Proc. Natl. Acad. Sci. USA Vol. 91, pp. 8945-8949, September 1994 Immunology Monoclonal antibody-superantigen fusion proteins: Tumor-specific agents for T-cell-based tumor therapy MIKAEL DOHLSTEN*t, LARS ABRAHMStNt, PER BJ6RK*, PETER A. LANDO*, GUNNAR HEDLUND*t, GORAN FORSBERG*, THOMAS BRODIN*, NICK R. J. GASCOIGNE§, CECILIA F6RBERGt, PETER LINDf, AND TERJE KALLAND*t¶ *Pharmacia Oncology Immunology, Lund, Sweden; tThe Wallenberg Laboratory, Department of Tumor Immunology, University of Lund, Lund, Sweden; *Pharmacia Bioscience Center, Stockholm, Sweden; and §The Scripps Research Institute, Department of Immunology, La Jolla, CA 92037 Communicated by Hans J. Maller-Eberhard, April IS, 1994 (receivedfor review October 18, 1993)

ABSTRACT The bacterial superantigen staphylococcal We recently demonstrated that chemical conjugation ofmAb enterotoxin A (SEA) is an extremely potent activator of T against colon carcinomas with the bacterial SAg staphylo- lymphocytes when presented on major histocompatibility com- coccal enterotoxin A (SEA) targeted T cells to lyse MHC plex (MHC) class H molecules. To develop a tumor-specific class II tumor cells in vitro (6) and induced tumor-suppressive superantigen for cancer therapy, we have made a recombinant lymphokines (7). We have now expressed in Escherichia coli fusion protein of SEA and the Fab region of the C215 mono- a recombinant fusion protein of SEA and a Fab fragment of donal antibody specific for human colon carcinoma cells. SEA a colon carcinoma-reactive mAb. Fab-SEA treatment of as part of a fusion protein showed a >10-fold reduction in mice carrying tumors expressing the relevant antigen resulted MHC class II binding compared to native SEA, and accord- in 85-99%o suppression of tumor growth in the absence of ingly, the affinity of the FabC215-SEA fusion protein for the overt systemic side effects. We believe that tumor-specific C215 tumor antigen was 100-fold stronger than to MHC class SAgs should be ofconsiderable interest as therapeutic agents 1I molecules. The FabC215-SEA fusion protein efficiently against cancer. targeted T cells to lyse C215+ MHC class II- human colon carcinoma cells, which demonstrates functional substitution of MATERIALS AND METHODS the MHC class fl-dependent presentation of SEA with tumor specificity. Treatment of mice carrying B16 melanoma cells Protein Reagents. Recombinant SEA was expressed in E. expressing a transfected C215 antigen resulted in 85-99% coli and purified to homogeneity. The hybridomas secreting inhibition of tumor growth and allowed long-term survival of the C215 (IgG2a) and C242 (IgGl) mAbs, reacting with animals. The therapeutic effect was dependent on antigen- human colon cancer, were produced at Pharmacia (8, 9). E. specific targeting of the FabC215-SEA fusion protein, since coli HB101 was used as host during the DNA construction native SEA and an antigen-irrelevant FabC242-SEA fusion work (10). protein did not influence tumor growth. The results suggest Cloning ofthe cDNAs Encoding the Murine Antibodies C215 that Fab-SEA fusion proteins convey superantigenicity on and C242. PCR was performed on C215 hybridoma cells by tumor cells, which evokes T cells to suppress tumor growth. using the total RNA isolation kit (Ginna/Biotecx Laborato- ries, Friendswood, TX). Reverse transcriptase (from Molo- The therapeutic use of naked monoclonal antibodies (mAbs) ney murine leukemia virus, Superscript, GIBCO/BRL) and in human epithelial cancer has met with limited success (1). random hexamer primers were used to make cDNA. The The amount of mAb found to accumulate in the tumor is primers Mhvp-7 (11) and Mushcp-21 (5'-CAATTTTCT- generally low, tumor cells display extensive heterogeneity in TGTCCACCTTGGTGCT) were used to isolate the heavy antigen expression, and the effector mechanisms responsible (H)-chain DNA. Similarly, the primers Mkvp-7 (11) and for tumor-cell destruction are insufficient (1, 2). Recent Muslcp-11 (5'-TGTGTCCCGGGATACAGTTGGTGCAG- progress in the molecular understanding ofT-cell recognition CATCAGCCC) were used to isolate the K-chain DNA. The has provided insight into the role of T cells in the response C242 hybridoma cell line was used as source material for against allogeneic and neoplastic tissues. The vigorous and cloning ofthe cDNAs encoding the immunoglobulin H chain destructive response against an allograft involves a high and the K chain. Polyadenylylated RNA was extracted from frequency of T lymphocytes that, via the T-cell receptor hybridoma cells, converted to double-stranded cDNA, and (TCR), recognizes peptide antigens presented in the context cloned into the phage A-based vector Uni-ZAP (Stratagene). of major histocompatibility complex (MHC) molecules. In Positive clones were used to prepare cDNA containing contrast, the frequency of T cells responding to a tumor is pBluescript SK(-) plasmids by phagemid excision and prop- generally low and insufficient to interfere with tumor growth. agation. The resulting cDNA-containing plasmids were char- This suggests that an attractive approach for immunotherapy acterized by restriction enzyme mapping, and the nucleotide would be to target a high frequency of T cells to the tumor sequences of inserts from selected plasmids were deter- area. We have therefore developed an antibody-based ther- mined. apy that provides tumor cells with superantigenicity. Super- Assembly of the Fab-SEA Gene Fusion Segment. The fol- antigens (SAgs) are a family of bacterial and viral proteins lowing pieces were used: (i) A Not I-Mlu I fragment con- that activate a high frequency of T cells to cytokine release taining the ribosomal binding site and most of the signal and cell-mediated cytotoxicity (3-5). Bacterial SAgs bind peptide encoding a portion of the gene from E. coli outer with high affinity to MHC class II molecules and subse- membrane protein A as translation and secretion signals for quently interact with T cells expressing particular sequences in the variable (V) region ofthe TCR ,/ chain (TCR V(3) (3-5). Abbreviations: SEA, staphylococcal enterotoxin A; SAg, superan- tigen; MHC, major histocompatibility complex; CTL, cytotoxic T lymphocyte; TCR, T-cell receptor; mAb, monoclonal antibody; V, The publication costs ofthis article were defrayed in part by page charge variable; C, constant; H, heavy; V8, (8 chain V region. payment. This article must therefore be hereby marked "advertisement" ITo whom reprint requests should be addressed at: Pharmacia in accordance with 18 U.S.C. §1734 solely to indicate this fact. Oncology Immunology, Scheelevagen 22, S-223 63 Lund, Sweden. 8945 Downloaded by guest on September 29, 2021 8946 Immunology: Dohlsten et al. Proc. Natl. Acad. Sci. USA 91 (1994) the H chain. The introduction of the Mlu I site changed the Cell Lines. The human B-cell lymphoma Raji, the human second to the last codon from CAG to AAC. (ii) The H chain colon carcinomas SW620 and Colo2O5, the murine B16 V region gene was modified by introducing flanking BssHII melanoma (ATCC, MD), and CHO cells were cultured as and Xma I sites by PCR. The last base of the former site is described (6, 16). SEA-reactive T-cell lines (>99%o CD3+) the first base ofthe gene and the latter spans codons 12-14 of were established from peripheral blood lymphocytes as de- the constant (C) portion. (iii) The gene encoding the first H scribed (6). chain C region was obtained from mAb C242 gene. The Transfection with cDNA Encoding the C215 Antigen. The encoded protein is identical to this portion of a consensus expression vector pKGE839 containing the GA733-2 cDNA murine IgG1 chain. A Xma I-Rsr II fragment was used from (encoding the C215 antigen) and the neomycin-resistance codon 12 including the first Cys codon of the hinge-encoding gene (14) was transfected into B16 melanoma cells or CHO region followed by the tripeptide spacer Gly-Gly-Pro. (iv) cells using Transfectam (Sepracor, IBF, Villeneuve-la- The SEA gene included on a Rsr II-Sfi I fragment was taken Garenne, France) and electroporation, respectively. G418- from the plasmid pKP554. The SEA gene contained on this resistent clones were harvested and subjected to repeated cell plasmid is identical to the sequence published by Betley and sorting. The sorted CHO-C215 cells were kept as a stable cell Mekalanos (12), except for two TAA stop codons. (v) The line, and high-expressing clones were selected from the translation and secretion signals for the K chain were derived B16-C215 cells. Establishment of CHO cells expressing from a Sfi I-Mlu I fragment. This contained 40 bp from the HLA-DR has been described (16). staphylococcal protein A gene (13), including the ribosomal Cytotoxicity Assay. Cytotoxicity was measured at various binding site and the first 10 bp ofthe signal peptide sequence, effector/target cell ratios in 4-h 51Cr release assays as de- followed by 59 bases to compose a synthetic signal peptide. scribed (6, 16). (vi) The K chain V region gene was modified by introducing Immunohistochenical Staining. Tumor tissues from colon flanking Dsa I and Kas I sites by PCR. The last base of the cancer patients were snap-frozen in liquid nitrogen-cooled former site is the first base of the gene and the latter spans isopentane and 4-pm frozen sections were prepared. After codons 4-6 of the C portion. (vii) The gene encoding the K drying and fixation in acetone, the sections were rehydrated chain C domain was obtained from the mAb C242 gene, with and stained with FabC215-SEA and SEA followed by anti- the same Kas I site as above introduced and ending with a mouse immunoglobulin-biotinylated antibody or rabbit anti- TAA stop codon followed by a Xba I site. SEA-biotinylated antibody and avidin-biotinylated peroxi- Expression and Purification ofFab-SEA Fusion Proteins. E. dase complex (17). The slides were developed in diaminoben- coli K-12 UL635 (ara-14, xyl-7, AompT, T4R) was used for zidine (Sigma) and mounted in DPX medium (Sigma). expression of Fab-SEA. The fermentation was performed at In Vivo Tumor Models. C57BL/6 (H-2d) mice were ob- 25°C at pH 7 in a 7-liter fermenter (Chemap, Mannedorf, tained from Bomholtgaard (Ry, Denmark). For induction of Switzerland). At an OD600 of r50, transcription from the trc lung metastasis, 6- to 10-week-old C57BL/6 mice were promoter was induced by addition of isopropyl P-D- inoculated i.v. into the tail vein with 75,000 B16-C215 cells in thiogalactoside to 0.05 mM. The fermentation was inter- 0.2 ml of PBS containing 1% normal syngeneic serum. rupted at an D060 of -85 and the cells were separated by Treatment with Fab-SEA, Fab, SEA, or PBS (control) i.v. filtration. Medium containing Fab-SEA was incubated with was initiated 1 day after tumor inoculation followed by Q-Sepharose FF (Pharmacia), the gel was removed by suc- additional doses on days 3, 5, and 7 as indicated. Mice were tion filtration, and the filtrate was further clarified by cen- sacrificed after 3 weeks and lung metastases were counted. In trifugation at 20,000 x g. The Fab-SEA protein was then the intraperitoneal tumor model, 3 x 103 B16-C215 cells were affinity-purified on a protein G-Sepharose column. The injected i.p. and therapy with FabC215-SEA or control eluted fraction was further purified on a Mono S HR 5/5 vehicle was initiated i.v. 1 day later. The animals were column (Pharmacia) and fractions containing Fab-SEA were observed daily and sacrificed when moribound, and the pooled and passed through a PD-10 column (Pharmacia) with presence of tumor was confirmed by macroscopic examina- 10 mM sodium phosphate (pH 7.4)-buffered saline (PBS) as tion. elution buffer. Determination ofKd for Binding to the C215 and MHC Class ntigen. 251 labeling of SEA, FabC215-SEA, and C215 RESULTS mAb was done by the lactoperoxidase method (Amersham). Prokaryotic Expression of FabC215-SEA. The V region of The binding of the labeled protein (15-50 ,uCi/,ug; 1 Ci = 37 the H chain of the C215 antibody cDNA was ligated to the GBq) was as unlabeled protein. The Kd value for binding to first C region of a H chain gene fragment from a consensus the C215 antigen was determined by a sandwich RIA in which murine IgG1. This Fd gene fragment was then fused to the mAb KS1/4, recognizing an epitope on the GA733-2 protein SEA gene and expressed as a bicistronic transcription unit distinct from that recognized by mAb C215 (14), was used to with the gene encoding the V region ofthe C215 K light chain coat eight-well microplate strips. Tumor extract containing ligated to the first C region ofa consensus K chain. The vector C215 antigen (14) was incubated with the mAb-coated mi- pKP865 was used for expression ofthe FabC215-SEA fusion crotiter wells, 125I-labeled protein was added at appropriate protein in E. coli (Fig. 1). The fusion protein was purified to dilutions and incubated with the C215 antigen, and bound >95% homogeneity as determined by SDS/PAGE and re- radioactivity was measured in a y counter. To determine the verse-phase HPLC (data not shown). Under nonreducing Kd value for binding to MHC class II, a binding assay using conditions, the protein migrated as an 82-kDa band that Raji cells and 125I-labeled SEA or 125I-labeled Fab-SEA dissociated into the light chain (26 kDa) and Fd-SEA fusion fusion protein was used (6). protein (52 kDa) under reducing conditions. TCR a-Chain Binding Assay. The cell binding assay was Binding to the C215 Antigen, MHIC Class U, and TCR. performed as described (15). Briefly, [35S]methionine-labeled Saturation curves and Scatchard plots for binding of the Raji or Colo2O5 cells were coated with SEA or FabC215- FabC215-SEA fusion protein and the hybridoma mAb C215 SEA (2.8 !.&M) and then applied to a microtiterwell containing to the C215 antigen demonstrated Kd values of -2 nM and 1 an immobilized truncated recombinant murine TCR V(3 nM, respectively (Fig. 2A and B). Inhibition experiments chain. After incubation and washing (15), the bound cells with iodinated mAb C215 as a tracer confirmed that the were lysed and the radioactivity was determined. The num- monovalent FabC215-SEA protein and the bivalent hybrid- ber ofbound cells was calculated from the specific activity of oma mAb C215 display an -2-fold difference in binding labeled cells. The SD was routinely <15%. affinities (data not shown). Immunohistochemistry was used Downloaded by guest on September 29, 2021 Immunology: Dohlsten et al. Proc. Natl. Acad. Sci. USA 91 (1994) 8947

B1l iII chains was detected with FabC215-SEA presented on MHC class II+ Raji cells but not when presented on C215+ Colo2O5 o) ri cells (Fig. 3C). This difference was not due to insufficient amounts of FabC215-SEA exposed on Colo2O5 cells, since strong binding was seen with an immobilized anti-SEA mAb I; (Fig. 3D).

K aNS I C, h Cytotoxic T-Lymphocyte (CTL) Targeting of FabC215- pKP865 Iss IIII SEA. The fusion protein targeted SEA reactive CTLs to lyse the MHC class II-C215+ human colon carcinoma cell line Rfil SW620 (18) at 10 pM, whereas native SEA showed no effect, S1E even at 10 nM (Fig. 4A). No cytotoxicity was seen with native mAb C215 (data not shown). To -demonstrate that lysis of II MHC class II- target cells was dependent on expression of I.'. tumor we \mlal the relevant antigen, used CHO cells transfected Nto with a cDNA encoding the human C215 antigen. The CHO- C215 transfectant was highly sensitive to FabC215-SEA- FIG. 1. Outline of the expression vector pKP865 encoding the induced lysis at effector/target ratios of3:1 and 10:1, whereas FabC215-SEA fusion protein. The orientation of the genes is indi- the nontransfected parental CHO cell line remained resistant cated by arrows. The secretion ofthe H-chain-SEA fusion is directed by an OmpA-derived signal peptide, and K-chain secretion is directed even at an effector/target ratio of 30:1 (Fig. 4B). No cyto- by a synthetic signal peptide (signal peptides are hatched). The toxicity against CHO-C215 or CHO was induced by SEA transcription of the bicistronic mRNA is initiated from the trc alone. To analyze whether the reduced MHC class II binding promoter. The transcript ends at two phage fd transcription termi- of the fusion protein would influence functional presentation nators (indicated by solid boxes). To keep the basal level of expres- on a MHC class II+ target cell, we examined cytotoxicity sion low, the lacI gene was included on the plasmid and the against MHC class II+ C215- Raji B-lymphoma cells. SEA replication part from pBR322 was used. Km, kanamycin-resistance targeted cytotoxic T cells against Raji cells efficiently at 1 gene; CK and VK, K chain C and V region genes, respectively; CH1 pM, whereas similar effects were seen at 10 pM FabC215- and VH, H chain first C region and V region, respectively. SEA protein (Fig. 4C). Since SEA-induced cytotoxicity is influenced by expression of adhesion molecules on the target to confirm that the FabC215-SEA fusion protein retained the cell (16), we determined the effect of the fusion protein staining pattern of native mAb C215. FabC215-SEA (Fig. against target cells, differing only in the expression of trans- 3A) and mAb C215 (data not shown) showed homogeneous fected MHC class II or C215 molecules. The FabC215-SEA and strong staining of human colon carcinoma cells. In protein was -10-fold more potent against the CHO-C215 contrast, SEA stained exclusively cells in the stroma, prob- compared to the CHO-DR target cells (Fig. 4D). ably reflecting infiltrating MHC class II' leukocytes (Fig. In Vivo Tumor Therapy with FabC215-SEA. A syngeneic 3B). The MHC class U binding of the fusion protein was mouse tumor model for the analysis of the effects of analyzed against MHC class II+ Raji B-lymphoma cells. FabC215-SEA was constructed by transfection of B16 Scatchard analysis demonstrated that the Kd value for bind- mouse melanoma cells with a plasmid encoding the C215 ing of FabC215-SEA was 238 nM, whereas native SEA antigen and selection of stable B16-C215 transfectants. Ad- showed a Kd value of -.14 nM (Fig. 2 C and D). Competition ministration of FabC215-SEA fusion protein i.v. strongly experiments with fluorescein isothiocyanate-labeled SEA as reduced the number of B16-C215 lung metastases (Fig. SA). a tracer confirmed that the fusion protein displayed an For two out of four tested B16-C215 clones (clones D2 and -.'.15-fold lower binding affinity compared to native SEA (data 7.B6) >99% suppression ofthe number offormed metastases not shown). The FabC215-SEA protein may interact with was recorded and >50% of the animals became tumor free. three distinct structures, the C215 antigen, MHC class II Treatment of the two other clones resulted in :95% (clone molecules, and the TCR complex. To examine the TCR 5.A12) and 85% (clone B5) suppression oftumor growth. The interaction, we analyzed binding of the FabC215-SEA pro- effect of FabC215-SEA was dose-dependent with maximal tein to a TCR Vf33 chain. Binding to immobilized TCR V83 tumor inhibition at 640 pmol (50 ug) per mouse and strong

1.0 0.8 A. 1.2 B. 0.8

i 0. E O. 4km m 0.5 0 5 10 15 0 25 50 T (nM) T (nM) Kd=2.3 aM Kd=1.3 nM r= -0.97 r=40.98 It 0 0.5 1.0 I .5 0 0.5 1.0 B (nM) B (nM) 12a 0.018 0.1LO - C. 2. ; D. FIG. 2. Binding characteristics of FabC215-SEA 0 (A), mAb C215 (n), and SEA (e) to the C215 antigen 0.012 0.112 mgo (A and B) and MHC class II+ Raji cells (C and D). (A and B) Representative Scatchard plots and saturation 0 400 80( r=*\8 0 50 100 1M curves (Insets) for FabC215-SEA (A) and intact mAb A T (nM) T 0.006 A 0.1A6 (nJ) C215 (B) from a C215 antigen RIA. (C and D) Cor- Kd=288 DM Kd=13.7nM responding diagrams for FabC215-SEA (C) and SEA r. -0.95 (D) in the Raji cell assay. The Kd value and the 0 II regression coefficient, r, are shown. Each point rep- 0 0.5 1.0 1.5 2.0 2.5 0 1 2 resents the mean of triplicate samples. B, bound; F, B (M) B (nM) free. Downloaded by guest on September 29, 2021 8948 Immunology: Dohlsten et al. Proc. Natl. Acad Sci. USA 91 (1994)

A C 15- 12 - 9- T0 6 13 0-

Raji Raji Colo2O5 ,'ii control T7CR 3 TCR A FIG. 3. (A and B) Immunohistochemical analysis of binding to human colon carcinoma tissue. Tissue sec- 80- tions were stained with FabC215-SEA (A) or SEA (B) at 100 nM followed by secondary reagents. (C) Binding

0 60' :::1 ofa soluble TCR VP chain to FabC215-SEA presented l' B~~~~~~~~~~~~~~~a _ on MHC class II+ Raji and C215+ Colo2O5 cells. (C _ ::: - _ '' Radiolabeled cells were treated with FabC215-SEA or n0*8 40 _ 20 _ 2~~~~~ti _ SEA and compared for binding to TCR V133 chains _ ::: :: _ immobilized on plastic microtiter wells. Control refers O20 |||l _ A, lllll _ lsC to cell binding to uncoated wells. (D) Binding of an anti-SEA mAb to LL.- immobilized (a-SEA) FabC215-SEA 0 |: ': '' _ _ _ presented on radiolabeled Raji and Colo2O5 cells. Con- Raji Raii Colo205 trol refers to cell binding to uncoated wells. In CandD, control X-SEA a-SEA data for the cells bound are presented as cpm x 10-3. reduction in the number of B16-C215 lung metastases at 64 ibound and sacrificed 30-45 days after tumor inoculation pmol per mouse (Fig. SB). The treatment was well toleratedr_F -(Fig. I f 5F). as no change in body weight or any other overt side effects were noted in animals treated with these doses of FabC215- DISCUSSION SEA. Systemic immune activation was not sufficient to inhibit tumor growth since treatment with SEA was without Current strategies for immunotherapy include attempts to effect (Fig. 5C). The therapeutic effect was dependent on improve tumor-specific T-cell activation by vaccination with antigen-specific targeting, since a similarfusion protein based tumor cells transfected with genes encoding cytokines or on the irrelevant mAb C242 did not mediate significant costimulatory molecules, such as B7 (19, 20). These ap- anti-tumor effects (Fig. 5D). The FabC242-SEA fusion pro- proaches have proven highly effective in experimental animal tein showed a similar MHC class II-dependent binding and models, but they may be limited by the scarcity of T-cell T-cell activation as FabC215-SEA (data not shown). More- epitopes expressed on human tumor cells. In contrast, nu- over, treatment with the FabC215 fragment alone failed to merous tumor-associated epitopes have been defined on induce a significant anti-tumor effect (Fig. 5E). The human tumors by using mAbs. This led us to use tumor- FabC215-SEA fusion protein also mediated strong anti- reactive mAbs for targeting of the extremely potent bacterial tumor effects when the tumor was growing i.p. The survival SAg SEA. In this paper we have used gene-transfer experi- of mice carrying B16-C215 cells i.p. was prolonged after ments to show that the specificity in Fab-SEA targeting treatment with 64 or6.4 pmol ofFabC215-SEA i.v. (Fig. 5F). against MHC class II- tumor cells is exclusively defined by A substantial fraction of the treated mice were long-term the antigen reactivity ofthe mAb. This result is in conformity survivors (>90 days), whereas all untreated mice were mor- with earlier observations that SAg may interact with TCR

50 - C SW620 80 Raji 60 30 / FabSEA 40- 10 1-- ~~SEA 20 .5 i-14 -13-12 - x 0 -14 -13 -12 -11 -10 -9 -14 -13 -12 -1 1 -10 0 FIG. 4. FabC215-SEA fusion protein directs CTLs against MHC class II-C215+ tumor cells. (A) Effects of I0- P the FabC215-SEA fusion protein (o) or SEA (A) 'HO-C215 against the MHC class II- human colon carcinoma cell line SW620 in a 4-h Cr release assay at an effector/ target ratio of 30:1. (B) Effect of FabC215-SEA (4 nM) at various effector/target ratios against CHO-C215 'HO-DR transfectants or untransfected control CHO cells (dashed line). (C) FabC215-SEA and SEA targeting of 'HO CTLs against MHC class II+ Raji cells at an effector/ target ratio of 30:1. (D) Dose-response curves of i Tv FabC215-SEA-induced CTLs targeted against CHO- 3 10 100 ~9 C215, CHO-DR, and CHO cells at an effector/target Effector/target ratio log[compound] ratio of 30:1. Downloaded by guest on September 29, 2021 Immunology: Dohlsten et al. Proc. Natl. Acad. Sci. USA 91 (1994) 8949

1I treated mice, which indicate that therapy with FabC215-SEA PBS is well tolerable. Several observations suggest that local 120 Il 1 °'' iFt1-ah s} !i 60- antigen-specific targeting is essential for the observed anti- 90. _._ ... ~~~~~~~~~~~~~~~~~~~.... tumor effects. Anti-tumor effects in the B16-C215 model were 40 - induced by FabC215-SEA but not by either moiety sepa- rately. The possibility that the observed difference of 0.) 30}-- FabC215-SEA and SEA was due to a prolonged serum . half-life of the fusion was use ( :. .- _- 0 larger protein excluded by the 1< l3. 1 1 D2)' 12- 6.4 64 640 of the nonspecific FabC242-SEA fusion protein. This indi- cates that systemic T-cell activation is not sufficient for

2 5;( anti-tumor effects and favors a role for antigen-specific I _- 1n1 F- targeting. Moreover, FabC215-SEA treatment effectively 'Z. '0 120---. suppressed the growth of B16-C215 tumor cells, whereas 7_ parental B16 cells remained unaffected (data not shown). ;- 1 (} i .- ( Fab-SEA fusion proteins represent another strategy for 00 mAb-based immunotherapy and allow high-frequency target- ing ofpseudospecific T cells to the tumor. The results suggest ; ', that Fab-SEA fusion proteins have properties that merit ( ...... _ .: .._.. further evaluation for treatment of human tumor diseases. {I 6f.4 614 0ufl I ) (,.4 6 4 640 . . ! N.R.J.G. is a Scholar of the Leukemia Society of America. This work was funded in part by National Institutes of Health Grant I ',}1,l GM46134 to N.R.J.G. . I

SP-1i 60.- 1. Riethmuller, G. & Johnson, J. P. (1992) Curr. Opin. Immunol. 4, 647-655. 4t A-- 2. Foon, K. A. (1989) Cancer Res. 49, 1621-1639. r 4H1 3. Scherer, M. T., Ignatowicz, L., Winslow, G., Kappler, J. & 20- Marrack, P. (1993) Annu. Rev. Cell Biol. 9, 101-128. 4. Johnson, H. H., Russel, J. K. & Pontzer, C. H. (1991) FASEB O6. 4 6-4 J. 5, 2706-2711. 0I 6..4 64 gill. 30( ai 61 90 5. Kalland, T., Hedlund, G., Dohlsten, M. & Lando, P. A. (1991) s--:al- -1 Curr. Top. Microbiol. Immunol. 174, 81-92. 6. Dohlsten, M., Hedlund, G., Akerblom, E., Lando, P. A. & FIG. 5. FabC215-SEA therapy of B16-C215 metastases. (A-E) Kalland, T. (1991) Proc. Natl. Acad. Sci. USA 88, 9287-9291. Number of lung metastases from experiments with six to eight mice 7. Dohlsten, M., Sundstedt, A., Bjorklund, M., Hedlund, G. & per group (mean ± SD). (A) Effect of four injections of 640 pmol (50 Kalland, T. (1993) Int. J. Cancer 54, 482-488. jLg) of FabC215-SEA or control vehicle on lung metastases formed 8. Larson, L. N., Johansson, C., Lindholm, L. & Holmgren, J. by the B5, 5.A12, D2, and 7.B6 B16-C215 cell clones. (B and C) (1988) Int. J. Cancer 42, 877-882. Effects of five injections of various doses of FabC215-SEA (e) or 9. Johansson, C., Nilsson, 0. & Lindholm, L. (1991) Int. J. SEA (o) on lung metastases. (D and E) Effects of four injections of Cancer 48, 757-763. the relevant FabC215-SEA protein (e) are compared to the nonspe- 10. Boyer, H. W. & Roulland-Dussoix, D. (1969) J. Mol. Biol. 41, cific FabC242-SEA control fusion protein (A) (D) or the FabC215 459-472. fragment (o) (E). The FabC215 fragment was prepared by cleavage of C215 with 11. Jones, S. T. & Bendig, M. M. (1991) Biotechnology 9, 88-89. mAb immobilized papain and purified by protein A 12. Betley, M. J. & Mekalanos, J. J. (1988) J. Bacteriol. 170, chromatography. (F) Effect of FabC215-SEA therapy on survival of C57BL/6 mice carrying B16-C215 cells i.p. An i.v. administration of 34-41. FabC215-SEA at 64 pmol (thick solid line) or 6.4 pmol (thin solid 13. Uhldn, M., Guss, B., Nilsson, B., Gatenbeck, S., Philipson, L. line) or control vehicle (dotted line) was given on days 1, 3, and 5. & Lindberg, M. (1984) J. Biol. Chem. 259, 1695-1702. Statistical analysis was performed with Mann-Whitney U test. **, P 14. Bjork, P., Jonsson, U., Svedberg, H., Larsson, K., Lind, P., < 0.01; ***, P < 0.001. Dillner, J., Hedlund, G., Dohlsten, M. & Kalland, T. (1993) J. Biol. Chem. 268, 2432-2441. molecules in the absence ofMHC class 11 (7, 21). The binding 15. Gascoigne, N. R. J. & Ames, K. T. (1991) Proc. Natl. Acad. affinity of FabC215-SEA for the C215 antigen is 100-fold Sci. USA 88, 613-616. stronger than for HLA-DR molecules. Despite this, targeting 16. Gjorloff, A., Hedlund, G., Kalland, T., Sansom, D., Fischer, ofCTLs H., Trowsdale, J., Sjogren, H. 0. & Dohlsten, M. (1992) by FabC215-SEA is only 10-fold less effective when Scand. J. Immunol. 36, 243-250. FabC215-SEA is presented on HLA-DR+ cells compared to 17. Brodin, T. N., Jansson, B., Hedlund, G. & Sjogren, H. 0. C215+ cells. Direct binding experiments of a soluble TCR (1989) J. Histochem. Cytochem. 37, 1013-1024. chain to FabC215-SEA presented on target cells exposing 18. Dohlsten, M., Hedlund, G., Segrdn, S., Lando, P. A., Herr- MHC class II+ or C215+ molecules further underlined that man, T., Kelly, A. P. & Kalland, T. (1991) Eur. J. Immunol. 21, the contribution of MHC class II molecules increases the 1229-1233. affinity to the TCR, in agreement with models of TCR-SAg- 19. Fearon, E. R., Pardoll, D. M., Itaya, T., Golembeck, P., MHC class II complex (22). The therapeutic effect of Levitsky, H. I., Simons, J. W., Karasuyama, H., Vogelstein, FabC215-SEA was analyzed in vivo by using four B16-C215 B. & Frost, P. (1990) Cell 60, 397-403. 20. W. tumor clones. All untreated animals had many lung metas- Chen, I., Ashe, S., Bradey, A., Hellstrom, I., Hellstrom, K. E., Ledbetter, J. A., McGowan, P. & Linsley, P. (1992) Cell tases, whereas FabC215-SEA-treated animals had none or 71, 1093-1096. few metastases. Dose-response studies showed 85-99% sup- 21. Fleischer, B., Gerardy-Schan, R., Metzroth, B., Carrel, S., pression of tumor growth at 640 pmol of FabC215-SEA and Gerlach, D. & Kdhler, W. (1991) J. Immunol. 146, 11-17. substantial tumor suppression at a 64 pmol per mouse. No 22. Woodland, D. L. & Blackman, M. A. (1993) Immunol. Today overt side effects and normal body weight were seen in 14, 208-212. Downloaded by guest on September 29, 2021