Two mouse monoclonal anti-anti-idiotopic antibodies (anti-anti-Id, Ab3), AF14 and AF52, were prepared by immunizing BALByc mice with rabbit polyclonal antiidiotypic antibodies (anti-Id, Ab2) raised against antibody D1.3 (Ab1) specific for the antigen hen egg lysozyme. AF14 and AF52 react with an ‘‘internal image’’ monoclonal mouse anti-Id antibody E5.2 (Ab2), previously raised against D1.3, with affinity constants (1.0 3 109 M21 and 2.4 3 107 M21, respectively) usually observed in secondary responses against protein antigens. They also react with the antigen but with lower affinity (1.8 3 106 M21 and 3.8 3 106 M21). This pattern of affinities for the anti-Id and for the antigen also was displayed by the sera of the immunized mice. The amino acid sequences of AF14 and AF52 are very close to that of D1.3. In particular, the amino acid side chains that contribute to contacts with both antigen and anti-Id are largely conserved in AF14 and AF52 compared with D1.3. Therapeutic immunizations against different pathogenic antigens using anti-Id antibodies have been proposed. Our experiments show that a response to an anti-Id immunogen elicits anti-anti-Id antibodies that are optimized for binding the anti-Id antibodies rather than the antigen. The complementarity determining regions (CDR) of Ab display great structural diversity and constitute a vast array of potential antigenic determinants. Under appropriate experimental conditions, these determinants give rise to immune responses and are called idiotypic (1, 2). Idiotypes of Ab are the sum of idiotopes (Id) or antigenic determinants characterized experimentally by the reaction of an anti-idiotopic (anti-Id) antibody (Ab2) with the Id (Ab1). In most cases, idiotypes have been shown to be associated, partially or entirely, with the CDR of Ab molecules (3). The observation that external antigens and anti-Id Ab can competitively bind to specific Ab has led to proposals that anti-Id Ab may carry an ‘‘internal image’’ (2) of the external antigen. Functional mimicry of ligands of biological receptors by anti-Id Ab has been described in several systems (4). The potential for mimicking external antigens has led to proposals (5, 6) to use anti-idiotypic Ab as surrogate antigens. Recent reviews (7–9) have discussed this topic from a structural viewpoint. Indeed, anti-Id Ab have been used in exploring therapeutic immunizations against different pathogenic antigens. A report from this laboratory compared the threedimensional structures of the complexes between an Ab1antigen and an Ab1-anti-Id and provided a concrete example of how an anti-Id Ab can structurally mimic an external antigen (10, 11). In this system, the Ab1 was the mAb D1.3 specific for the antigen, hen egg lysozyme (HEL). The three-dimensional crystal structure of the complex between the Fv fragment of D1.3 with HEL has been determined at the resolution of 1.8 Å (12). Subsequently, the three-dimensional structure of a complex between the Fv from D1.3 and the anti-Id Fv from mAb E5.2 (the Ab2; BALByc IgG1, k) was determined (10, 11) at the nominal resolution of 1.9 Å. With these high resolution structures, it was possible to assess antigen mimicry by the anti-Id. In this system, the affinity of the anti-Id for the Ab1 (1.4 3 109 M21) was close to that of the Ab1 for HEL (2.7 3 108 M21). A comparison of the three-dimensional structures of the two complexes showed that, of the 18 D1.3 residues that make chemical contacts with the anti-Id and the 17 that contact the antigen, 13 contact both. These 13 residues of D1.3 contribute 75% (687 Å2) of the contacting area with the anti-Id and 87% (675 Å2) of the contacting area with the antigen. Six of the 12 interface hydrogen bonds in the D1.3–antigen complex are superimposable with hydrogen bonds in the D1.3–anti-Id complex. Furthermore, the positions of the atoms by which the antigen contacts D1.3 (Ab1) are close to those of the anti-Id that contact D1.3. It is by this bonding pattern that it can be said that the anti-Id (E5.2) mimics the antigen (HEL) although it does not provide an exact ‘‘topological image’’ of the antigen (10, 11). The results outlined in the preceding paragraphs were obtained by a purely structural approach. To test how well the anti-Id E5.2 mimics the antigen (HEL) physiologically, mice were immunized with E5.2. In the immune sera of some mice (BALByc and C57BL), anti-HEL Ab were detected. By this test, the anti-Id (E5.2) behaved immunologically like a typical ‘‘internal image’’ Ab, validating the observations made on its structural mimicking of the antigen, HEL (10). We present results of a study of an idiotypic chain involving D1.3, E5.2, and two newly produced anti-anti-Id mAbs, AF14 and AF52. These anti-anti-Id mAbs were obtained using rabbit polyclonal Ab against the Ab1 D1.3 (Fig. 1). AF14 and AF52 mimic the Ab1 D1.3 in their ability to bind both the antigen and the anti-Id E5.2. To understand how well anti-Id Ab can act as antigen surrogates, we analyze those binding reactions and present structural data on AF14 and AF52. MATERIALS AND METHODS Preparation of Polyclonal Anti-Id Ab. Two New Zealand Black rabbits were immunized with purified monoclonal D1.3 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1997 by The National Academy of Sciences 0027-8424y97y948697-5$2.00y0 PNAS is available online at http:yywww.pnas.org. Abbreviations Ab1, antibody specific for external antigen; Ab2, antiidiotypic antibody; Ab3, anti-anti-idiotypic antibody; HEL, hen egg lysozyme; CDR, complementarity determining regions; VH, VL, the variable domains of the heavy (H) and light (L) Ig (Ig) polypeptide chains; Id, idiotype or idiotope. ‡To whom reprint requests should be addressed. e-mail: poljak@ umbi.umd.edu.