Vaccines prepared with sialyl-Tn and sialyl-Tn trimers using the 4-(4-maleimidomethyl)cyclohexane-1-carboxyl hydrazide linker group result in optimal antibody titers against ovine submaxillary mucin and sialyl-Tn-positive tumor cells.
Sialyl-Tn (STn) is an O-serine- or O-threonine-linked disaccharide [NeuAcalpha(2-->6)GalNAcalpha-O-Ser/Thr) expressed on mucins of most types of adenocarcinoma as single STn or clustered STn [STn(c)] epitopes. Though STn is expressed on some normal tissues it is relatively tumor-specific, especially in the clustered conformation. Clinical trials with STn-keyhole limpet hemocyanin (KLH) conjugate vaccines, prepared using reductive amination with a two-carbon linker group, have resulted in high titers against STn but lower titers against natural forms of STn (ovine submaxillary mucin, or tumor cells). To obtain antibodies of more appropriate specificity, we attempted to prepare STn(c)-KLH conjugates to establish their immunogenicity in mice in preparation for clinical trials; however, conjugation efficiency was poor when the same two-carbon linker was used, presumably because of steric hindrance. STn-KLH and STn(c)-KLH conjugates were prepared using the regular two-carbon or the recently developed more efficient longer heterobifunctional 4-(4-maleimidomethyl)cyclohexane-1-carboxyl hydrazide (MMCCH) linkers, and the resulting immunogenicities in mice were compared. The highest titers against STn were seen with the STn-KLH conjugate with the two-carbon linker, and the highest titers against STn(c) were seen with STn(c)-KLH with the MMCCH linker. Conjugation with MMCCH resulted in the highest conjugation efficiency (yield) and the highest titers against ovine submaxillary mucin and STn-positive tumor cells, and is the method of choice for the preparation of STn(c) vaccine for clinical trials.
Ragupathi
G
G
Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA. ragupatg@mskcc.org
Howard
L
L
Cappello
S
S
Koganty
R R
RR
Qiu
D
D
Longenecker
B M
BM
Reddish
M A
MA
Lloyd
K O
KO
Livingston
P O
PO
D000818:Animals; D000912:Antibodies, Neoplasm / Q000096:biosynthesis* / Q000097:blood; D015295:Antigens, Tumor-Associated, Carbohydrate / Q000276:immunology*; D019496:Cancer Vaccines / Q000138:chemical synthesis / Q000276:immunology* / Q000494:pharmacology; D002240:Carbohydrate Sequence; D003432:Cross-Linking Reagents / Q000737:chemistry*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006433:Hemocyanins / Q000276:immunology / Q000494:pharmacology; D018796:Immunoconjugates / Q000737:chemistry / Q000276:immunology* / Q000494:pharmacology; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000097:blood; D007075:Immunoglobulin M / Q000096:biosynthesis / Q000097:blood; D008301:Maleimides / Q000737:chemistry*; D051379:Mice; D008969:Molecular Sequence Data; D009077:Mucins / Q000276:immunology*; D009374:Neoplasms, Experimental / Q000276:immunology* / Q000378:metabolism / Q000628:therapy; D012756:Sheep; D013363:Submandibular Gland / Q000276:immunology / Q000378:metabolism
C113054:4-(4-N-maleimidomethyl)cyclohexane-1-carboxyl hydrazide; D000912:Antibodies, Neoplasm; D015295:Antigens, Tumor-Associated, Carbohydrate; D019496:Cancer Vaccines; D003432:Cross-Linking Reagents; D018796:Immunoconjugates; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D008301:Maleimides; D009077:Mucins; C055491:sialosyl-Tn antigen; D006433:Hemocyanins; C032808:keyhole-limpet hemocyanin