Chapter 15
Oligosaccharide Antigen Conjugation to Carrier Proteins
to Formulate Glycoconjugate Vaccines
Brittany R. Smith and Zhongwu Guo
Abstract
Conjugation, that is, covalent linkage, to immunological proteins is a common strategy to address the low
immunogenicity issue of carbohydrate antigens in vaccine development. This chapter describes an easy and
efficient method for oligosaccharide–protein conjugation employing dicarboxylic acid linkers. In this
regard, a free amino group is introduced to an oligosaccharide antigen to facilitate coupling with the
bifunctional linker upon reaction with its corresponding disuccinimidyl ester. The resultant monosuccinimidyl ester of the oligosaccharide antigen then reacts with the free amino groups of a carrier protein to
provide the desired oligosaccharide–protein conjugate.
Key words Oligosaccharide, Carrier protein, Dicarboxylic acid linker, Conjugation, Glycoconjugate,
Vaccine
1 Introduction
Tumor-associated carbohydrate antigens (TACAs) are promising
target molecules for the development of therapeutic cancer vaccines since TACAs are overexpressed on the cancer cell surface
[1, 2]. However, on their own, TACAs are poorly immunogenic.
To overcome this limitation, TACAs are usually conjugated to an
immunostimulatory carrier molecule to improve their immunogenicity [3–5]. The carrier molecule is usually a protein, such as
keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA),
ovalbumin (OVA), or human serum albumin (HSA) [3–5]. Alternatively, it can be a synthetic small molecule, such as lipids (e.g.,
monophosphoryl lipid A [MPLA] [6]).
Many methods have been developed for conjugating TACAs to
carrier molecules [7, 8]. Among them, one of the most common is
through alkylation and acylation of the free amino groups of carrier
proteins. In this regard, the commonly utilized linkers include
alkylating diethyl squarate [9, 10] and acylating dicarboxylic anhydrides (succinic anhydride, glutaric anhydride) [11] or activated
Blaine A. Pfeifer and Andrew Hill (eds.), Vaccine Delivery Technology: Methods and Protocols, Methods in Molecular Biology,
vol. 2183, https://doi.org/10.1007/978-1-0716-0795-4_15, © Springer Science+Business Media, LLC, part of Springer Nature 2021
305
Oligosaccharide Antigen Conjugation to Carrier Proteins
to Formulate Glycoconjugate Vaccines
Brittany R. Smith and Zhongwu Guo
Abstract
Conjugation, that is, covalent linkage, to immunological proteins is a common strategy to address the low
immunogenicity issue of carbohydrate antigens in vaccine development. This chapter describes an easy and
efficient method for oligosaccharide–protein conjugation employing dicarboxylic acid linkers. In this
regard, a free amino group is introduced to an oligosaccharide antigen to facilitate coupling with the
bifunctional linker upon reaction with its corresponding disuccinimidyl ester. The resultant monosuccinimidyl ester of the oligosaccharide antigen then reacts with the free amino groups of a carrier protein to
provide the desired oligosaccharide–protein conjugate.
Key words Oligosaccharide, Carrier protein, Dicarboxylic acid linker, Conjugation, Glycoconjugate,
Vaccine
1 Introduction
Tumor-associated carbohydrate antigens (TACAs) are promising
target molecules for the development of therapeutic cancer vaccines since TACAs are overexpressed on the cancer cell surface
[1, 2]. However, on their own, TACAs are poorly immunogenic.
To overcome this limitation, TACAs are usually conjugated to an
immunostimulatory carrier molecule to improve their immunogenicity [3–5]. The carrier molecule is usually a protein, such as
keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA),
ovalbumin (OVA), or human serum albumin (HSA) [3–5]. Alternatively, it can be a synthetic small molecule, such as lipids (e.g.,
monophosphoryl lipid A [MPLA] [6]).
Many methods have been developed for conjugating TACAs to
carrier molecules [7, 8]. Among them, one of the most common is
through alkylation and acylation of the free amino groups of carrier
proteins. In this regard, the commonly utilized linkers include
alkylating diethyl squarate [9, 10] and acylating dicarboxylic anhydrides (succinic anhydride, glutaric anhydride) [11] or activated
Blaine A. Pfeifer and Andrew Hill (eds.), Vaccine Delivery Technology: Methods and Protocols, Methods in Molecular Biology,
vol. 2183, https://doi.org/10.1007/978-1-0716-0795-4_15, © Springer Science+Business Media, LLC, part of Springer Nature 2021
305