Developments in CRM197 Glycoconjugates for Anticancer Vaccines

The concept of using glycoconjugates to induce immunological responses to a carbohydrate hapten was reported in 1929 by Avery and Goebel [1]. However, glycoconjugate vaccines would not be revisited as therapeutic and prophylactic agents against bacterial infections until the 1970’s and early 1980’s [2-4]. Prior to these interests in glycoconjugates, licensed antibacterial vaccines consisted of isolated capsular polysaccharides which were efficacious in adults but failed to induce protection in high risk populations such as infants and children [5,6]. Glycoconjugate vaccines consist of a bacterial capsular polysaccharide conjugated to an immunogenic carrier molecule such as keyhole limpet hemocyanin (KLH), diptheria toxin (DT), tetanus toxoid (TT), cross-reactive material 197 (CRM197), as well as others [7-9]. This conjugate vaccine strategy is necessary due to the T cell independent nature of carbohydrates that have nominally elicited low affinity, short lived IgM antibodies with the exception of zwitterionic polysaccharides (ZPSs) [10-13]. Glycoconjugates are able to effectively bind to the MHCI and/or MHCII molecule on antigen presenting cells (APCs). This cellular interaction with the carbohydrate conjugate allows for the development of T cell mediated responses, and consequently leads to helper T cells (Th) which induce antibody isotype switching and immunological memory [14].


Introduction
The concept of using glycoconjugates to induce immunological responses to a carbohydrate hapten was reported in 1929 by Avery and Goebel [1]. However, glycoconjugate vaccines would not be revisited as therapeutic and prophylactic agents against bacterial infections until the 1970's and early 1980's [2][3][4]. Prior to these interests in glycoconjugates, licensed antibacterial vaccines consisted of isolated capsular polysaccharides which were efficacious in adults but failed to induce protection in high risk populations such as infants and children [5,6]. Glycoconjugate vaccines consist of a bacterial capsular polysaccharide conjugated to an immunogenic carrier molecule such as keyhole limpet hemocyanin (KLH), diptheria toxin (DT), tetanus toxoid (TT), cross-reactive material 197 (CRM197), as well as others [7][8][9].
This conjugate vaccine strategy is necessary due to the T cell independent nature of carbohydrates that have nominally elicited low affinity, short lived IgM antibodies with the exception of zwitterionic polysaccharides (ZPSs) [10][11][12][13]. Glycoconjugates are able to effectively bind to the MHCI and/or MHCII molecule on antigen presenting cells (APCs). This cellular interaction with the carbohydrate conjugate allows for the development of T cell mediated responses, and consequently leads to helper T cells (Th) which induce antibody isotype switching and immunological memory [14].
One of the more recent clinically successful carrier proteins in antibacterial glycoconjugates is CRM 197 [15]. CRM 197 is a nontoxic mutant of DT that has a single point mutation at position 52, which substitutes a glutamic acid residue with glycine [16]. The toxicity of DT is generated within a eukaryotic cell in which DT has ADP-ribosyltranferase activity towards elongation factor 2 (EF-2) causing a halt to protein synthesis, and consequently initiates cellular apoptosis. The single point mutation in CRM 197 limits this cytotoxic mechanism. Aside from being an inherently nontoxic protein, CRM 197 also lacks lysine residues within its T cell epitope, meaning chemical conjugation of haptens will not likely effect T cell interactions [17] Vaccines and Diagnostics, Inc.) [6,15].
The success of these bacterial glycoconjugate vaccines has helped develop similar strategies against certain epithelial cancers.
Complementary to bacterial pathogens, cancer cells express a unique carbohydrate fingerprint as a result of genetic mutations that effect glycosyltransferase enzymes and chaperone proteins [18]. These unique tumor-associated carbohydrate antigens (TACAs) have been investigated within many glycoconjugate vaccines in conjunction with other approaches to involve immunological stimulation [7][8][9].
Unfortunately, these investigations have had limited clinical success as compared to the bacterial glycoconjugate vaccines.

TACA-CRM 197 Glycoconjugates
One of the first preclinical works on TACA-CRM 197 conjugates was published by Perico et al. [25] which described the synthesis of Galα) of the Globo H hexasaccharide [19]. Their linker strategy began with an allyl group at the reducing end which was oxidized to a methyl ester in 3 steps. The resulting methyl ester was then reacted with ethylene diamine which was designed to react with a bifunctional bis-hydroxysuccinimidyl ester of adipic acid. This rather long linker was then used to conjugate to CRM 197 's many lysine residues resulting in an 11:1 ratio of hapten to protein. This conjugate, in comparison to CaMBr1-KLH conjugate, exhibited lower titers but was credited to give a more specific response towards  [20], an analogue of α-galactosylceramide (C1) named C34 [21][22][23][24][25], and a combination of complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA) [25]. The adjuvant C34 was used for the majority of the studies due to potent immune activating effects. As an analogue of C1, C34 targets the CD1d receptor on dendritic cells which has downstream effects to promote T cell activation, proliferation, IL-4 production, IFN-γ production, and an enhanced cytotoxicity [21]. These effects, stemmed from the CD1d-glycolipid-T cell receptor complex, promote a Th1 skew in T cell populations which are proposedly favored for anticancer responses. Another variable is the antigen loading within these glycoconjugates. Dose response curves for many successful vaccines created a Gaussian distribution with the most effective doses being within the low to medium range [26].
This was shown with Perico and coworkers as their CRM 197 conjugate gave good results at a dose of 2.5 μg rather than 0.5 μg or 12.5 μg suggesting a tolerogenic effect of the conjugate [19,20]. Similarly, Wong and coworkers demonstrated a Gaussian distribution of immunological responses when comparing the hapten to protein ratio [21,22]. Best results were observed with ratios of 5.1 and 4.7 rather than lower or higher ratios. These results suggest that there is a "Goldilocks" dosage for these glycoconjugates which also implies an immune tolerance at a high enough dose. The phenomenon involved with these observed dose response curves has been described as carrier induced epitope suppression (CIES).
CEIS involves a pre-existing immunity to the carrier which holds potential to suppress the immune response. These mechanisms may involve pre-existing antibodies that can bind to the conjugate causing steric hinderance, promotion of anti-carrier specific B cells over anti-hapten B cells, competition for resources when anti-carrier B cells over populate anti-hapten B cells thus reducing hapten specific B cells from T cell help, and the production of regulatory T cells by the carrier [27,28]. This phenomenon is clearly observed in trials pertaining to anti-bacterial glycoconjugate vaccines where vaccines are concomitantly administered or serially administered [29,30]. Due to the clinical success of these anti-bacterial glycoconjugate vaccines and because these vaccines are usually administered concomitantly, investigations on vaccine interactions and coadministrative effects have revealed both enhanced and limited efficacy of these vaccines [28]. One such study by Borrow and coworkers involved the co-administration and the sequential administration of TT and CRM 197 conjugates which showed an enhanced immune response when conjugates were coadministered rather than administered sequentially, suggesting enhanced efficacy of a vaccine in the presence of another carrier [31]. In addition, another study performed by Dagan and coworkers involved the co-administration of a pneumococcal vaccine with an Hib vaccine using only TT as a carrier or both TT and CRM 197 [29].
When both conjugate vaccines utilized TT a bystander interference effect was observed resulting in decreased efficacy. However, this effect was not observed when both TT and CRM 197 was used.

Conclusion
Although CRM 197 has had clinical success in antibacterial glycoconjugate vaccines, CRM 197 glycoconjugates against cancer has had limited success. Most efforts to increase efficacy of TACA based glycoconjugate vaccines have been focused on creating nonnatural analogues of the antigen, creating multivalent displays of a single antigen as well as multivalent displays of different antigens, discovering better adjuvants, and naturally occurring antibody recruiting epitopes [7][8][9]. However, there are limited studies that have observed co-administration effects of TACA-CRM 197 conjugates as compared to antibacterial CRM 197 glycoconjugates.
The co-administrative effects in bacterial glycoconjugate vaccines are still not well understood, but there is consensus that there are both positive and negative effects and that these effects differ in vulnerability [28]. Concomitant vaccine effects may also be translated into preclinical research involved with  conjugates where the co-administration of TACA conjugates could have a profound effect on observed immunity. Following the few examples herein, using CRM 197 in combination with another immunogenic carrier may have beneficial effects and enhance immunity towards a single hapten structure and reduce tolerogenic effects observed in TACA-CRM 197 preclinical studies [19][20][21][22]. Such examples could include the combination of KLH, TT, or ZPSs [10][11][12][13]32,33].