Sandra Santulli-Marotto, Smita K. Nair, Chris Rusconi, Bruce Sullenger and Eli Gilboa ²

Center for Genetic and Cellular Therapies, Department of Surgery, Duke University Medical Center, Durham, North Carolina 27710

² To whom requests for reprints should be addressed, at Department of Surgery, Box 2601, Duke University Medical Center, Durham, NC 27710. Phone: (919) 684-6465; Fax: (919) 681-7970;
E-mail: e.gilboa@cgct.duke.edu

The potency of cancer immunotherapy can be enhanced by administration of high-avidity ligands specific to receptors expressed on T cells. Antibodies or cytokines are the main agents used in such capacity. Antibody-mediated inhibition of cytotoxic T cell antigen-4 (CTLA-4) function in mice augments antitumor
immunity and could serve as an important adjunct in cancer immunotherapy. However, antibody-based therapy used in the setting of chronic diseases such as cancer poses significant cost, manufacturing, and regulatory challenges. Here we describe the development of RNA aptamers that bind CTLA-4 with high affinity and specificity. These aptamers inhibit CTLA-4 function in vitro and enhance tumor immunity in mice. Moreover, assembly of the aptamers into tetrameric forms significantly enhances their bioactivity in vitro and in vivo. These results demonstrate that aptamers can be used to manipulate the immune system for therapeutic applications and that multivalent versions of aptamers may be particularly potent agents in vivo.

INTRODUCTION

Activation of naïve T cells is dependent on the delivery of at least two signals by the antigen-presenting cells, an antigen-specific signal via the T-cell receptor and a second signal via costimulatory molecules (1) . CD28, expressed on the cell surface of resting and activated T cells, and its counterreceptors B7-1 and B7-2 expressed on antigen-presenting cells are a major source of costimulatory signals to T cells (2). CTLA-4 (3) is a second high-affinity receptor for the B7 family members that is expressed on activated, but not resting, T cells. However, unlike CD28, CTLA-4 engagement delivers a negative signal, attenuating T-cell responses by raising the threshold of signals needed for T-cell activation (3, 4, 5) . This is consistent with the observations that CTLA-4-deficient mice develop a fatal lymphoproliferative disorder (6 , 7) and that blocking CTLA-4 signaling in vitro with Ab leads to enhanced T-cell receptor and CD28-dependent proliferation of T cells (8 , 9) . CTLA-4 is constitutively expressed on CD25⁺CD4⁺ regulatory T cells (10 , 11) , but the functional role of CTLA-4 in this subset of T cells is at present unclear (10, 11, 12, 13, 14) .

In murine studies, blockade of CTLA-4 function in vivo enhanced antitumor T cell-dependent immunity. Treatment of mice with a-CTLA-4 Ab led to the rejection of immunogenic transplanted tumors but had little or no effect on weakly or nonimmunogenic tumors (15 , 16) . Rejection of nonimmunogenic tumors, including pre-established tumors, was achieved if CTLA-4 blockade was used in combination with an immunization protocol (17, 18, 19) or with low-dose chemotherapy(20) under the conditions that neither treatment alone was effective. These observations reinforce the view that CTLA-4 blockade in vivo facilitates the antigen-dependent expansion of T cells by blocking inhibitory signals delivered by CTLA-4. CTLA-4 blockade therefore may serve as a useful adjunct to immunotherapy in the setting of cancer or infectious diseases.

Ab-based therapy, especially in the setting of chronic diseases such as cancer requiring repeated administration of antibodies over a long period of time, poses significant cost, manufacturing, and regulatory challenges. Affinity-based screening of short RNA libraries can be used to isolate aptamers, which bind to their targets with exquisite specificity and high avidity (21 , 22) . In this study, we describe the isolation of aptamers that bind to murine CTLA-4 and block function. Generation of tetravalent derivatives significantly enhanced the bioactivity of the CTLA-4 aptamers in vitro and in vivo. Compared with monoclonal antibodies, the cell-free chemical manufacturing process and the regulatory approval process of aptamers are simple. Aptamers have exhibited little or no immunogenicity in animal and human studies (23) and, because of their small size, exhibit superior tissue penetrance (24) . The isolation of CTLA-4-specific aptamers is the first example of using aptamers to manipulate the immune system.

MATERIALS AND METHODS

 Animals and Antibodies.
C57BL/6, C3H, and BALB/c mice were purchased from Charles Rivers Laboratories (Raleigh, NC). In conducting the research described in this paper, we adhered to the "Guide for the Care and Use of Laboratory Animals" as proposed by the Laboratory Animal Resources Commission on Life Sciences, National Research Council. The facilities at the Duke vivarium are fully accredited by the American Association for Accreditation of Laboratory Animal Care.

Purified hamster CD3 clone 500A2 and hamster CD28 clone 37.51 with no azide and low endotoxin were
purchased from PharMingen (San Diego, CA). The a-CTLA-4-producing hamster hybridoma 9H10 was a gift from J. Allison (University of California at Berkeley, Berkeley, CA). Cells were grown in a hollow-fiber culture system, and supernatants were routinely harvested and quantitated for Ab production. Purification of a-CTLA-4 Ab was carried out using protein G columns (Amersham Pharmacia Biotech, Piscataway, NJ), dialyzed into PBS, and then filter sterilized using 0.22 µM syringe filters (Schleicher & Scheull, Keene, NH). a-CTLA-4 and isotype-control Fab fragments were prepared and purified using the ImmunoPure Fab Preparation kit from Pierce (Rockford, IL). Control hamster immunoglobulin was purchased from Jackson ImmunoResearch (West Grove, PA).

In Vitro Proliferation Assays.
The ability of the aptamers to inhibit CTLA-4 function was determined by quantitating their effect on purified lymph node T-cell proliferation. Superficial inguinal, axillary, and mesenteric lymph nodes were aseptically harvested from female BALB/c mice 6–10 weeks of age and then teased into single-cell suspensions. Indirect enrichment of T cells was accomplished using the Murine T Cell Enrichment mixture with the StemSep system (StemCell Technologies, Vancouver, British Columbia, Canada). Analysis of the purified T cells was carried out using flow cytometry on a FACScalibur (Becton-Dickinson, San Jose, CA), and enriched T cells were consistently >95% pure (data not shown). Purified T cells were seeded at 10⁵ cells/well into U-bottomed, 96-well plates coated with 0.1 µg/ml a-CD3, and a-CD28 Ab was added at 10 µg/ml. a-CTLA-4 or isotype-matched control hamster IgG (Jackson Immunoresearch, West Grove, PA) was used at 20 µg/ml, or Fab fragments were used at 100 µg/ml, and aptamers were added at 200 nM to a final culture volume of 200 µl/well in complete RPMI + 10% fetal bovine serum. Aptamers were sterilized using 3-mm 0.22 µM syringe filters (Schleicher & Scheull). Cultures were incubated in replicates of 3–10 in 5% CO2 for 72 h and then pulsed with [³H]thymidine for 12–17 h before harvesting using a Tometec harvester (Perkin-Elmer Life Sciences, Boston, MA) onto glass filtermats (Perkin-Elmer Life Sciences) and counted using a scintillation counter (Perkin-Elmer Life Sciences). Each aptamer was tested in a minimum of three separate assays.

Tumor Immunotherapy Studies.
The F10.9 clone of the B16 melanoma of C57BL/6 origin is a highly metastatic, poorly immunogenic, and a low class I-expressing cell line (25) . GM-CSF producing B10/F10.9 tumor cells were described previously (26) . The murine MBT-2 cell line, derived from a carcinogen-induced bladder tumor in C3H mice (27) , was obtained from Dr. T. Ratliff (Washington University, St. Louis, MO). Cells were maintained in DMEM supplemented with 10% FCS, 25 mM HEPES, 2 mM L-glutamine, and 1 mM sodium pyruvate. Murine precursor-derived DCs were generated from bone marrow progenitors as described previously (28) . At day 0, mice were implanted with 5 x 10⁴ tumor cells s.c. and immunized with 10⁶ irradiated, GM-CSF-producing B16/F10.9 cells, irradiated MBT-2 cells, or TERT mRNA-transfected DCs as described previously (29) . On days 3 and 6, mice were re-immunized in the right flank and received Ab or aptamer in PBS i.p. Per injection, mice received 667 pmol of Ab, 500-4500 pmol of monomeric aptamers, or 26–50 pmol of tetrameric aptamers. Tumor growth was evaluated every other day starting on day 6. Mice were sacrificed once the tumor size reached 15–20 mm.

RESULTS

We used iterative in vitro selection techniques to screen an RNA-based combinatorial library of >10¹⁴ species for those members capable of binding murine CTLA-4 with high affinity. To ensure that the resultant aptamers would be stable in cell culture and in vivo, the starting library contained 2'-fluoropyrimidines. The selection was carried out for nine rounds (M9) against murine CTLA-4/Fc fusion protein, after which no further increase in affinity was seen. The RNAs present in round 9 were converted to cDNAs, cloned, and sequenced. (For additional details, see Supplementary Methods online.)

Oligonucleotide-based Aptamers Inhibit CTLA-4 Function in Vitro and in Vivo.
The amplification products from round 9 revealed limited sequence diversity, with eight unique sequences represented multiple times (Fig. 1A) , indicating that the selection was approaching an end point. Clones representing each sequence (M9-1, -5, -8, -9, -14, and -15) exhibited high affinity binding to CTLA-4 with K_ds ranging from 10 to 70 nM (Supplementary Table 1 online and data not shown). The ability of the CTLA-4 binding aptamers to interfere with CTLA-4 function was tested in vitro. In this assay, purified T cells are suboptimally stimulated to proliferate by incubation with a-CD3 and a-CD28 antibodies. Consistent with the function of CTLA-4 to attenuate T-cell proliferation, incubation with an a-CTLA-4 Ab, but not with an isotype control Ab, resulted in an enhancement of T-cell proliferation (8 , 9) . Several aptamers inhibited CTLA-4 function comparably or better than the a-CTLA-4 Ab (Fig. 1 A, M9-8, M9-9, and M9-14 and data not shown), whereas other RNA species did not inhibit CTLA-4 function, despite the fact that they bound to CTLA-4 (Fig. 1A , M9-15 and data not shown). We selected aptamer M9-9 for further study because it was consistently the most potent inhibitor of CTLA-4 function (Fig. 1A and data not shown) and exhibited the highest affinity binding to CTLA-4 of the clones tested (Supplementary Table 1 online).

Fig. 1. In vitro functional characterization of CTLA-4 binding aptamers.

A, sequence of eight clones obtained after nine rounds of selection (M9).
The frequency of each clone represented in the M9 pool is indicated in parentheses. In vitro T-cell proliferation assays were performed using mouse T cells enriched from lymph nodes stimulated with limiting concentrations of immobilized a-CD3 and soluble a-CD28 Ab in the presence of a-CTLA-4 Ab or aptamers (see "Materials and Methods"). Inhibition of a-CTLA-4 function is reflected in increased proliferation of T cells in the presence of Ab or aptamers. Isotype control and a-CTLA-4 Ab were used at 20 µg/ml (133 nM) and aptamers at 200 and 400 nM. T-cell proliferation was enhanced in the presence of a-CTLA-4, but not isotype Ab, and was enhanced in a dose-dependent manner in the presence of M9-8, M9-9, and M9-14, but not M9-15, aptamers. Bars, SD.

B, inhibition of CTLA-4 function in vitro by Del 60, a 36-nucleotide-long synthetic, truncated derivative of M9-9.
A model for the predicted secondary structure of the Del 60 aptamer shows the proposed CTLA-4 binding site. A proliferation assay was performed as above except that Fab fragments of isotype control and CTLA-4 Ab were used at 100 µg/ml (2000 nM). Bars, SD.

C, inhibition of CTLA-4 with Del 60 and a control aptamer, M8G-28del 69, which binds to CTLA-4 but did not inhibit its function in previous experiments (data not shown).
Where indicated, the Del 60 aptamer solution was preincubated with 2-fold excess CTLA-4/Fc or human IgG, followed by protein G-coated magnetic beads before addition to the T-cell cultures. The assay was performed with five replicates/condition. Bars, SD.

Murine studies have shown that rejection of tumors can be achieved if Ab-mediated CTLA-4 blockade is used in combination with vaccination under conditions that neither treatment was effective alone (17, 18, 19) . In Fig. 2 , the ability of the CTLA-4 binding aptamers to impact on tumor growth was tested in the poorly immunogenic B16/F10.9 melanoma model (25) used in the previous studies (17, 18, 19) . Mice were implanted with B16/F10.9 tumor cells and either treated with PBS or immunized with irradiated, GM-CSF-secreting B16/F10.9 (F10.9-GM) tumor cells. The F10.9-GM-immunized groups were injected i.p. with either Ab or aptamer as indicated in the figure. In this experimental system, immunization alone had no impact on tumor growth (Fig. 2 , mice immunized and treated with isotype Ab compared with the nonimmunized PBS-treated group, and data not shown). As seen previously, treatment with a-CTLA-4 Ab, but not isotype control Ab, led to a significant delay in tumor growth. Treatment with a-CTLA-4 Ab alone had no impact on tumor growth (data not shown). Treatment of the immunized mice with the Del 60 aptamer also inhibited tumor growth, whereas treatment with the nonfunctional, CTLA-4-binding M8G-28del 69 aptamer was ineffective (P = 0.04). However, the Del 60 aptamer-mediated inhibition of tumor growth shown in Fig. 2 (and data not shown) necessitated the administration of high doses of aptamer, 3–5 nmol/injection, compared with 0.667 nmol of a-CTLA-4 Ab. This may reflect the limited bioavailability of the aptamers in vivo because in vitro the aptamers were as
effective or superior to a-CTLA-4 Ab when used at similar molar concentrations of binding sites (Fig. 1) .

Fig. 2. Inhibition of tumor growth in mice treated with the CTLA-4-binding Del 60 aptamer.

C57BL/6 mice were implanted with 5 x 10⁴ B16/F10.9 melanoma tumors cells in the left flank and either
treated with PBS or immunized with 1 x 10⁶ irradiated, GM-CSF-secreting B16/F10.9 (F10.9-GM) tumor cells on days 0, 3, and 6 after tumor implantation. Ab (0.667 nmol/injection) or aptamer (4.5 nmol/injection) was administered i.p on days 3 and 6. M8G-28del 69 is a truncated derivative that binds to CTLA-4 but does not inhibit CTLA-4 function in vitro (Fig. 1C and data not shown). Median time to tumor onset was 12 days for Del 60/Scram, 13 days for isotype-control Ab, and 17 days for Del 60 and a-CTLA-4 Ab treatment groups. The difference between the control aptamer and Del 60 groups was statistically significant by log-rank test (P = 0.04).

Fig. 3. Derivation of tetravalent Del 60 aptamers.

A, Del 60 tetramer.
Two oligonucleotides were used to generate a linker that acts as a scaffold to anchor four Del 60 monomers. Four Del 60 monomers were bound to the linker through single-stranded regions of the linker as shown. The double-stranded region of the linker consisted of 20 bp, which formed two complete helical turns and placed two Del 60 monomers in the same plane at a distance of 68–74 Å, capable of binding two CTLA-4 molecules.

B, proposed interaction of B7-1 and CTLA-4 deduced from crystal structure (33) .
CTLA-4 exists on the cell surface as a dimer (34) and is cross-linked by a pair of B7-1 molecules that span 46–60 Å. The distance between the two CTLA-4 dimers when cross-linked by B7-1 is 77Å.

Fig. 4. In vitro functional characterization of the Del 60 tetramers.

Analysis was performed as described in Fig. 1 . The concentrations of tetramer and monomer are indicated. T-cell proliferation above the line indicates CTLA-4 inhibition. Enhancement at 0.4 nM Del 60 tetramer
was statistically significant compared with either control (P < 0.01) or 8nM Del 60 monomer (P < 0.001) using Student’s t test. Bars, SD.

Fig. 5. Inhibition of tumor growth in mice treated with Del 60 monomer and tetramer.

A, B16/F10.9 melanoma model as described in Fig. 2 .
Mice were monitored for the appearance of palpable tumors at the site of tumor implantation. 667 pmol of a-CTLA-4 Ab, 500 pmol of Del 60 monomer, and 50 pmol of Del 60 tetramer were injected on days 3 and 6. Median time to tumor onset was 20 days for Del 60 tetramer, 21 days for a-CTLA-4 Ab, and 14 days for Del 60/Scram tetramer and isotype-control treatment groups. The difference between Del 60 and Del 60/Scram tetramer treatment groups was statistically significant by log-rank test (P = 0.02). Overall significance was P < 0.0001 by the Kruskal-Wallis test.

B, MBT-2 bladder tumor model.
Experimental design is essentially as described in A except that nonmodified, irradiated MBT-2 tumor cells were used to immunize the mice (see "Materials and Methods"). Median time to tumor onset was 24 days for Del 60 tetramer, 14 days for Del 60/Scram tetramer, and 17 days for both CTLA-4 and isotype-control treatment. The difference between the Del 60 and Del 60/Scram tetramer treatment groups was statistically significant with by the log-rank test (P = 0.01).

Fig. 6. Del 60 tetramer-mediated enhancement of tumor immunity in tumor-bearing mice immunized with TERT mRNA-transfected DCs.

C57BL/6 mice were implanted with B16/F10.9 melanoma tumors cells s.c. on day 0 and immunized with TERT mRNA-transfected DCs 2, 9, and 17 days after tumor cell implantation. As indicated, 50 pmol of Del 60 tetramer or the control Del 60/Scram tetramer were administered to mice on days 3, 10, and 17. Five mice were used in each treatment group. Mice were monitored for the appearance of palpable tumors at the site of tumor implantation. The log-rank test (Mantel-Haenszel test) was used to determine the differences between individual groups. Relative to the TERT + Del 60 tetramer group, Ps were 0.006 for TERT and 0.04 for TERT + Del 60/Scram tetramer groups. Relative to Del 60 tetramer treatment group, Ps were 0.005 for the actin group, 0.03 for the TERT group, and 0.15 for the TERT + Del 60/Scram tetramer group.

DISCUSSION

This study represents the first demonstration of using aptamers to manipulate the immune system in vivo. Using affinity-based in vitro selection methods, we isolated short oligonucleotide aptamers that bind murine CTLA-4 with high affinity and specificity (Supplementary Table 1 online) and interfere with its function in vitro
(Fig. 1) and in vivo (Fig. 2) . However, compared with the CTLA-4 Ab, more aptamer was required to elicit an effect in vivo (inhibition of tumor growth, Fig. 2 ) than in vitro (enhancement of T-cell proliferation; Fig. 1 ). These observations suggest that whereas the avidity of the aptamers to their CTLA-4 targets is comparable if not superior to that of the CTLA-4 Ab, the bioavailability of the aptamers in vivo is significantly reduced. Consequently, the need to administer high doses of aptamer, 10–15 nmol/mouse in this study, was challenging, and by extrapolation, would limit its clinical use.

In vivo bioactivity of aptamers can be enhanced by increasing the avidity of the aptamers to their target or by extending their persistence in circulation, or both. Because CTLA-4 also regulates the function of autoreactive T cells (6 , 7) , we reasoned that increasing the circulation half-life of CTLA-4 inhibitors could increase the risk of autoimmune pathology. We therefore chose to improve the in vivo bioactivity of the aptamers by increasing their avidity to CTLA-4 but not their in vivo circulating half-life. Here, we show that the in vivo bioactivity of the CTLA-4 aptamers can be significantly enhanced by generating tetravalent derivatives (Del 60 tetramer; Fig. 3A ). Consequently, the amount of aptamer required to elicit a biological effect in vivo was dramatically reduced from 3–5 nmol/injection to 50 pmol/injection (Figs. 2 , 5 , and 6 , and data not shown). Although not easy to demonstrate directly, the superior bioactivity of the Del 60 tetramer in vivo is most likely attributable to enhanced avidity to CTLA-4. This interpretation is consistent with the in vitro functional studies (Fig. 4) and the observations that tetrameric, but not monomeric, forms of MHC class I peptides bind to cell surface T-cell receptors with high avidity (32) . The method used in this study to generate tetrameric aptamers using an oligonucleotide scaffold is, however, inefficient, especially for clinical use. For future studies and clinical applications, simple and cost efficient chemical coupling methods using solid-phase phosphoramidite coupling chemistry, flexible polyamine linkers, and other methods could be developed to generate multimeric aptamers
with defined valencies (38 , 39) .

Aptamers represent a new class of reagents that could potentially replace the use of antibodies or cytokines to
manipulate the immune system in vivo. Using in vitro selection methods, bioactive aptamers with specificity and avidity comparable or superior to that of antibodies can be isolated for any target (for examples, see Refs. 40, 41, 42, 43, 44, 45 ; reviewed in Refs. 21 , 22 ). The isolation of CTLA-4-specific aptamers is the first example of using aptamers to manipulate the immune system and can be used to isolate ligands to other immunological targets of interest such as CD40, 4-1BB, OX40, B7H1, or transforming growth factor-ß receptor.

Aptamers are synthetic chemicals and not biologicals; hence, manufacturing and especially the regulatory approval process should be much more favorable compared with protein-based clinical reagents. Aptamers can also be chemically modified to enhance their stability, bioavailability, and function (24 , 30 , 31) . Importantly, using procedures whereby selection rounds alternate between two related targets, such as human and murine CTLA-4, aptamers can be isolated with cross-species specificities (44) and hence can be tested in preclinical animal models for bioactivity and toxicity prior to clinical applications.

FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supplementary data for this article are available at Cancer Research Online: http://cancerres.aacrjournals.org/cgi/content/full/63/21/7483

2 To whom requests for reprints should be addressed, at Department of Surgery, Box 2601, Duke University Medical Center, Durham, NC 27710. Phone: (919) 684-6465; Fax: (919) 681-7970;
E-mail:    e.gilboa@cgct.duke.edu

3 The abbreviations used are: CTLA-4, cytotoxic T cell antigen-4; Ab, antibody; GM-CSF,
granulocyte/macrophage-colony stimulating factor; DC, dendritic cell; TERT, telomerase reverse transcriptase.

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