International Immunology. Vol 4. No.3, pp.

417-420

© 1992 Oxford University Press

Heteroconjugate antibodies enhance cell-mediated anti-herpes simplex virus immunity in vivo Lesley M. Voss1-2, Chella S. David1, Stephen D. Showalter4, Carlos V. Paya1-3, and Paul J. Leibson1-3 Departments of immunology and 2Pediatrics, and the 3Division of Infectious Diseases, Mayo Clinic and Foundation, Rochester, MN 55905, USA "•Bio-Molecular Technology, Inc., Frederick, MD 21701, USA

Abstract Heteroconjugate antibodies are generated by covalently linking two mAbs with different specificities. When antl-CD3 mAb, capable of activating effector T cells, is coupled to anti-herpes simplex virus (HSV) mAb, which binds HSV antigens on vlrally-lnfected target cells, the resulting heteroconjugate antibody can be used In vitro to enhance antl-HSV immunity. Specifically, these heteroconjugate antibodies can augment antl-HSV immunity among lymphocytes previously lacking cytotoxicity against HSV-infected target cells. However, the efficacy of these specialized reagents in enhancing antl-HSV Immunity In vivo has not been determined. We report here that anti-HSV heteroconjugates used In an adoptive transfer murine model of HSV-1 infection inhibited HSV replication In vivo and Improved long-term survival. These results demonstrate that heteroconjugate antibodies have a potential therapeutic role in enhancement of anti-HSV immunity. Introduction Despite reductions in mortality with the use of antiviral chemotherapeutic agents, herpes simplex virus (HSV) remains a significant cause of morbidity in neonates and in those with impaired T cell immunity (1,2). In these patients, an insufficient T cell response to HSV predisposes them to overwhelming viral infection. A potential strategy to augment anti-HSV immunity is to redirect non-HSV specific T lymphocytes to directly kill infected cells. When mAbs capable of activating immune cells are covalently linked to mAbs specific for tumor-associated antigens, these heteroconjugate antibodies can enhance anti-tumor immunity in vitro (3,4). Recent studies by our group (5) and others (6,7) also indicate a role for antiviral heteroconjugates in augmenting virus-specific immunity in vitro. However, the capacity of these reagents to effectively inhibit viral replication in vivo and to subsequently affect survival remains undetermined. We describe here in vivo testing of heteroconjugate antibodies [e.g. anti-CD3 x anti-HSV-1 glycoprotem D (gD)] in an adoptive transfer model of HSV-1 infection. Our results indicate the effectiveness of heteroconjugate antibodies in reducing viral replication and in improving long-term survival in this murine model.

Methods Monoclonal and heteroconjugate antibodies OKT3 mAb reacting with the e chain of the human CD3 complex was kindly provided by Ortho Diagnostic Systems (Raritan, N J). The anti-Fc-yRIII mAb 3G8, specific for the Fc receptor (CD16) expressed on human NK cells, neutrophils, and activated macrophages, was generously provided by Dr D. M. Segal (Experimental Immunology Branch, National Cancer Institute/ National Institutes of Health, Bethesda, MD). Isolation and characterization of mAb specific for HSV-1 glycoprotein C (gC; mAb 19S) and HSV-1 gD (mAb 4-S) have previously been described (8). Heteroconjugate antibodies were generated by crosslinking either anti-CD3 mAb (OKT3) or anti-Fc^RIII (3G8) with either anti-HSV-1 gC or anti-HSV-1 gD using the heterobifunctional reagent succinimidyl-3-(2-pyridyldithio)propionate (Pierce, Rockford, IL), as previously described (5). Isolation and characterization of clonal human T cell lines Ficoll-Hypaque fractionated peripheral blood mononuclear cells were stained for 30 mm at 4°C with fluorescein-conjugated

Correspondence to: P. J. Leibson Transmitting editor: M. J. Bevan

Received 24 October 1991, accepted 20 November 1991

Downloaded from http://intimm.oxfordjournals.org/ at University of Sussex on July 29, 2015

Key words: adoptive transfer, anti-CD3 mAb, immunotherapy, viral antigens

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Enhancement of anti-HSV immunity (sham-treatment) or with CD8 + human T cells (4 x lOVmouse) with or without heteroconjugate antibodies (0.06 /xg/mouse). At 24 h later, the mice were inoculated i.p. with the KOS strain of HSV-1 (7 x 10" p.f.u./mouse). After an additional 48 h, the mice were sacrificed, and their spleens were removed and weighed. After homogenizing and sonicating the spleens, the amount of live virus was quantifed by plating serial dilutions onto confluent monolayers of RSG. The monolayers were overlaid with 0 5% methylcellulose and incubated for 72 h at 37°C. Plaques were then counted after fixing the cultures with a methanol:glacial acetic acid solution (3:1) and staining with 1 % crystal violet (9).

anti-CD8 mAb (Ortho Diagnostics, Raritan, NJ), washed, and sorted on a FACS IV cell sorter (Becton-Dickmson, Mountain View, CA). The separated population was >95% viable and > 9 8 % CD8 + upon re-analysis. Clonal T cell lines were then isolated from the sorted cells using the method previously described (5). The T cell line utilized in these studies was not HSV-specific, and the surface phenotype was CD3 + , CD4", CD8 + , CD2 + , and HLA-DR + . HSV replication in neonatal mice Three day old suckling B10.M (H-21) mice were randomly assigned to a treatment group and inoculated i.p. with PBS alone

Survival in HSV-infected neonatal mice

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Statistical analysis Splenic HSV titers, after logarithmic transformation (to normalize the data), were compared by using two-tailed r-tests. Survival rates at indicated time points were compared using two-tailed Fisher's exact test.

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Fig. 1. Inhibition of HSV replication in mice treated with T cells and heteroconjugate antibodies In two experiments (A and B), neonatal B10 M mice were injected i.p. with either PBS alone (sham-treatment), CD8 + human T cells alone (4 x 104/mouse), anti-CD3 x anti-HSV-1 gC heteroconjugate antibody alone (0.06 ^g/mouse), or T cells and heteroconjugate antibody. In both experiments measurement of HSV titers in the spleens indicated that treatment with the combination of T cells and heteroconjugate antibody reduced HSV viral replication (P < 0.006 and P < 0.004, respectively).

Results and discussion Reduced viral replication in mice treated with heteroconjugate antibodies and T cells The heteroconjugate antibodies evaluated in these experiments consisted of anti-CD3 mAb, capable of broadly stimulating effector T cells, covalently coupled to anti-HSV mAb, which specifically binds HSV-1 envelope gC or gD Since both gC and gD are required for HSV-1 infectivity (10) and since

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Post-HSV Inoculation, days Fig. 2. Prolonged survival in mice treated with T cells and heteroconjugate antibodies. (A) Neonatal B10.M mice were injected i.p. with either PBS alone (sham-treatment) or CD8 + human T cells (4 x Iff'/mouse) and anti-CD3 x anti-HSV-1 gC heteroconjugate antibody (0.12 /ig/mouse). After injection with HSV (10 p.f.u./mouse), survival was increased in the mice receiving the combined T cell/heteroconjugate antibody therapy (P

Heteroconjugate antibodies enhance cell-mediated anti-herpes simplex virus immunity in vivo.

Heteroconjugate antibodies are generated by covalently linking two mAbs with different specificities. When anti-CD3 mAb, capable of activating effecto...
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