were washed twice and mixed at a 1:1 ratio. Cells were incubated in a solution containing 1 µg of the indicated-fusion protein for 30 min at 37°C to allow conjugate formation. FACS analysis was performed for the detection of the conjugates.
IL-2 assay
IL-2 activity of the fusion proteins was determined by a standard T cell proliferation assay measuring the survival of the IL-2-dependent T cell line CTLL-2 by 3,[4,5-dimethythiazol-2yl]-2,5-diphenyltetrazoliumbromide, thiazolyl blue (25) (MTT, Sigma, St. Louis, MO). Briefly, after IL-2 depletion for 24 h, 103 CTLL-2 cells were added to each well of a 96-well flat-bottom plate with various concentrations of recombinant human IL-2 (obtained from Biological Resource Branch, Nation Cancer Insititute, Bethesda, MD) or fusion protein. After 24 h, MTT was added at 5 mg/ml and incubated for 4 h. Color crystals produced by viable cells were dissolved in isopropanol and 0.04 N HCL, and their absorbance was read at 570–630 nm. All samples were tested in duplicate.
Cytotxicity assay
Target cells were incubated at 37°C with 150 µCi of 51Cr sodium chromate for 90 min. Cells were washed three times and resuspended in complete RPMI. For the assay, 51Cr-labeled target cells (104) were incubated with varying concentration of effector cells either in the presence or absence of the indicated fusion proteins (0.1 µg/well) in a final volume of 200 µl in U-bottomed 96-well microtiter plates. Supernatants were recovered after 6 h of incubation at 37°C, and the percent specific lysis was determined by the formula: percent specific lysis = 100 x (experimental release - spontaneous release)/(maximum release - spontaneous release). The role of perforin was assessed by chelating Ca2+ with EGTA (4 mM) and adding 3 mM MgCl2 to the media.
Adoptive transfer model
Six- to eight-week-old female C.B.-17 scid/scid mice were used for tumor implantation. Mice were injected with the transplantable NCI-H1355 tumor cell line. On day 0 animals were implanted subcutaneosly with 106 cells. Tumors were allowed to develop for 7 days before receiving CTLs. On day 7 after tumor cell inoculation, animals were randomly divided into groups of 3–5, and A2-restricted CTLs specific for Her-2/neu or Clone-4 CTL specific for the KdHA peptide were injected i.p. at 50 x 106 cells per animal. Control animals were injected with PBS only. On day 14, animals were injected again with a second equivalent dose of CTLs. From day 7 to day 21 animals were injected daily i.p. with 10 µg of neu-Ab-IL-2 fusion protein, 10,000 U of rIL-2, or PBS. Tumor growth was monitored every 5 days, and growth rates were determined by caliper measurements in two diameters.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The fusion protein encoding the C6.5 single chain Fv was first
fused to the human CL chain and then linked to IL-2 (referred to as
neu-Ab-IL-2; see Fig. 1
A). The
CL chain was included to provide a spacer between the Ab and the IL-2
to allow independent folding of each domain. The neu-Ab and
37-38-Ab-IL-2 fusion proteins were constructed for control experiments
as described in Fig. 1. The neu-Ab was composed of the single chain Fv
linked to the CL. The 37-38-Ab-IL-2 molecule has specificity for the
influenza HA. All three fusion proteins were purified by anti-CL
affinity columns from supernatants of appropriately transfected myeloma
cells as described in Materials and Methods.
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FACS analysis was used to determine whether the neu-Ab-IL-2 fusion
protein had retained both the ability to bind Her-2/neu and to be
recognized by anti-IL-2 Ab. As shown in Fig. 2, both the neu-Ab and neu-Ab-IL-2
constructs bound to the Her-2/neu expressing OV5 cell line, but not to
LG-2 cells that do not express Her-2/neu, as determined using a
FITC-labeled anti-CL Ab (Fig. 2). Once bound to the OV5 cells, the
neu-Ab-IL-2 fusion protein was also detectable using anti-IL-2 Ab,
indicating that the IL-2 domain remained structurally intact. To
determine whether the IL-2 retained its ability to bind to the IL-2R,
the fusion protein was tested for its ability to bind CTLL-2 cells that
express high levels of IL-2R. As shown in Fig. 3, binding was detected by the
neu-Ab-IL-2 fusion protein but not with the neu-Ab. Thus, the
constructs retain the ability to bind both Her-2/neu and IL-2R.
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, 1 µg of fusion protein corresponds
to 1200 IU of IL-2 activity.
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Considering the ability of the fusion protein to bind both T cells
and tumor cells expressing Her-2/neu, it was of interest to determine
whether the presence of the fusion protein could induce formation of
stable conjugates between these two types of cell lines. To determine
if this was the case, a CTL line specific for an A2-restricted
Her-2/neu peptide 773 (19) and OV5 tumor cells, which express Her-2/neu
but not A2, were individually stained with different color fluorescent
dyes, washed, and then mixed in a 1:1 ratio in the presence of 1 µg
of neu-Ab or neu-Ab-IL-2 (Fig. 5). Stable
conjugates between T cells and tumor cells were formed only in the
presence of neu-Ab-IL-2, demonstrating that this molecule can bridge
noncognate effector and target cells and form heteroconjugates by
binding the IL-2R on T cells and Her-2/neu on the tumor.
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It was previously demonstrated that Ab-IL-2 fusion protein could
enhance the cytotoxic activity of LAK cells, tumor infiltrating
lymphocytes, and activated CD8+ T cells against tumors
cells in vitro (26). In these previous studies, in vitro cytotoxicity
was equivalent when either soluble IL-2 or the fusion protein was added
to the mixture of the tumor and effector cells in vitro. Therefore, it
was assumed that the fusion protein was mimicking the activity of
soluble IL-2 by enhancing LAK activity in vitro. To determine what
contribution the specificity of the CTL may have under these
conditions, we compared the ability of the fusion protein to promote
the lysis of Her-2/neu expressing tumor cells, NCI-H1355, by either
tumor-specific CTL, or CTL with specificity for an irrelevant peptide.
The p773 CTL line can specifically lyse the A2+,
Her-2/neu+ NCI-H1355 cells (19). As shown in Fig. 6A, this lysis could be
enhanced in the presence of IL-2 or the neu-Ab-IL-2 fusion protein. The
Kd-restricted Clone-4 TCR CTL, which does not specifically
recognize NCI-H1355 cells, could also lyse these targets when in the
presence of the neu-Ab-IL-2 fusion protein or IL-2 (Fig. 6B). Thus, as previously reported, the fusion protein could
enhance cytotoxicity against tumor cells by activated-CD8+
CTL, regardless of the specificity of the TCR.
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We wished to determine the mechanism of killing mediated by the
neu-Ab-IL-2, particularly because it was not directed through the TCR.
The two major pathways for T cell-mediated killing are perforin and Fas
(27). It is known that perforin requires Ca2+ to mediate
killing and is inhibited by EGTA (28). As shown in Fig. 7B, killing of OV5 cells by
the Kd restricted Clone-4 TCR CTL was not inhibited by the
presence of EGTA, suggesting that the killing was probably mediated by
the Fas pathway. To confirm that killing was Fas mediated, CTLs
obtained from gld+/+ mice (FasL positive) or
gld-/- mice (FasL negative) Clone-4 TCR mice were
compared. As shown in Fig. 7C, in the presence of the
neu-Ab-IL-2, the Clone-4 TCR CTLs were able to kill the OV5 target
cells whereas the Clone-4 TCR gld/gld CTLs could not, thus
demonstrating that killing required the presence of FasL. The neu-Ab
protein could not induce lysis. These data demonstrate that the
noncognate killing induced by the neu-Ab-IL-2 fusion protein is
mediated through the Fas-FasL pathway.
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, OV5 tumor cells were killed by the
CD4+ K3-Th1 clone in the presence of neu-Ab-IL-2. Thus, the
fusion protein enables both CD8+ and Th1-CD4+ T
cells to kill Her-2/neu-expressing tumor cells.
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Our original goal in producing the fusion protein was to use it to
replace IL-2 to enhance the activity of tumor-specific CD8+
T cells in vivo. To determine whether the neu-Ab-IL-2 fusion protein
could perform this function in vivo, we used a model in which human
tumor cells were transferred into SCID mice. NCI-H1355 tumor cells
1 x 106 (A2+, Her-2/neu+)
were implanted s.c. into SCID mice and tumors were allowed to establish
for 7 days at which time a small tumor mass was palpable at the site of
injection. At day 7, animals were randomly divided into groups
(3–5/group) and injected i.p. with 50 x 106 of the
Her-2/neu-specific p773 CTLs or the KdHA-specific Clone-4
TCR CTLs. These T cells were preincubated with the indicated fusion
protein before adoptive transfer into the animals. On day 14, mice were
treated with a second equivalent dose of CTLs. Animals also received 10
µg daily of neu-Ab-IL-2 fusion protein from day 7 to day 21. As shown
in Fig. 9A, tumor growth was
delayed in mice that were treated with p773-CTL. However, in the
presence of the neu-Ab-IL-2 fusion protein or rIL-2 (in amounts
equivalent to that delivered by the fusion protein), complete rejection
of the tumor was observed. Thus, IL-2 or the fusion protein could
significantly enhance the ability of Her-2/neu-specific CTL to reject
tumor cells.
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B, Clone-4 TCR CTL alone did
not retard tumor growth. Thus, the CTL specificity was important for
inhibition of tumor growth. However, if mice received Clone-4 TCR CTLs
followed by injection with neu-Ab-IL2, a significant reduction in tumor
growth was evident. (Fig. 9B). In contrast, treatment of
control animals with Clone-4 TCR CTL in combination with neu-Ab fusion
protein plus rIL-2 did not prevent tumor growth. Thus, conjugate
formation between effector and target cells, as promoted by the fusion
protein, appears to be required for tumor rejection by
non-tumor-specific CTL in vivo. To determine whether CTLs require
preincubation with Ab-IL2 fusion protein to achieve optimum tumor
elimination, treatment with the fusion protein was delayed until
36 h after the CTLs were first injected. As can be observed in
Fig. 9B, the T cells do not need to be preincubated with
fusion protein to be effective in eliminating tumor. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RIII receptors (30). In this
report, our focus was on the activity of the IL-2 portion of the
molecule that was engineered to be devoid of Fc domains. Thus, its
activity on effector cells was mediated solely through binding to the
IL-2R on T cells. Additionally, there have been no studies of the
significance of TCR specificity to the activity of fusion proteins in
vivo. The tumor target Ag selected for these studies was Her-2/neu. We had developed several CD8+ T cell lines specific for peptide epitopes of this tumor-associated Ag presented by A2 molecules and we wished to determine how effective such CTL would be in eliminating human tumor cell lines that express this epitope. To test this hypothesis, we obtained a single chain Her-2/neu- specific Ab and used the variable regions to construct a neu-Ab-IL-2 fusion protein. The molecule that was produced maintained both its IL-2 activity and Ab binding specificity, as demonstrated by antigenicity and biological activity.
Based on the current results, there appear to be several advantages in the use of fusion proteins as opposed to systemic application of rIL-2 in monitoring tumor-specific effectors in vivo. First, as demonstrated by FACS analysis, the fusion protein can stably bridge effector cells bearing IL-2R with tumor cells. This could serve to concentrate activated T cells that are not necessarily tumor specific, yet express high levels of IL-2R at the tumor site. Next, as recently described by Esser et al. (31), engagement of IL-2R on T cells can trigger Fas-mediated lysis. Indeed, this has been proposed to be the mechanism of activation of LAK cells. The significance of Fas in the current studies was supported by our findings that FasL-deficient CTL could not kill in the presence of the neu-Ab-IL-2 fusion protein. By directing cells expressing IL-2R to the tumor, the fusion protein allows non-tumor-specific, activated T cells to lyse the tumor through the Fas-mediated pathway. Indeed, both activated-CD8+ T cells and TH1-CD4+ cells were able to mediate lysis in vitro. This finding suggested the possibility of using non-tumor-specific T cells such as Clone-4 TCR cells to control tumor growth in vivo. It is anticipated that CD4+ T cells would also be capable of eliminating tumors in vivo. Future experiments will examine whether CD4+ T cells alone are also sufficient to eradicate tumors and will assess localization of each subset at the tumor site.
Our results demonstrate that despite the ability of Ab-IL-2 to mediate
Fas killing, tumor-specific T cells have an advantage over
non-tumor-specific T cells in eliminating tumors in vivo. There are
several possible reasons why tumor-specific T cells are more effective.
First, the homing of tumor-specific T cells to the tumor site might be
more efficient than that of non-tumor- specific cells. Second, there is
the additional targeting of the effector cell through TCR specificity
so that the T cell has the ability to lyse through both perforin- and
Fas-mediated mechanisms. Third, triggering the T cell through the TCR
leads to secretion of cytokines such as TNF-
and IFN-. These may
enhance the lytic capacity of the effectors through FasL by increasing
both Fas and class I MHC on the target cells, thus augmenting
cytolysis. We confirmed this by treating tumor cells with TNF- and
IFN-, which augmented the expression of Fas on the target cells,
increasing the susceptibility of lysis by non-tumor-specific T cells in
the presence of the Ab-IL-2 fusion protein (data not shown). It is
possible that cytokines may be administered in vivo to augment Fas
expression on tumors.
Ab-IL-2 fusion proteins could represent an alternative to the use of bifunctional Abs that bridge T cells with tumors for redirecting the lytic activity of T cells to a tumor site (32). These two strategies differ in that bifunctional Abs trigger T cells through their TCR, whereas IL-2 fusion proteins trigger through IL-2R. This difference may have important consequences in terms of T cell viability, as it is known that activation induced cell death requires signaling through the TCR. It will be of interest to compare the longevity of tumor-specific and non-tumor-specific T cells in vivo in this model.
The current findings add to the growing list of the pleiotropic effects of Ab-targeted IL-2 therapy in eliminating tumors. Importantly, they suggest alternative strategies for applying these fusion proteins. For example, these therapies may be used in conjunction with vaccination to tumor-associated Ag, or alternatively, in cases in which these T cells or Ag may be unavailable, fusion proteins may be used in conjunction with vaccination to unrelated Ag that could serve to establish a reservoir of activated T cells that could then be targeted by fusion proteins to the tumor microenviroment. Such activated T cells could provide a source of cytokines required for up-regulation of Fas on the tumor, thereby enhancing their sensitivity to Fas-mediated killing. Future studies are required to evaluate various elements of these hypotheses.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Linda A. Sherman, The Scripps Research Institute, 10550 North Torrey Pines Road, IMM-15, Rm. R201, La Jolla, CA 92037. E-mail address:
3 Abbreviations used in this paper: LAK, lymphokine activated killer cells; HA, hemagglutinin; CL, constant lambda; L, ligand.
Received for publication July 20, 1998. Accepted for publication September 8, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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