Tumors Protective Immune Responses Against Delivery Of

The Journal of Immunology

Delivery of Antigen to CD40 Induces Protective Immune Responses against Tumors1

Karoline W. Schjetne,2 Agnete B. Fredriksen,2,3 and Bjarne Bogen3

Ligation of CD40 induces maturation of dendritic cells (DC) and could be a useful target for vaccines. In this study, we have constructed two types of Ab-based vaccine constructs that target mouse CD40. One type is a recombinant Ab with V regions speciﬁc for CD40 and has deﬁned T cell epitopes inserted into its C region. The other type is a homodimer, each chain of which is composed of a targeting unit (single-chain fragment variable targeting CD40), a dimerization motif, and an antigenic unit. Such proteins bound CD40, stimulated maturation of DC, and enhanced primary and memory T cell responses. When delivered i.m. as naked DNA followed by electroporation, the vaccines induced T cell responses against MHC class II- restricted epitopes, Ab responses, and protection in two tumor models (myeloma and lymphoma). Two factors apparently contributed to these results: 1) agonistic ligation of CD40 and induction of DC maturation, and 2) delivery of Ag to APC and presentation on MHC class II molecules. These results highlight the importance of agonistic targeting of Ag to CD40 for induction of long-lasting and protective immune responses. The Journal of Immunology, 2007, 178: 4169–4176.

accines should not only deliver Ag to APC, but should and adhesion molecules, and up-regulation of cytokine production. also induce maturation of the APC so that they can po- Consistent with a crucial importance of CD40L-CD40 interaction V tently stimulate T cells. Dendritic cells (DC)4 are in induction of immune responses, mice and humans that lack increasingly considered to be the most important APC (1). Matu- CD40 or CD40L (CD154) genes have reduced Ab production and ration of DC initiates up-regulation of MHC class II and costimu- Ig-class switching, and are unable to mount effective responses latory molecules, and migration to secondary lymphoid tissues against infectious agents. where they activate T cells. In the absence of maturation, as under Recently, the importance of activating DC via CD40 ligation steady-state conditions, DC may rather induce T cell tolerance was demonstrated in a series of studies, where DEC205-targeted than activation (2). Consistent with such a model, T cell toler- Ab-Ag fusion proteins induced tolerance to the Ag unless an ag- ance was replaced by memory T cell responses when Ag-loaded onistic anti-CD40 mAb was coadministered (4, 9, 10). Thus, ag- DC received an additional maturation signal such as ligation of onistic mAbs against CD40 appears to be an effective substitute for CD40 on DC (3, 4). Th cells in maturing DC. In this study, we have exploited this CD40 is a member of the TNF family and is expressed by a finding and designed two types of Ig-like vaccine constructs that range of cells, including DC, macrophages, B cells, fibroblasts, agonistically target CD40 on APC for delivery of Ag. One type epithelial cells, and endothelial cells (5). During a normal T cell resembles Ab molecules and has Ag introduced as defined T cell response, CD40 on DC is engaged by CD40L, which is transiently epitopes in the C region. The other type carries larger parts of Ag expressed on activated CD4ϩ Th cells. CD40 molecules are expressing both B and T cell antigenic determinants. These CD40- thereby cross-linked, which induces DC maturation and enable specific molecules elicited Ag-specific immune responses and in- them to efficiently present Ag to T cells (6–8). The changes in the duced protection in two different tumor models (myeloma and DC are not fully understood, but probably involve a combination lymphoma). of improved Ag processing, increased expression of costimulatory Materials and Methods Institute of Immunology, University of Oslo and Rikshospitalet-Radiumhospitalet Construction of CD40-specific vaccine constructs Medical Center, Oslo, Norway Troybodies. CD40-specific troybodies were constructed essentially as de- Received for publication April 21, 2006. Accepted for publication January scribed previously (11, 12). Rearranged V(D)J region genes of the H and 22, 2007. L chain were PCR amplified from cDNA synthesized from mRNA ex- The costs of publication of this article were defrayed in part by the payment of page tracted from FGK-45 cells (13), cloned, sequenced, and inserted into ex- charges. This article must therefore be hereby marked advertisement in accordance pression vectors encoding either a complete H chain (pLNOH2) or L chain with 18 U.S.C. Section 1734 solely to indicate this fact. (pLNO␬). Primers used were as follows (restriction enzyme sites are un- Ј CD40 Ј CD40 1 This work was funded by the Research Council of Norway, Norwegian Cancer derlined): 5 VH , ggtgtgcattcc gag gtg cag ctg gtg gag; 3 VH , gacg Ј CD40 Society, and Multiple Myeloma Research Foundation. tacgactcacc tga gga gac tgt gac cat gac; 5 VL , ggtgtgcattcc gac act gta ctg acc cag tct cc; 3ЈV CD40, gacgtacgttctactcacg ttt caa ttc cag ctt gg. The 2 K.W.S. and A.B.F. contributed equally to this work. L T cell epitope ␭2315 (91–101) from L chain of M315 (14) was introduced 3 Address correspondence and reprint requests to Dr. Agnete B. Fredriksen, Institute into the CH gene of pLNOH2 by site-specific mutagenesis (11, 15) ex- of Immunology, University of Oslo and Rikshospitalet-Radiumhospitalet Medical ␥ ␥ changing loop 6 (loop F-G) of CH1 in the C region of human 3(h 3). The Center, N-0027 Oslo, Norway; E-mail address: [email protected] or Dr. vector encoding the 4-hydroxy-3-iodo-5-nitrophenylacetic acid (NIP)-spe- Bjarne Bogen, Institute of Immunology, University of Oslo and Rikshospitalet-Ra- diumhospitalet Medical Center, N-0027 Oslo, Norway; E-mail address: bjarne.bogen@ cific control Ab was as described previously (11). medisin.uio.no Vaccibodies. The CD40-specific V(D)J genes from the FGK-45 hybrid- 4 Abbreviations used in this paper: DC, dendritic cell; h␥3, human ␥3; NIP, 4-hy- oma were modified by PCR to provide linker sequences (bold). The prim- Ј CD40 Ј CD40 droxy-3-iodo-5-nitrophenylacetic acid; scFv, single-chain fragment variable; ers were as follows: 5 VL , as described above; 3 VL , gcc aga gcc 3 3 Ј CD40 [ H]Thd, [ H]thymidine; wt, wild type. acc tcc gcc aga tcc gcc tcc acc ttt caa ttc cag ctt gg; 5 VH , ggc gga ggt Ј CD40 ggc tct ggc ggt ggc gga tcg gag gtg cag ctg gtg gag; 3 VH , as described Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 above. The VL and VH PCR products were combined to a single-chain www.jimmunol.org 4170 TARGETING CD40 INDUCES PROTECTIVE IMMUNE RESPONSES fragment variable (scFv) format by PCR SOEing and inserted into the Immediately following injection, electroporation was performed using a targeting unit of the pLNOH2-vaccibody expression vectors encoding a caliper electrode as described previously (23). dimerization unit (hinge and CH3 from hIgG3) and an antigenic unit (scFv315 or scFvA20) as described previously (16). The vectors encoding In vivo detection of primed APC in draining lymph nodes the NIP-specific controls were as described previously (16). BALB/c mice were injected i.m. with 50 ␮g of plasmids and electropo- Production of vaccine protein rated. Eight days later, draining (lumbar and sacral) and nondraining (mes- enteric) lymph node cells were treated with collagenase and DNase, irra- Transient and stable transfections were performed in HEK293E cells es- diated (20 Gy), and 5 ϫ 105 cells/well were incubated with polarized sentially as described previously (16). All recombinant Abs were affinity ␭2315-specific Th2 cells (2 ϫ 104). After 48 h, the cultures were pulsed purified from cell supernatant by use of anti-human C␬ Ab (A8B5)-con- with 1 ␮Ci of [3H]Thd. Incorporated [3H]Thd was measured after 16 h. jugated, NP-lysin conjugated, or by DNP-lysin-conjugated Sepharose columns. Measure T cell proliferation in vivo Vaccibodies. DNA-vaccinated BALB/c mice were adoptively transferred Mice, cells, and mAbs with 1.5 ϫ 107 lymph node cells from ␭2315-specific TCR transgenic mice, BALB/cABom (H-2d) mice were obtained from Taconic Farms. The ␭2315- and BrdU incorporation was performed as described previously (16). specific TCR-transgenic mice on a BALB/c background (17) and the ␭2315- Troybodies. Adoptively transferred BALB/c mice (1.5 ϫ 107 lymph node specific TCR-transgenic mice on a C.B-17 scidϪ/Ϫ background have been cells) were protein vaccinated (s.c. injection on right flank, 100 ␮g/mouse), described previously (18). The studies have been reviewed and approved and BrdU incorporation was performed as described previously (12). by the National Animal Research Authority. NS0, HEK293E, and ␥ MOPC315 cells (19) were obtained from American Type Culture Collec- IFN- ELISPOT tion (ATCC). The A20 B lymphoma cell line (20) was a gift from S. Buus Adoptively transferred BALB/c mice (3 ϫ 106 TCR transgenic CD4ϩ T (Institute for Medical Microbiology and Immunology, Copenhagen, Den- cells/mouse) were protein vaccinated (s.c. injection on right flank, 100 ␭ 315 ϩ mark). The 2 -specific CD4 T cell clone 7A10B2 (21) has been de- ␮g/mouse) or DNA vaccinated. After 3 wk, spleens were removed and the scribed previously. Rat anti-mouse CD40 FGK-45 hybridoma (13) was number of IFN-␥ secreting, peptide-specific T cells in fresh splenocyte a gift from J. Andersson (Basel Institute of Immunology, Basel, Swit- preparations was determined by ELISPOT essentially as described previ- zerland), and the immature DC cell line (D2SC/1) (22) was provided by ously (23). Briefly, 96-well nitrocellulose plates (MultiScreen; Millipore) P. Ricciardi-Castagnoli (University of Milan-Bicocca, Milan, Italy). were coated with mAbs specific for mouse IFN-␥ (AN-18). Splenocytes ϫ 6 ␭ 315 Flow cytometry were plated in triplicates (1 10 cells/well) and synthetic peptide ( 2 aa 89–107) was added. Biotinylated anti-mouse IFN-␥ mAb (XMG1.2) Spleen cells were stained with CD40-specific vaccine proteins, or NIP- and streptavidin alkaline phosphatase (Sigma Chemical) was used for de- specific control proteins, and detected with anti-human IgG3 (troybodies) tection. Spots were developed by adding alkaline phosphatase conjugate (Southern Biotechnology Associates) or HP6017 (vaccibodies) (ATCC). substrate solution (50 ␮l) (Bio-Rad). Spots were counted electronically allophycocyanin-conjugated CD19 (BD Pharmingen) was used to stain B with a Zeiss KS-ELISPOT-401 instrument. The number of spots per 105 cells. splenocytes is shown. Each point represents two mice. CD69 expression Tumor challenge experiments The ␭2315-specific TCR transgenic mice on a SCID background were in- BALB/c mice were immunized by naked DNA as described above. Two jected i.m. with 50 ␮g of naked DNA and electroporated. At day 10 after weeks later, mice were injected s.c. on the right flank with MOPC315 vaccination, draining and nondraining lymph node CD4ϩ T cells were myeloma cells (1.6 ϫ 105) or A20 B cell lymphoma cells (1.2 ϫ 105). Ten analyzed for CD69 expression. Cells were analyzed on a FACSCalibur mice were included in each group and tumor growth was observed bi- (BD Biosciences). weekly. Tumor avoidance curves and statistical analyses were generated with GraphPad Prism 4.0 software. Agonistic ligation of CD40 on splenic B cells Splenocytes (1 ϫ 105) were cultured with CD40-specific troybodies or Results vaccibodies, the corresponding nontargeted controls, or FGK-45 (all 10 Mouse CD40-specific vaccine constructs bind agonistically ␮g/ml) in medium containing IL-4 (20 U/ml) for 3 days. Incorporation of to CD40 [3H]thymidine ([3H]Thd) was measured for the last 16 h. We engineered two different vaccine constructs, both based on Maturation of DC and IL-12 secretion Ab structure. troybodies (11) are recombinant Ab molecules The immature splenic DC line D2SC/1 cells (22) or immature bone mar- with V regions that target surface molecules on APC (herein row-derived DC cultured with IL-4 and GM-CSF were incubated with CD40). In addition, troybodies carry T cell epitopes introduced CD40-specific troybodies, the corresponding nontargeted troybody (10 ␮g/ into a loop connecting ␤-strands in Ig-C domains (Fig. 1A and ml), LPS (10 ng/ml), or medium alone for 48 h before the cells were Table I). As negative controls we used nontargeted troybodies stained with Abs specific for MHC class II, CD86, or CD54 and CD11c (BD Pharmingen). Secretion of IL-12p40 from bone marrow DC incubated with V regions specific for the hapten NIP. These should not be for 24 h with CD40-specific troybodies, the corresponding nontargeted able to bind to surface molecules on APC, but still harbor the troybody (10 ␮g/ml), LPS (10 ng/ml), or medium alone was measured by ␭2315 T cell epitope in their C regions. In addition, CD40-spe- ELISA (OptEIA; BD Biosciences). cific Ab molecules lacking the epitope (wild-type (wt) h␥3) ELISA were used (Table I). Vaccibodies (16) are homodimeric fusion proteins, each chain Wells were coated with NIP-BSA or recombinant mouse CD40/Fc Ig chi- consisting of a scFv that target APC (herein CD40), a dimerization meric protein (R&D Systems) and detected with biotinylated HP6017 (anti- ␥ human IgG, Fc-region) (Zymed Laboratories). Anti-Id315 Ab was detected motif (shortened hinge and CH3ofh 3), and an antigenic unit in ELISA as described previously (16). (herein scFv from MOPC315 plasmacytoma or A20 B cell lymphoma) (Fig. 1A and Table I). Nontargeted vaccibodies with In vitro T cell proliferation specificity for the hapten NIP, and with the corresponding an- T cell proliferation assays were performed essentially as described (12) tigenic units as described above (Table I), served as negative 315 315 using either polarized ␭2 -specific Th2 cells or cloned ␭2 -specific controls. CD4ϩ T cells (7A10B2) (both 2 ϫ 104 cells/well) as responder cells and irradiated BALB/c splenocytes (5 ϫ 105/well) as APC. We first investigated whether the cloned V regions from the FGK-45 hybridoma were able to bind to CD40 after they had been DNA immunization cloned and produced as proteins in the troybody and vaccibody Naked DNA immunization was performed essentially as described previ- formats. Splenocytes were stained with CD40-specific proteins or ously (16). As a negative control, mice were injected with NaCl alone. the nontargeted NIP-specific controls. Indeed, the CD40-specific The Journal of Immunology 4171

specific vaccine constructs, the corresponding nontargeted NIP- specific controls, FGK-45 mAb, or isotype-matched control Ab (rat IgG1). CD40-specific proteins induced a strong proliferation of splenocytes that was comparable to that of the FGK-45 mAb, whereas nontargeted constructs and isotype-matched rat IgG1 failed to do so (Fig. 2A). Ability of CD40-specific vaccine proteins to induce DC maturation was examined by using an immature DC line (D2SC/1) as well as bone marrow-derived DC. CD40-specific troybodies triggered up-regulation of MHC class II, CD86, and CD54 to a level similar to that achieved by LPS (Fig. 2B). By contrast, the nontargeted NIP-specific proteins had no effect compared with medium alone. Furthermore, incubation of bone marrow-derived DC with the CD40-specific protein induced secretion of IL-12 (Fig. 2C). These results indicate that the agonistic activity previously reported for the FGK-45 mAb was maintained when transposing the V regions to the CD40-specific vaccine proteins. Targeting Ag to CD40 enhances in vitro proliferation of Ag-specific CD4ϩ T cells and requires a physical link between V regions and Ag In the troybody molecules, the CD4ϩ ␭2315 T cell epitope was introduced into the C region of h␥3, whereas in the vaccibody molecules the ␭2315 CD4ϩ T cell epitope is located in its original 315 position in the CDR3 loop of the VL region in the scFv fragment. Both vaccine proteins were tested in in vitro T cell proliferation assays. Irradiated BALB/c splenocytes were used as APC, and either polarized ␭2315-specific Th2 cells from TCR transgenic mice (Fig. 3A, upper panel) or cloned ␭2315-specific Th1 cells (Fig. 3A, lower panel) were used as responders. The dose-response curves demonstrate that the CD40-specific vaccine proteins were 100–1,000 times more efficient at activating Ag-specific CD4ϩ T cells when compared with the corresponding nontargeted (NIP- specific) controls or synthetic peptide, respectively (Fig. 3A). The results above could be explained by activation of DC by FIGURE 1. A, Schematic figure of mouse CD40-specific vaccine mol- agonistic CD40 stimulation, increased loading of MHC class II ecules; troybodies (left) and vaccibodies (right). Both vaccine constructs molecules by targeting Ag to CD40, or both. To distinguish be- are based on Ig structure and target CD40, either via V regions on H and tween these possibilities, we did an experiment in which cultures L chains (troybodies) or scFv (vaccibodies) (light shade). The Ag is in received a mixture of anti-CD40 Ab lacking the T cell epitope in troybodies, a defined T cell epitope (dark star), whereas vaccibodies carry the C region and NIP-specific control Ab with the ␭2315 epitope. complete scFv derived from Ig produced by B cell tumors (dark shade). troybodies include h␥3 and human C␬, whereas vaccibodies include a In this experimental situation, the CD40 specificity and the T cell ␥ epitope resided on separate molecules and were thus unlinked. shortened hinge and CH3 domain from h 3 (open) that cause disulphide bond formation and homodimerization. See Table I for details. B, Staining Such physical uncoupling of CD40 specificity and T cell epitope of CD19ϩ splenocytes with CD40-specific troybodies (left) and vaccibod- abolished T cell responses. These results demonstrate a require- ies (right) (filled histograms), or corresponding nontargeted NIP-specific ment for a physical link between CD40-specific V regions and the vaccine proteins (open diagrams). C, Binding of CD40-specific or NIP- T cell epitope for enhancement of T cell responses (Fig. 3B). specific troybodies and vaccibodies to CD40-Ig fusion protein (left)or The results of Fig. 3A could be extended to human cells because NIP-BSA (right) in ELISA. a mouse mAb to human CD40 (clone 5C3) was ϳ100–1,000 times more efficient at stimulating a mouse C␬40Ϫ8-specific/DR4-restricted CD4ϩ T cell clone (25) than was isotype-matched IgG1,␬ molecules bound much better to gated CD19ϩ B cells expressing mAb (data not shown). CD40 (Fig. 1B) compared with the nontargeted controls. Further- Targeting CD40 in vivo induces priming of APC and activation more, the CD40-specific proteins bound to CD40-Ig fusion protein ϩ but not NIP-BSA in ELISA, whereas the converse was true for the and proliferation of Ag-specific CD4 T cells NIP-specific controls (Fig. 1C). To investigate whether CD40-specific vaccine proteins are in fact Ligation of CD40 has been reported to induce maturation of DC delivered to APC in vivo, DNA encoding CD40-specific vacci- (3). Moreover, the anti-CD40 FGK-45 mAb has previously been bodies with scFv315 were injected i.m. in the quadriceps of reported in several different studies to be agonistic, i.e., induce BALB/c mice, immediately followed by electroporation to in- proliferation of spleen cells (13), up-regulate CD86 and CD40 ex- crease uptake and expression of plasmids (16, 26). Eight days after pression on ex vivo DC from lymph nodes (4), and secretion of injection, the draining (sacral and lumbar) and nondraining lymph IL-12 and IFN-␥ from lymph node cells (24). To examine whether nodes were removed and used as APC in an in vitro proliferation the cloned CD40-specific V regions had maintained their ability to assay using polarized ␭2315-specific Th2 cells as responders. The agonistically bind to CD40, we incubated splenocytes with CD40- results show that CD40-specific vaccibodies primed APC in the 4172 TARGETING CD40 INDUCES PROTECTIVE IMMUNE RESPONSES

Table I. Vaccine constructs used in this studya

Nameb Speciﬁcityc C Regiond Age MHC Restrictionf Reference

Vaccibodies CD40 315 ϩ ␥ d (Fv Fv )2 CD40 Hinge CH3h 3 M315 I-E Present study CD40 A20 ϩ ␥ d (Fv Fv )2 CD40 Hinge CH3h 3 A20 K Present study NIP 315 ϩ ␥ d (Fv Fv )2 NIP Hinge CH3h 3 M315 I-E 16 NIP A20 ϩ ␥ d (Fv Fv )2 NIP Hinge CH3h 3 A20 K 16 Troybodies ␣CD40.L6-␭2315 CD40 h␥3 ␭2315(91–101) I-Ed Present study ␣NIP.L6-␭2315 NIP h␥3 ␭2315(91–101) I-Ed 11 Control Ab ␣CD40.wt CD40 h␥3 None Present study FGK-45 CD40 Rat ␥1 None 13

a See Fig. 1A for schematic overview. b The names indicate the specificity and the Ag introduced. L6 denotes that the T cell epitope replaced loop 6 (loop F-G) connecting ␤-strands in the ␥ CH1 domain of h 3. ()2 indicates homodimers. c CD40-specific V regions were cloned from hybridoma FGK-45 (13). NIP-specific V regions corresponds to that of the hybridoma B1-8 (34). d ␥ ␥ The homodimerization motif is composed of hinge exon 1 and 4 and the CH3 domain from h 3 C region (16). Troybodies have complete h 3C region. e The Ag in vaccibodies were cloned as scFv corresponding to monoclonal Ig produced by MOPC315 myeloma (19) or the A20 B cell lymphoma (20). The T cell epitope in troybodies were from ␭2315 L chain (14) and replaced the 4-aa sequence naturally occurring in L6 of h␥3 (15, 35). Amino acids are indicated. f ␭ 315 MHC restriction of defined idiotypic T cell epitopes (aa 91–101 of the V 2 fragment of M315 (17), aa 106–114 of VH of A20 (36)) is indicated. draining, but not in the nondraining lymph node (Fig. 4A). By APC. The primed APC evidently stimulated T cells because in contrast, nontargeted (NIP-specific) vaccibodies failed to prime TCR transgenic mice on a SCID background, where virtually all T APC. A likely explanation for these data is that transfected muscle cells express a ␭2315-specific TCR, a major fraction of the CD4ϩ cells synthesize and secrete CD40-specific vaccibodies that target T cells expressed the T cell activation marker CD69 10 days after DNA injection of the CD40-specific construct. By contrast, the nontargeted vaccine (NIP-specific) did not induce expression of CD69 (Fig. 4B). We proceeded to investigate whether CD40-specific vaccine constructs could induce proliferation of CD4ϩ T cells in vivo. First, CD40-specific vaccibody constructs with scFv315 were tested. BALB/c mice were immunized with naked DNA encoding CD40-specific vaccine construct, or nontargeted controls, and electroporated. On day 7, mice were adoptively transferred with ␭2315-specific TCR transgenic lymph node cells so that Ag-specific T cells constituted ϳ1–2% of total CD4ϩ T cells in the recipient. On day 10, BrdU was given and the mice were sacrificed and analyzed on day 13. We found that in mice immunized with CD40-specific vaccibodies, Ͼ66% of the ␭2315-specific CD4ϩ T cells had incorporated BrdU in draining lymph nodes by day 14, whereas mice immunized with NIP-specific vaccibodies, or CD40- specific vaccibodies with an irrelevant scFv in the antigenic unit, showed negligible incorporation of BrdU compared with reconsti- tuted mice injected with NaCl alone (Fig. 4C). We also tested whether CD40-specific troybody injected as protein in PBS without adjuvant could induce T cell activation. In- deed, s.c. injection of 100 ␮g of CD40-specific troybody protein in BALB/c mice adoptively transferred with ␭2315-specific TCR transgenic lymph node cells induced a significant incorporation of BrdU into ␭2315-specific CD4ϩ T cells in draining lymph nodes 6 days after protein immunization. Mice that had been injected with the corresponding nontargeted NIP-specific troybodies or CD40- specific Ab lacking the epitope showed a much lower incorpora- FIGURE 2. Cloned CD40-specific V regions in troybodies and vacci- tion of BrdU (Fig. 4D). When titrated amounts of CD40-specific bodies bind agonistically and induce maturation of DC. A, Splenocytes troybodies were injected, the percentage of BrdU-positive ␭2315- were cultured with CD40-specific vaccine constructs or controls in medium specific CD4ϩ T cells gradually decreased, as would be expected supplemented with IL-4. Proliferation (incorporation of [3H]Thd) was mea- (data not shown). These findings demonstrate that CD40-specific sured. B, The immature DC line D2SC/1 (left) or immature bone marrow- derived DC (middle and right) were incubated with CD40-specific troy- vaccine constructs induce potent T cell activation in vivo, either bodies (thick line), NIP-specific troybodies (thin line), LPS (hatched line), when delivered as naked plasmid DNA or as purified protein. or medium alone (filled) for 48 h before staining for MHC class II, CD54 CD40-specific proteins induce memory T cell responses and CD86 expression on CD11c-gated cells. C, Cultured immature bone morrow-derived DC were incubated for 24 h with the indicated CD40- The ␭2315 epitope is known to be a very weak Ag (21), and DNA specifc troybodies and controls before IL-12p40 was measured. vaccination with CD40-specific vaccibodies induced only minor The Journal of Immunology 4173

FIGURE 4. CD40-specific vaccine constructs prime APC with Ag in vivo and induce activation and proliferation of Ag-specific CD4ϩ T cells. A, BALB/c mice were injected i.m. with plasmids encoding CD40-specific vaccibodies with scFv315, the corresponding nontargeted vaccibodies, CD40-specific vaccibodies with irrelevant scFvA20, or NaCl. Muscle cells were immediately electroporated after injection. After 10 days, draining and nondraining lymph nodes were removed, irradiated, and used as APC FIGURE 3. CD40-specific vaccine proteins to CD40 enhance prolifer- in an in vitro T cell proliferation assay using polarized ␭2315-specific Th2 ϩ ation of Ag-specific CD4 T cells in vitro: a physical linkage between the cells as responders. B, ␭2315-specific TCR-transgenic SCID mice were im- targeting V regions and the T cell epitope is required. A, Irradiated BALB/c munized as described in A. On day 10, draining lymph nodes were stained splenocytes were cultured with titrated amounts of CD40-specific troybod- for CD69 expression. C, BALB/c mice were immunized as described in A, ies (left column), CD40-specific vaccibodies (right column), the corre- adoptively transferred (day 7) with lymph node cells from ␭2315-specific sponding nontargeted controls, or ␭2315 synthetic peptide. Polarized ␭2315- TCR transgenic mice, and given BrdU (day 11). Three days later, draining ϩ specific Th2 cells (upper row, proliferation) or cloned ␭2315-specific CD4 lymph nodes were analyzed for incorporation of BrdU into gated ␭2315- Th1 cells (lower row, IFN-␥ production) were used as responder T cells. B, specific GB113ϩF23.1ϩCD4ϩ T cells. D, BALB/c mice adoptively trans- Irradiated BALB/c splenocytes were cultured with titrated amounts of ferred with lymph node cells from ␭2315-specific TCR transgenic mice CD40-specific troybodies, the corresponding nontargeted troybodies, were injected s.c. with 100 ␮g of CD40-specific troybody protein express- CD40-specific Ab with wt h␥3 C region, or a mixture of both the nontar- ing the ␭2315 epitope, the corresponding nontargeted troybodies, CD40- geted troybodies and the CD40-specific Ab with wt h␥3 C region (unlinked specific control Ab with wt h␥3 C region, or PBS alone. The mice were ϩ condition). Cloned ␭2315-specific CD4 Th1 cells were used as responder given BrdU and 6 days later, gated ␭2315-specific CD4ϩ T cells in draining T cells. lymph nodes were analyzed for incorporation of BrdU as described in C. responses in normal mice (Fig. 5A). Therefore, to study whether CD40-specific molecules could induce memory T cell responses, had been immunized with CD40-specific vaccine constructs com- we resorted to mice adoptively transferred with lymph node cells pared with the controls (Fig. 5B). Thus, targeting Ag to CD40 in from ␭2315-specific TCR transgenic mice so that Ag-specific T vivo induced memory T cell responses in contrast to delivery of a cells constituted ϳ1–2% of total CD4ϩ T cells in the recipient. nontargeted vaccine. Adoptively transferred BALB/c mice were either protein-injected s.c. with CD40-specific troybodies with ␭2315 epitope, the corre- CD40-specific DNA vaccines induce Abs specific for tumor Ag sponding nontargeted NIP-specific troybodies, CD40-specific Ab The current vaccibodies contain tumor-specific scFv from mono- without the epitope (␣CD40.wt), or PBS alone (Fig. 5B, left), or clonal Ig derived either from a mouse multiple myeloma DNA immunized i.m. with CD40-specific vaccibodies or controls (MOPC315) or lymphoma (A20) as antigenic units. Such scFv (Fig. 5B, right). Three weeks later, the spleens were removed. express serologically defined Id determinants in addition to T cell Upon restimulation in vitro with ␭2315 peptide, the number of IFN- epitopes. To investigate whether CD40-specific vaccibodies could ␥-producing splenocytes was significantly increased when mice induce production of tumor-specific anti-Id Ab, normal BALB/c 4174 TARGETING CD40 INDUCES PROTECTIVE IMMUNE RESPONSES

FIGURE 6. CD40-speciﬁc vaccines induce protection against multiple myeloma and B cell lymphoma. BALB/c mice were immunized with naked DNA encoding CD40-speciﬁc vaccibodies with scFv antigenic units from MOPC315 or A20, the corresponding nontargeted controls, or NaCl alone, in combination with electroporation (10 mice in each group). Two weeks later, the mice were challenged with a lethal dose of MOPC315 myeloma cells (A) or A20 B cell lymphoma cells (B) s.c.

that because the troybody molecules only express short T cell epitopes, their ability to induce Ab was not investigated.

CD40-specific DNA constructs induce protection against the MOPC315 myeloma and the A20 B cell lymphoma Next, we wanted to test whether immunization with CD40-specific vaccine constructs could induce protection against challenges with FIGURE 5. CD40-specific vaccines induce Th1 memory T cells and Ab cancer cells. Multiple myeloma and B lymphoma cells express responses. A, BALB/c mice were immunized i.m. with DNA encoding monoclonal Ig with V regions that function as tumor-specific Ag CD40-specific vaccibodies. After 3 wk, spleens were removed and restim- called Id. Normal BALB/c mice were injected i.m. with naked ␭ 315 ␥ ulated with 2 peptide in an IFN- -specific ELISPOT assay. B, BALB/c DNA encoding CD40-specific vaccibodies with the scFv from the mice were adoptively transferred with ␭2315-specific TCR transgenic ϩ MOPC315 myeloma, the nontargeted control, CD40-specific vac- CD4 T cells and either injected s.c. with a single dose of CD40-specific A20 troybody protein expressing the ␭2315 epitope, the corresponding nontar- cibody with scFv , or NaCl alone. All mice were electroporated. geted troybodies, CD40-specific troybodies with wt h␥3 C region, or PBS Two weeks after immunization, the mice were challenged with a alone (left). Alternatively, BALB/c mice were DNA immunized i.m. with lethal dose of MOPC315 cells s.c. Immunization with the CD40- 315 CD40-specific vaccibodies with scFv315, NIP-specific vaccibody with specific DNA vaccine expressing scFv induced protection in scFv315, CD40-specific vaccibody with irrelevant Ag (scFvA20), or NaCl Ͼ50% of the mice, whereas all mice in the other groups developed .(Indi- tumors within 25 days (Fig. 6A ,ء .right). IFN-␥-producing spleen cells were detected in ELISPOT) cate statistically significant differences (p Ͻ 0.01). C, BALB/c mice were In a second tumor model, similar groups of immunized mice injected i.m. with naked DNA encoding CD40-specific vaccibodies with were challenged with A20 lymphoma cells. The results (Fig. 6B) 315 A20 either scFv or scFv , nontargeted controls, or NaCl alone, followed by show that Ͼ50% of the mice immunized with CD40-specific DNA electroporation. Levels of serum Ab against the M315 Id (anti-Id315) were vaccine with scFvA20 were protected, whereas mice injected with measured on the indicated time points (six mice in each group). vaccine constructs not targeting CD40, or expressing the irrelevant scFv315 Ag, were not (Fig. 6B). Thus, CD40-specific vaccines with tumor-specific scFv induced Id-specific tumor resistance. mice where injected i.m. with DNA-encoding CD40-specific vaccibodies with either scFv315 or scFvA20. Mice injected with CD40- Discussion specific vaccibodies containing scFv315 produced high amounts of Ligation of CD40 is essential for activation of DC (3) and may tumor-specific anti-Id Ab. Mice injected with the corresponding release immature DC from suppression by CD4ϩCD25ϩ regula- nontargeted vaccibody, or with anti-CD40 vaccibody with tory T cells (27). Under physiological conditions, CD40 is bound scFvA20, produced no anti-Id315 Ab (Fig. 5C). On day 28, Abs by CD40L expressed on activated CD4ϩ T cells. However, ago- were predominately of IgG1 isotype, while at later time points nistic anti-CD40 mAb can substitute for CD40L. Thus, adminis- IgG1 and IgG2a were equally expressed (data not shown). Note tration of agonistic anti-CD40 mAb in combination with Ag The Journal of Immunology 4175 loaded via DEC-205 on DC resulted in memory and protective plasmacytoma and A20 lymphoma tumors used are major ob- CD4ϩ and CD8ϩ T cells responses, whereas in the absence of stacles to therapeutic vaccination of tumor-bearing mice. Nev- anti-CD40 mAb, tolerance was induced (4, 9). In these previous ertheless, the results suggest that targeting tumor Ag to CD40 studies, anti-CD40 mAb and delivery of Ag to DEC205 was un- on APC could be a valuable approach to eradicate minimal linked. In this study, we show that a single molecule can deliver residual disease after conventional treatment, e.g., after high- both an activation signal through CD40, as well as the Ag to APC. dose chemotherapy and reconstitution of the immune system by This strategy induced potent Ab and memory T cell responses, and bone marrow transplantation. protective immunity in two tumor models. The Ab-based vaccine constructs are quite flexible molecules. The Ig-like vaccine constructs used in this study target CD40 by troybodies have been successfully equipped with T cell epitopes in agonistic V regions cloned from the FGK-45 hybridoma (13). The several different loops both in CH1, CH2, and CH3 domains current two types of recombinant Ab-based vaccines differ, how- (M. Flobakk, J. B. Rasmussen, E. Lunde, G. Berntzen, T. E. ever, in their ability to bind to FcRs. The troybody molecule ex- Michaelsen, B. Bogen, and I. Sandlie, manuscript in preparation). presses a complete C region of h␥3 and binds to FcRs (28). In Moreover, epitopes up to 37 aa, proline-rich gluten epitopes, and contrast, the vaccibody molecule lacks the CH2 domain, and there- tandemly linked T cell epitopes have been successfully incorpo- fore the FcR binding site, and should only enter APC via their V rated (G. Tunheim, K. W. Schjetne, I. B. Rasmussen, L. M. Sollid, regions. So, because vaccibodies were efficient in the present ex- I. Sandlie, and B. Bogen, submitted for publication). As for vac- periments, targeting of CD40 is efficient in itself and does not cibodies, they can in their antigenic unit express large Ag-like scFv require simultaneous engagement of FcRs on APC. from myeloma patients (M. Frøyland, K. M. Thompson, T. Gedde- Our experiments demonstrated a need for physical linkage be- Dahl, A. B. Fredriksen, and B. Bogen, submitted for publication), tween the Ag and the CD40-specific V regions to activate Ag- fragment C of tetanus toxin (G. Tunheim, K. M. Thompson, A. B. specific T cells in vitro. This finding is in agreement with previous Fredriksen, T. Espevik, K. W. Schjetne, and B. Bogen, submitted findings demonstrating that efficiency of vaccine molecules con- for publication), hemagglutinin from influenza virus (G. Tunheim, sisting of scFv linked to chemokines (i.e., MCP, IFN-␥-inducible H. von Boehmer, and B. Bogen, unpublished data), and gp120 of protein-10) is dependent on a physical link between the Ag (scFv) HIV and streptavidin (A. B. Fredriksen, K. M. Thompson, D. and targeting unit (chemokine) (29). However, the requirement for Barouch, and B. Bogen, unpublished data). From our current work a physical linkage between the Ag and the maturation signal in the described above, it appears that there is a large potential for which present experiments superficially appears to contradict recent in Ag that the vaccine protein can accommodate. vivo studies demonstrating induction of long-lasting T cell responses even when anti-CD40 mAb was not linked to the Ab-Ag Acknowledgments fusion protein (4, 9, 10). The difference might be explained by the We thank Hilde Omholt, Peter Hofgaard, Mona Lindeberg, and Tom-Ole fact that in the latter case, the recombinant Ab-Ag fusion was Løvås for excellent technical assistance. targeted to DEC205, an efficient endocytic receptor expressed on DC (30), but lacking the ability to induce a maturation signal. Disclosures Thus, linkage between the CD40-targeting unit and the Ag might B. Bogen and A. B. 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