Transgenic Mice Potent Humoral Responses in Human CD64
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Targeting Weak Antigens to CD64 Elicits Potent Humoral Responses in Human CD64 Transgenic Mice This information is current as Tibor Keler, Paul M. Guyre, Laura A. Vitale, Karuna of October 1, 2021. Sundarapandiyan, Jan G. J. van de Winkel, Yashwant M. Deo and Robert F. Graziano J Immunol 2000; 165:6738-6742; ; doi: 10.4049/jimmunol.165.12.6738 http://www.jimmunol.org/content/165/12/6738 Downloaded from References This article cites 30 articles, 19 of which you can access for free at: http://www.jimmunol.org/content/165/12/6738.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on October 1, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Targeting Weak Antigens to CD64 Elicits Potent Humoral Responses in Human CD64 Transgenic Mice Tibor Keler,1* Paul M. Guyre,† Laura A. Vitale,* Karuna Sundarapandiyan,* Jan G. J. van de Winkel,‡ Yashwant M. Deo,* and Robert F. Graziano* Previous studies have documented that targeting foreign Ags to IgG Fc␥R leads to enhanced Ag-specific responses in vitro and in vivo. However, the ability to overcome immunologic nonresponsiveness by targeting poorly immunogenic Ags to Fc␥R has not been investigated. To address this question in a simple model, we immunized transgenic mice expressing human CD64 (Fc␥RI) and their nontransgenic littermates with Fab derived from the murine anti-human CD64 mAb m22. The m22 Fab served as both the targeting molecule and the Ag. We found that only CD64-expressing mice developed anti-Id titers to m22. Furthermore, chemically linked multimers of m22 Fab, which mediated efficient internalization of the human CD64, were significantly more potent than monomeric m22 F(ab )2 at inducing anti-Id responses. In all cases, the humoral responses were specific for m22 Id and Downloaded from (did not react with other murine IgG1 Fab fragments. Chemical addition of a second murine Fab (520C9 anti-human HER2/neu to m22 Fab multimers demonstrated that IgG1 and IgG2a anti-Id titers could be generated to 520C9 only in the CD64-expressing mice. These results show that targeting to CD64 can overcome immunological nonresponsiveness to a weak immunogen. There- fore, targeting to CD64 may be an effective method to enhance the activity of nonimmunogenic tumor vaccines. The Journal of Immunology, 2000, 165: 6738–6742. aul and co-workers showed more than 25 years ago that Id determinants of the Igs expressed by B cell tumors can serve http://www.jimmunol.org/ Abs could enhance Ag-specific immune responses (1). as tumor-specific Ags, but are weakly immunogenic. Induction of P Subsequently, it has been shown that targeting Ags to Fc anti-Id responses usually requires CFA, cytokines such as GM- receptors for IgG (Fc␥R) expressed by APCs can potentiate acti- CSF, and/or conjugation to highly immunogenic carrier proteins vation of cultured T cells (2–9) and promote immune responses such as keyhole limpet hemocyanin (15–18). Therefore, murine when targeted in vivo (10, 11). Three classes of leukocyte Fc␥R Ab Id can serve as an appropriate model of a poorly immunogenic have been defined molecularly, Fc␥RI (CD64), Fc␥RII (CD32), tumor Ag in mice. Previously, Heijnen and co-workers have re- and Fc␥RIII (CD16), and their relative contributions to beneficial ported on a transgenic mouse model in which the human CD64 and pathogenic mechanisms are being elucidated (12, 13). transgene is expressed and regulated in a pattern consistent with by guest on October 1, 2021 Using bispecific Abs that simultaneously bind Ag and Fc␥RII/ the expression and regulation of CD64 on human APCs (11, 19). Fc␥RIII on the surface of APCs, Snider and Segal demonstrated With this model they demonstrated enhance IgG responses to a the ability of these Fc␥Rs to greatly enhance Ag processing and CD64-targeted humanized Ab. In this report we extend those stud- presentation to T cells in vitro (3) and Ag-specific Ab responses in ies by investigating the humoral responses to murine idiotypic Ags vivo (10). More recently, Wernersson et al. (14) have shown that targeted to CD64. IgG enhancement of Ag-specific Ab responses is drastically re- duced in FcR ␥-chain knockout mice that do not express functional Materials and Methods ␥ ␥ ␥ Ј Ј Fc RI or Fc RIII, implicating one or both of these Fc Rinthe Preparation of F(ab )2 and F(ab )3ϩ Ag ␥ enhancing effect. When mice that were deficient in Fc RIII were Ј ␥ F(ab )2 of the anti-Fc RI mAb m22 (IgG1) (20) and the murine anti-human tested in this system, Ab enhancement of the immune response was HER2/neu 520C9 (21) were prepared by pepsin digestion and purified by comparable to that in wild-type mice. This result suggested that flowing through a protein A column followed by Superdex 200 (Pharmacia, Fc␥RI was sufficient to mediate the enhanced response. Collec- Piscataway, NJ) gel filtration chromatography. To make multimeric Ј Ј tively these studies have clearly demonstrated that targeting model F(ab )3ϩ molecules, m22 F(ab )2 was treated with a 20-fold molar excess of sulfo-succinimidyl 4-N-maleimidomethyl-cyclohexane-1-carboxylate immunogenic Ags to Fc␥R on APCs, and particularly CD64, re- Ј Ј (Pierce, Rockford, IL). Separately, m22 F(ab )2 or 520C9 F(ab )2 was re- Ј Ј Ј sults in enhanced immune responses. However, it is not clear duced to Fab with 2-ME amine. The Fab were then added to m22 F(ab )2- whether this approach would effectively induce immune responses maleimide and incubated for2hatambient temperature. Purification of the to weakly immunogenic tumor Ags. multimers was performed by Superdex 200 gel filtration chromatography. ϫ Ј To make the m22 520C9 F(ab )3ϩ multimers, equimolar amounts of Ј Ј Ј m22 Fab and 520C9 Fab were added to the m22 F(ab )2-maleimide. The control Abs m32.2, anti-human CD64 (murine IgG1), A77; anti-human CD89 (murine IgG1), H22; and anti-human CD64 (humanized IgG1), were *Medarex, Inc., Annandale, NJ 08801; †Department of Physiology, Dartmouth Med- purified from supernatant of hybridomas or transfected myeloma cells. ical School, Lebanon, NH 08801; and ‡Department of Immunology and Medarex Europe, University Hospital Utrecht, Utrecht, The Netherlands Modulation of CD64 Received for publication June 2, 2000. Accepted for publication September 13, 2000. Peritoneal macrophages were elicited in human CD64 transgenic and non- The costs of publication of this article were defrayed in part by the payment of page transgenic littermates with 1 ml of 3% Brewer’s thioglycolate (Difco, De- charges. This article must therefore be hereby marked advertisement in accordance troit, MI) for 4 days before recovery by peritoneal lavage. The macro- with 18 U.S.C. Section 1734 solely to indicate this fact. Ј phages were incubated in polypropylene microtiter plates with m22 F(ab )2 1 Ј Address correspondence and reprint requests to Dr. Tibor Keler, Medarex, Inc., or m22 F(ab )3ϩ at varying concentrations for2hat37°C (active group) 1545 Route 22 East, Annandale, NJ 08801. E-mail address: [email protected] or at 4°C (control group). Cells were harvested after being placed on ice for Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00 The Journal of Immunology 6739 15 min and then washed and incubated on ice with 32.2 anti-CD64 Ab conjugated to FITC, which binds to a site on CD64 that is distinct from the m22 epitope (20). Samples were washed, and fluorescence was measured using a FACScalibur (Becton Dickinson, San Jose, CA). Immunization of mice The mice employed for these studies were 5- to 12-wk-old females from the human CD64-transgenic FVB/N line 1852 previously described by Heinen et al. (11, 19). We used F1 mice from heterozygous mating such that approximately 50% of each litter expressed human CD64. For each experiment human CD64-expressing mice were matched with their non- transgenic littermates as controls. All immunizations were performed i.p. using the Ribi Adjuvant System (Sigma, St. Louis, MO) in a final volume of 200 l/mouse. Mice were immunized with 25 g of Ag three or four Ј times at approximately 2-wk intervals. Blood was drawn for analysis 1 wk FIGURE 1. Anti-Id response to m22 F(ab )2 immunizations. Human after each immunization. CD64-expressing mice and their nontransgenic littermates were given four i.p. injections of m22 F(abЈ) (25 g/mouse/dose) in Ribi adjuvant. Blood Analysis of anti-Id responses 2 was collected from each animal 1 wk after the last immunization, and the Anti-Id responses were evaluated by sandwich immunoassays. Microtiter plasma was tested for anti-m22-Id IgG by ELISA. Each circle represents an Ј plates were coated with F(ab )2 from relevant or control Abs. Nonspecific individual mouse. adsorption was blocked with 5% BSA in PBS. Samples of plasma from mice were diluted in PBS and incubated on the plates for 1–2 h at 37°C.