Proc. Natl. Acad. Sci. USA Vol. 88, pp. 4040-4044, May 1991 Immunology Antibodies that neutralize human I8 interferon biologic activity recognize a linear epitope: Analysis by synthetic peptide mapping (multiple peptide synthesis/"pepscan"/monoclonal antibodies) PHILIP N. REDLICH*t, PAUL D. HOEPRICH, JR.t§, C. BUDD COLBYt, AND SIDNEY E. GROSSBERG*¶ Departments of *Microbiology and tSurgery, The Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226; and tTriton Biosciences, 1501 Harbor Bay Parkway, Alameda, CA 94501 Communicated by Duard L. Walker, December 28, 1990 (received for review June 12, 1990) ABSTRACT The location of biologically relevant epitopes T-cell epitopes important in the biologic activity of viral and on recombinant human fi interferon in which Ser-17 replaces plasmodial proteins (22-26). Cys-17 (rh[Ser17JIFN-_l) was evaluated by testing the immuno- To map the location of epitopes identified by a panel of reactivity of antibodies against 159 sequential, overlapping mAbs raised against recombinant Ser-17-substituted hIFN-p8 octamer peptides. Three monoclonal antibodies (mAbs) that (rh[Ser17]IFN-f3) (17, 18), we determined their reactivity with neutralize rh[Ser'7]IFN-fl biologic activity, designated Al, A5, sequential, overlapping octamer peptides spanning the entire and A7, bound to peptides spanning only residues 3948, length ofthe rh[Ser17]IFN-,8 molecule. The panel consisted of whereas nonneutralizing mAb bound less specifically at mul- neutralizing (A) and nonneutralizing (B) mAbs that identify tiple sites near the amino terminus. The immunoreactivity of three functionally and immunochemically distinct epitopes, peptides spanning residues 40-47 that contained a series of designated sites I, Il, and III. Spatially distinct epitopes I and single amino acid substitutions suggested that residues 41-43 II are recognized by mAbs Al (or A5) and A7, respectively, (Pro-Glu-Glu) and 46 (Gln) are important for the binding of whereas site III is recognized by the nonneutralizing B mAbs. neutralizing mAbs. The reactivity of mAbs to larger synthetic Site I- and II-directed mAbs also neutralize the antiprolifer- peptides containing rh[Ser7J]IFN-P sequences from residue 32 ative activity of rh[Ser17]IFN-P and block binding to its cell through residue 56 was evaluated. All mAbs except A7 reacted receptors (17, 18). We report here that neutralizing mAbs with synthetic peptides representing rh[Ser17JIFN-P residues recognized a unique, linear epitope in the amino-terminal 32-47, 40-56, and 32-56, but only mAbs Al and AS bound to region of hIFN-,8 spanning residues 39-48; synthetic pep- the core peptide composed of residues 40-47. Peptide 32-56 tides that include this region inhibited the binding and neu- effectively blocked the binding of mAbs Al and AS to tralizing activity of site I-directed mAbs. rh[Ser17]IFN-13 and markedly inhibited their neutralizing ac- tivity. Biologic activity ofthe peptides was undetectable. Rabbit MATERIALS AND METHODS antisera raised against peptides 32-47 and 40-56 recognized Reagents, IFN Bioassay, and Radioiodination. Murine rh[Ser'7JIFN-,B but did not neutralize its antiviral activity. mAbs to rh[SerI7]IFN-f3 were produced, characterized, and Thus, structure-function analysis by peptide mapping has purified as described (17, 18). Purified neutralizing [Al permitted the identification of a linear epitope recognized by (IgGl), A5 (IgA), and A7 (IgG2a)] and nonneutralizing [B2 neutralizing antibody on a biologically active cytokine. We (IgG2b) and B7 (IgG2a)] mAbs as well as neutralizing rabbit conclude that the region spanning residues 32-56 is of major antisera (Lee Biomolecular Laboratories, San Diego) were importance in the expression of the biologic activity of human used in the epitope analyses. Anti-peptide rabbit antisera IFN-B. were raised by injections of peptide-keyhole limpet hemocy- anin conjugates in Freund's adjuvant and purified by ammo- The interferons (IFNs) constitute a family of cytokines that nium sulfate precipitation. Bioassays for the measurements possess multiple activities, including antiviral, antitumor, of antiviral, antiproliferative, and antibody-neutralizing ac- cell growth regulatory, and immunoregulatory properties (1). tivities have been detailed (17). Radioiodination of The mechanism of action of IFN that allows the expression rh[Ser'7]IFN-,B was achieved without loss in antiviral activity of these diverse biologic activities is not well understood. (17). One approach to study IFN action is to identify domains and Peptide Synthesis. Duplicate sets of 159 octamer peptides structural properties that are associated with biologic activ- representing the entire sequence of rh[Ser17]IFN-18 in a ities. Previous structure-function studies have utilized ge- sequential and overlapping manner were synthesized on netically altered IFN gene products (2-8), peptide mapping of prederivatized polyethylene pins (Cambridge Research Bio- large fragments (9-14), and immunochemical mapping with chemicals, Valley Stream, NY) arranged with a format and monoclonal antibodies (mAbs) (15-18). Such analyses of spacing that superimposes the chambers of a 96-well plate as human a and p interferon (hIFN-a and -4) molecules have reported (27). An amino acid replacement set analysis was yielded inconclusive results, implicating large regions near performed on a selected octamer sequence by the synthesis the amino terminus (2, 3, 7, 9, 15), carboxyl terminus (11, 16), on the pins of octapeptides in which each residue was or both (4-6, 8, 10), as domains responsible for biologic substituted by the other 19 commonly occurring amino acids, activity. The screening of short, sequential overlapping pep- while the remainder of the sequence was kept intact (20). tides for antibody reactivity as described by Geysen et al. (19, Antibody reactivity toward the solid-phase octapeptides was 20), also known as "pepscan" (21), permits the simultaneous scanning of entire molecules for linear immunoreactive Abbreviations: IFN, interferon; IFN-a and IFN-,8, a and f3 inter- epitopes. This approach has been effective in mapping B- and ferons; hIFN-a and -,3, human IFN-a and -a; rhlSer17]IFN-1B, recombinant hIFN-,8 in which Ser-17 replaces Cys-17; mAb, mono- clonal antibody. The publication costs of this article were defrayed in part by page charge §Present address: Applied Biosystems, 850 Lincoln Centre Drive, payment. This article must therefore be hereby marked "advertisement" Foster City, CA 94404. in accordance with 18 U.S.C. §1734 solely to indicate this fact. $To whom reprint requests should be addressed. 4040 Downloaded by guest on September 29, 2021 Immunology: Redlich et al. Proc. Natl. Acad. Sci. USA 88 (1991) 4041 determined by an enzyme-linked immunosorbent assay were all well recognized by these neutralizing mAbs with (ELISA). Peptides representing selected sequences of little or no binding elsewhere. In contrast, nonneutralizing hIFN-f3 were synthesized using a Biosearch 9500 peptide mAbs B2 and B7 demonstrated reactivity throughout the synthesizer and conventional Merrifield chemistry with tert- amino terminus. Neutralizing rabbit antibody revealed two butyloxycarbonyl/benzyl protection strategy (28). Synthetic major areas of binding with a peak of reactivity near the peptides were cleaved from the solid support with anhydrous amino terminus around octapeptides with initial residues 37 HF and purified to homogeneity by reverse-phase HPLC; and 38. residue composition was confirmed by amino acid analysis. To analyze neutralizing mAb binding at the single amino ELISA. The peptide-pin-based ELISA was performed (27) acid level, an amino acid replacement-set analysis was per- by incubating the peptide pins in phosphate-buffered saline formed: peptides spanning residues 40-47 were synthesized containing 1% bovine serum albumin and 0.05% Tween 20 for to contain single amino acid substitutions and tested for 1 hr, followed by incubation with antibody overnight at 40C. immunoreactivity (Fig. 2). Amino acids substitutions for Bound antibody was detected by incubation with an appro- Pro-41, Glu-42, Glu-43, and Gln-46 led to a loss in reactivity priate antibody-horseradish peroxidase conjugate for 1 hr by mAbs Al and A7, whereas substitutions for the other four followed by substrate (2,2'-azino-di-[3-ethylbenzthiazoline- residues were generally well tolerated. In contrast, only sulfonic acid]). An unrelated mAb or enzyme conjugate alone Glu-43 and Gln-46 were found to be important for the served as controls. The reactivity of polyclonal antibody or specificity and binding of mAb AS. These data suggest that protein-A-purified mAb to rh[Ser'7]IFN-,3 was measured in a residues 41-43 and 46 are essential for the binding of neu- plate-based ELISA as described (17). Reactivity of antibody tralizing mAbs. In addition, the evaluation of the binding to synthetic peptides was determined by ELISA with alkaline specificities at the single amino acid level for mAbs Al and phosphatase conjugates (Sigma) specific for mouse IgG or A5 has revealed differences not detected by previous con- IgA or for rabbit antibody, as required. ventional epitope analyses that used neutralization studies, competitive binding assays, and chemical modification of RESULTS rh[Ser17]IFN-13 (17, 18). Immunoreactivity and Biologic Activity of Synthetic Pep- Screening of Antibody Reactivity to Octamer Peptides. tides. Since reactivity of short peptides may result from Overlapping, sequential octamer peptides representing the hydrophobic and/or charge interactions (29-31), peptides entire rh[Ser'7]IFN-,f amino acid sequence were treated with that contained residues beyond the core hIFN-,3 immunore- selected mAbs and neutralizing antisera to identify linear active sequence (residues 40-47) were synthesized and an- epitopes (Fig. 1). The antigenic profiles of the neutralizing alyzed for antibody reactivity and biologic activities. These mAbs (Al, AS, and A7) were nearly identical. One region peptides were composed of residues 40-47 (core peptide), composed of octapeptides with initial residues 39, 40, or 41 1.5 Al 1.0W Al (.5 11 ; 11:'~~~I!1ii~ ii 0.5 AS 0 0 :...!hi I5J.i.1 J 0.5 A7 A5 B2 _ 2.