A preclinical study comparing approaches for augmenting...

A preclinical study comparing approaches for augmenting the immunogenicity of a heptavalent KLH-conjugate vaccine against epithelial cancers

A preclinical study comparing approaches for augmenting the immunogenicity of a heptavalent KLH-conjugate vaccine against epithelial cancers
October 2003
Received: 22 November 2002 Accepted: 28 March 2003 Published online: 17 June 2003
Govind Ragupathi1 , Fusataka Koide1, Natarajan Sathyan1, Ella Kagan1, Maria Spassova2, William Bornmann2, Polly Gregor1, Celso A. Reis4, 5, Henrik Clausen4, Samuel J. Danishefsky3 and Philip O. Livingston1
Cancer Immunology, Immunotherapy
Publisher: Springer-Verlag GmbH
(1) Laboratory of Tumor Vaccinology, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA
(2) Synthetic Core facility, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA
(3) Bioorganic Chemistry, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA
(4) School of Dentistry, University of Copenhagen, Norre Alle 20, 2200 Copenhagen N, Denmark
(5) Institute of Molecular Pathology and Immunology, IPATIMUP, University of Porto, Rua Dr Roberto Frias s/n, 4200 Porto, Portugal
Abstract Previously using a series of monovalent vaccines, we demonstrated that the optimal method for inducing an antibody response against cancer cell-surface antigens is covalent conjugation of the antigens to keyhole limpet hemocyanin (KLH) and the use of a saponin adjuvant. We have prepared a heptavalent-KLH conjugate vaccine containing the seven epithelial cancer antigens GM2, Globo H, Lewisy, TF(c), Tn(c), STn(c), and glycosylated MUC1. In preparation for testing this vaccine in the clinic, we tested the impact on antibody induction of administering the individual conjugates plus adjuvant compared with a mixture of the seven conjugates plus adjuvant, and of several variables thought to augment immunogenicity. These include approaches for decreasing suppressor cell activity or increasing helper T-lymphocyte activity (low dose cyclophosphamide or anti-CTLA-4 MAb), different saponin adjuvants at various doses (QS-21 and GPI-0100), and different methods of formulation (lyophilization and use of polysorbate 80). We find that: (1) Immunization with the heptavalent-KLH conjugate plus GPI-0100 vaccine induces antibodies against the seven antigens of comparable titer to those induced by the individual-KLH conjugate vaccines, high titers of antibodies against Tn (median ELISA titer IgM/IgG 320/10,240), STn (640/5,120), TF (320/10,240), MUC1 (80/20,480), and globo H (640/40); while lower titers of antibodies against Lewisy (160/0) and only occasional antibodies against GM2 are induced. (2) These antibodies reacted with the purified synthetic antigens by ELISA, and with naturally expressed antigens on the cancer cell surface by FACS. (3) None of the approaches for further altering the suppressor cell/helper T-cell balance nor changes to the standard formulation by lyophilization or use of polysorbate 80 had any impact on antibody titers. (4) An optimal dose of saponin adjuvant, QS-21 (50 g) or GPI-0100 (1000 g), is required for optimal antibody titers. This heptavalent vaccine is sufficiently optimized for testing in the clinic.
Keywords Conjugate vaccines - Cancer vaccines - Polyvalent vaccines - Carbohydrates - Synthetic antigens - KLH
Abbreviations Ag antigen
BSA bovine serum albumin
CTLA-4 cytotoxic T-lymphocyte associated antigen-4
ELISA enzyme-linked immunosorbent assay
FACS fluorescence activated cell sorting
FCS fetal calf serum
GM-CSF granulocyte-macrophage colony stimulating factor
HSA human serum albumin
IP intraperitoneal
KLH keyhole limpet hemocyanin
MBS m-maleimidobenzoyl-N-hydroxysuccinimide ester
MFI mean fluorescence intensity
MMCCH 4-(4-maleimidomethyl) cyclohexane-1-carboxyl hydrazide
PBS phosphate buffered saline
Philip O. Livingston is a Paid Consultant and Shareholder in Progenics Pharmaceuticals Inc., Tarrytown, NY 10591, USA and Galenica Pharmaceuticals Inc., Birmingham, AL 35344, USA.
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