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).