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. Author manuscript; available in PMC: 2022 Nov 2.
Published in final edited form as: Angew Chem Int Ed Engl. 2021 Oct 4;60(45):24179–24188. doi: 10.1002/anie.202108610

Chemoenzymatic Synthesis of 9NHAc-GD2 Antigen to Overcome the Hydrolytic Instability of O-Acetylated-GD2 for Anticancer Conjugate Vaccine Development

Xuanjun Wu 1,2,3, Jinfeng Ye 1, Andrew T DeLaitsch 4, Zahra Rashidijahanabad 2,3, Shuyao Lang 2,3, Tayeb Kakeshpour 2, Yuetao Zhao 2,3,5, Sherif Ramadan 2,3,6, Paulo Vilar Saavedra 7, Vilma Yuzbasiyan-Gurkan 8, Herbert Kavunja 2,9, Hongzhi Cao 1, Jeffrey C Gildersleeve 4, Xuefei Huang 2,3,10
PMCID: PMC8545922  NIHMSID: NIHMS1739548  PMID: 34469031

Abstract

Ganglioside GD2 is an attractive tumor-associated carbohydrate antigen for anti-cancer vaccine development. However, its low immunogenicity and the significant side effects observed with anti-GD2 antibodies present significant obstacles for vaccines. To overcome these, a new GD2 derivative bearing an N-acetamide (NHAc) at its non-reducing end neuraminic acid (9NHAc-GD2) has been designed to mimic the 9-O-acetylated-GD2 (9OAc-GD2), a GD2 based antigen with a restricted expression on tumor cells. 9NHAc-GD2 was synthesized efficiently via a chemoenzymatic method and subsequently conjugated with a powerful carrier bacteriophage Qβ. Mouse immunization with the Qβ−9NHAc-GD2 conjugate elicited strong and long-lasting IgG antibodies, which were highly selective toward 9NHAc-GD2 with little cross-recognition of GD2. Immunization of canines with Qβ−9NHAc-GD2 showed the construct was immunogenic in canines with little adverse effects, paving the way for future clinical translation to humans.

Keywords: cancer, glycoconjugates, 9NHAc-GD2, synthesis design, vaccine

Graphical Abstract

graphic file with name nihms-1739548-f0001.jpg

A potential anticancer vaccine was produced by the conjugation of bacteriophage Qβ virus-like particle with the chemoenzymatically synthesized 9-NHAc-GD2 mimic of the 9-O-acetylated GD2 antigen. The conjugate produced robust and long-lasting IgG responses in mice and canines with no observed adverse effects.

INTRODUCTION

Anti-cancer vaccines are exciting strategies to pursue for cancer treatment and prevention.[1–2] For a successful vaccine, antigen design and selection are critical. Disialoganglioside GD2 is an attractive tumor-associated carbohydrate antigen (TACA), which is overexpressed on a wide range of tumors such as neuroblastoma, lymphoma, melanoma, and osteosarcoma.[3–6] With the recognition of the important roles of GD2 in enhanced cell proliferation, adhesion, and invasion by tumor cells, an anti-GD2 monoclonal antibody (mAb), Dinutuximab, has been developed as the FDA approved first-line therapy against high-risk neuroblastoma,[7] suggesting that if sufficient levels of anti-GD2 antibodies can be achieved, patients can have a better prognosis. However, a significant concern of GD2 based antibody therapy is that anti-GD2 antibodies are frequently associated with serious side effects including severe neuropathic pain and perceived pain in response to touch (allodynia).[8–10] This is presumably due to the recognition of low levels of GD2 on normal tissues such as peripheral nerves by the antibodies at the therapeutic concentrations.

Parallel to anti-GD2 mAb development, GD2 has been investigated as a potential anti-cancer vaccine.[11–13] As GD2 is only weakly immunogenic, even repeated administration of GD2 can only lead to low levels of low affinity IgM antibodies. To enhance antibody production, GD2 has been conjugated with an immunogenic protein carrier, the keyhole limpet hemocyanin (KLH), and transformed to the lactone form, which successfully induced anti-GD2 IgG antibodies.[14] Phase I clinical trial of the KLH-GD2 conjugate as part of a bivalent vaccine showed encouraging outcomes and safety in 15 neuroblastoma patients.[11] In phase II clinical trials, higher anti-GD2 IgG titers were associated with improved survival of patients.[12] Thus, there is a conundrum that high levels of anti-GD2 antibodies need to be induced by vaccination to enhance treatment outcome, while at the same time there are concerns regarding the potential adverse effects that may emerge as encountered in anti-GD2 antibody therapy.

As an alternative to targeting GD2, acetylated GD2 may be appealing since the hydroxyl groups of gangliosides can be acetylated in nature. For example, the GD2 antigen can bear an acetyl group at the 9-O position of its non-reducing end N-acetyl neuraminic acid (Neu5Ac). Similar to GD2, the 9-O acetylated GD2 (9OAc-GD2) is expressed highly on GD2-positive tumor cells.[15–18] Importantly, it is not found on human peripheral nerve fibers,[19] suggesting that anti-9OAc-GD2 antibodies have the potential to be less toxic compared to the corresponding anti-GD2 antibodies. Therefore, 9OAc-GD2 can be an attractive antigenic target for anti-cancer vaccine design. However, to date, no such vaccines have been reported.

Targeting 9OAc-GD2 in vaccine development can be a challenging task. Synthesis of OAc-GD2 is not trivial, which has not been accomplished to date. It is known that the 9-OAc group is labile even at neutral pH, which tends to migrate to other OH groups such as the 7-OH of the neuraminic acid or be cleaved to release the free hydroxyl group.[20] In order to address this issue, we hypothesize that the GD2 derivative bearing an N-acetamide at the C-9 position (9NHAc-GD2) can be a suitable surrogate of 9OAc-GD2. If anti-9NHAc-GD2 antibodies can be induced through vaccination, they can potentially be highly selective toward tumor cells reducing the adverse effects associated with anti-GD2 antibodies. In order to elicit powerful and long-lasting IgG responses against 9NHAc-GD2, a suitable protein carrier needs to be utilized to conjugate with the antigen. Herein, we report the first synthesis of 9NHAc-GD2 glycan via an efficient chemoenzymatic strategy and its conjugation to a virus-like particle bacteriophage Qβ as a potential anti-cancer vaccine (Figure 1).

Figure 1.

Figure 1.

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Schematic diagram of the proposed Qβ−9NHAc-GD2 conjugate for eliciting robust IgG antibodies against 9OAc-GD2 positive cancer.

RESULTS AND DISCUSSION

Chemoenzymatic synthesis of GD2 glycan 1 and 9NHAc-GD2 glycan 3, and conformational analysis of 9NHAc-GD2 vs 9OAc-GD2

To facilitate the vaccine design, we started with the synthesis of GD2 pentasaccharide 1 (Scheme 1) via chemical glycosylation following a similar route as our prior synthesis of GM1 and GM2 glycans.[21–22] However, significant difficulties were encountered in the stereoselective formation of the neuraminic acid-α2,8-neuraminic acid linkage in GD2. As a result, we switched to a chemoenzymatic strategy for the synthesis.

Scheme 1.

Scheme 1.

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The structures of GD2 glycan 1, 9OAc-GD2 glycan 2, and 9NHAc-GD2 glycan 3.

The chemoenzymatic synthesis of GD2 started from the chemically prepared azidopropyl functionalized lactoside (LacβProN3) 4,[23] which was treated with Pasteurella multocida α2,3-sialyltransferase (PmST1),[23] Neisseria meningitidis CMP-sialic acid synthetase (NmCSS)[24] in the presence of cytidine-5’-triphosphate (CTP) and Neu5Ac at pH 8.5 forming trisaccharide Neu5Acα2,3-LacβProN3 (GM3-ProN3) 5 in 92% yield. The GM3-ProN3 5 was incubated with Campylobacter jejuni α2,8-sialyltransferase (CjCstII), NmCSS, Neu5Ac, and CTP leading to GD3 tetrasaccharide 6 (GD3-ProN3) in 80% yield (Scheme 2). GD3-ProN3 6 was subject to Campylobacter jejuni β−1,4-N-acetylgalactosaminyltransferase (CjCgtA),[25] N-acetylgalactosamine (GalNAc), adenosine triphosphate (ATP), uridine triphosphate (UTP) and NahK/GlmU, a fusion enzyme of Bifidobacterium longum N-acetylhexosamine-1-kinase (BlNahK) and Escherichia coli N-acetylglucosamine1-phosphate uridyl transferase (EcGlmU), which successfully installed the GalNAc moiety forming the azide bearing GD2 pentasaccharide 7, the azido group of which was reduced via catalytic hydrogenolysis producing the GD2 amine 1 (Scheme 2).

Scheme 2.

Scheme 2.

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Synthesis of GD2 amine 1. Reagents and conditions: (a) 4 (1.0 equiv), Neu5Ac (1.2 equiv), CTP (1.5 equiv), MgCl2 (20 mM), Tris-HCl buffer (100 mM, pH 8.5), NmCSS, PmST1, 37 °C, 2 h; (b) 5 (1.0 equiv), Neu5Ac (1.2 equiv), CTP (1.5 equiv), MgCl2 (20 mM), Tris-HCl buffer (100 mM, pH 8.0), NmCSS, CjCstⅡ, 37 °C, 2 h; (c) 6 (1.0 equiv), GalNAc (1.2 equiv), ATP (1.3 equiv), UTP (1.3 equiv), MgCl2 (20 mM), Tris-HCl buffer (100 mM, pH 7.5), NahK/GlmU, CjCgtA, 37 °C, 12 h; (d) Pd(OH)2/C, H2, CH3OH/H2O (2:1).

Despite the successful synthesis of GD2, however, we were not able to obtain 9OAc-GD2 2 using a similar strategy, as the O-acetyl group was hydrolyzed during synthesis. This observation is consistent with the known hydrolytic instability of the O-acetyl moiety.[20] This prompted us to focus on the synthesis of 9NHAc-GD2 3. 9NHAc-Neu5Ac 8 was synthesized first,[26] and subsequently converted to CMP-9NHAc-Neu5Ac by NmCSS in the presence of CTP and MgCl2 at pH 8.5. CjCstII can well tolerate the modification at the C-9 position as incubation of 9NHAc-Neu5Ac 8, and GM3-ProN3 5 with CjCstII generated 9NHAc-GD3 9 in 91% yield (Scheme 3). The transfer of GalNAc to 9NHAc-GD3 9 by CjCgtA with subsequent catalytic hydrogenolysis yielded the 9NHAc-GD2 amine 3 (Scheme 3). Unlike 9OAc-GD2, the 9NHAc-GD2 3 was stable with prolonged storage in buffers without losing the acetyl moiety.

Scheme 3.

Scheme 3.

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Synthesis of 9NHAc-GD2 amine 3. Reagents and conditions: (a) 5 (1.0 equiv), 9NHAc-Neu5Ac 8 (1.2 equiv), CTP (1.5 equiv), MgCl2 (20 mM), Tris-HCl buffer (100 mM pH 8.0), NmCSS, CjCstⅡ, 37 °C, 8 h; (b) 9 (1.0 equiv), GalNAc (1.2 equiv), ATP (1.3 equiv), UTP (1.3 equive), MgCl2 (20 mM), Tris-HCl buffer (100 mM pH 7.5), NahK/GlmU, CjCgtA, 37 °C, 12 h; (c) Pd(OH)2/C, H2, CH3OH/H2O (2:1).

Molecular modelling studies were performed to compare the conformations of 9OAc-GD2 2 and 9NHAc-GD2 3. Molecular dynamics simulations were first used to sample the conformational space, which was followed by quantum mechanical calculation using the B3LYP/6–31G*/SMD//PM6/C-PCM level theory for geometry optimization and energy calculation. The lowest energy conformations identified for 9OAc-GD2 and 9NHAc-GD2 were highly similar (Figure S5). This is consistent with the literature report from the Chen group that sialylated glycan analogs bearing NHAc in the neuraminic acid unit have similar 3-dimensional conformations as the corresponding OAc derivatives.[26] Furthermore, substitutions of OAc with NHAc did not significantly impact the native interactions between the sialoglycans and their protein receptors.[26]

Synthesis of Qβ-GD2 and Qβ−9NHAc-GD2 conjugates

For the induction of strong and long-lasting antibody responses, it is critical that the GD2 antigen is conjugated to an immunogenic carrier. Bacteriophage Qβ virus-like particle (VLP), a powerful carrier for tumor associated carbohydrate antigens,[27–29] was investigated in GD2 based vaccine design. The amine groups of GD2 1 and 9NHAc-GD2 3 were converted to thiocyanate GD2 11 and 9NHAc-GD2 12 with thiophosgene[30] (Scheme 4), which were then added to bacteriophage Qβ virus-like particles in K-Phos buffer (0.1 M, pH = 8) overnight at 37 °C to give Qβ-GD2 conjugate 13 and Qβ−9NHAc-GD2 conjugate 14 (Scheme 5a). Mass spectrometry analysis of the resulting particles showed that the numbers of 9NHAc-GD2/GD2 per Qβ particle were 250 on average per particle (Figure S1). To benchmark the performance of Qβ conjugates, KLH-GD2 conjugate was also synthesized, as KLH-GD2 is actively undergoing clinical trials.[12] GD2 pentasaccharide 1 was conjugated with KLH to give 15, which contained an average of 513 copies of GD2 per KLH (Scheme 5b).

Scheme 4.

Scheme 4.

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Synthesis of GD2 11 and 9NHAc-GD2 12. Reagents and conditions: a) thiophosgene, H2O/CHCl3 (2:3 v/v) 95% for 11 and 95% for 12.

Scheme 5.

Scheme 5.

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Synthesis of (a) Qβ-GD2 conjugate 13 and Qβ−9NHAc-GD2 conjugate 14, and (b) KLH-GD2 conjugate 15.

Qβ−9NHAc-GD2 conjugate 14 elicited the highest titers of IgG antibodies compared with Qβ-GD2 conjugate 13 and KLH-GD2 conjugate 15

With Qβ-GD2, Qβ−9NHAc-GD2, and KLH-GD2 conjugates in hand, immunological studies were performed. C57BL/6 mice were immunized with these conjugates (3.3 nmol of GD2) using complete Freund’s adjuvant (CFA) as the adjuvant. Two booster injections were administered to mice on days 14 and 28 with the incomplete Freund’s adjuvant (IFA). On day 35, the sera were collected. To analyze the levels of induced anti-9NHAc-GD2 antibodies and avoid the interference of anti-Qβ antibodies, the pentasaccharides 11 and 12 were conjugated with bovine serum albumin (BSA) respectively as a multivalent platform to generate BSA-GD2 conjugate 16 and BSA-9NHAc-GD2 conjugate 17 (Scheme S1). The average numbers of 11 or 12 per BSA were 4 as characterized by MALDI-TOF MS (Figure S3). Enzyme linked immunosorbent assay (ELISA) were performed using the conjugates 16 and 17 to analyze the levels of anti-9NHAc-GD2 antibodies generated in post-immune sera from mice immunized with 13 and 14. Strong anti-9NHAc-GD2 responses were elicited by 14 with a mean IgG titer of 1,012,515 ELISA units (Figure 2), while the IgM titers remained at background levels. In comparison, the mean anti-GD2 IgG titer induced by Qβ-GD2 13 was 56,646. As CFA cannot be applied in humans due to its toxicity, to facilitate future translation, MPLA, a TLR-4 agonist approved by FDA for usage in human patients to enhance immune responses,[31] was used for immunological evaluation of these conjugates. The Qβ conjugates also elicited strong anti-9NHAc-GD2/GD2 IgG responses with the MPLA adjuvant, similar to when Freund’s adjuvant was used. In contrast, only sera from 2 out of the 5 mice immunized with KLH-GD2 conjugate 15 showed appreciable anti-GD2 titers with an average IgG titer of 12,805 ELISA units (Figure 2a). The higher titers of anti-GD2 IgG induced by Qβ-GD2 than KLH-GD2 demonstrated Qβ is superior to KLH carrier for the production of anti-GD2 IgG antibodies. In addition, mice immunized with Qβ only or an admixture of Qβ with GD2 1 failed to induce anti-GD2 antibodies above the background (Figure 2a), suggesting it was critical to conjugate the GD2 antigen with the carrier covalently.

Figure 2.

Figure 2.

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(a) Titers of anti-9NHAc-GD2 IgG antibodies from mice immunized with the conjugate 14 against BSA-9NHAc-GD2 17, the conjugates 13 and 15 against BSA-GD2 conjugate 16 with either CFA/IFA or MPLA adjuvant. Each symbol represents one mouse (n = 3–9 mice for each group). (b) Changes of the titers of anti-9NHAc-GD2 IgG antibodies from Qβ−9NHAc-GD2 14 immunized mice over time. The black arrows indicate the vaccination. Mice were first immunized with three biweekly injections on days 0, 14, and 28. On day 242, a booster was administered. The IgG titers were determined with pooled sera. The statistical significance was determined through a two tailed t-test using GraphPad Prism. * p < 0.05, ** p < 0.01. ns: not significant.

An important parameter for an effective vaccine is the duration of immunity induced. The kinetics and persistence of anti-9NHAc-GD2 IgG antibodies in mice immunized with Qβ−9NHAc-GD2 14 were monitored over time (Figure 2b). Interestingly, on day 21 after the first immunization, high levels of anti-9NHAc-GD2 IgG antibodies were already produced, reaching 85% of those on day 35. On day 242, the mice received an additional booster vaccination. One week (day 249) after the booster, the average IgG antibody levels of the mice increased over 5 times compared to those on day 242, indicating the antibody responses could be boosted. On day 470, significant levels of IgG titers (30% of those of day 35) remained suggesting long lasting antibody responses were induced.

Glyco-microarray screening revealed highly selective recognition of 9NHAc-GD2 glycoform by antibodies induced by Qβ−9NHAc-GD2 conjugate 14

To gain a thorough understanding of glycan epitopes, the sera from mice immunized with the conjugates 13–15 were screened against a carbohydrate microarray.[32] This glycan array contained 738 components, which were prepared by printing a variety of glycans, O-linked glycopeptides, N-linked glycopeptides, glycolipid glycans, and glycoproteins on the slides (a full list of the library components is given in the SI Table S6). After incubation of the microarray with mouse serum and thorough washing to remove unbound antibodies, two fluorescently labeled secondary antibodies were added to the microarray to quantify the relative amounts of serum IgG and IgM antibody bound to individual array components. As there were minimal IgM signals to GD2 and related glycans, the analysis was mainly focused on IgG antibodies. IgG from Qβ−9NHAc-GD2 14 immunized mouse sera had excellent selectivity toward 9NHAc-GD2 among all 738 components on the microarray (Figure 3a). Consistent with ELISA results, these sera showed significantly stronger binding to 9NHAc-GD2 compared to Qβ-GD2 13 induced antibody binding to GD2 (Figure 3b). Only one mouse from the KLH-GD2 immunized group recognized GD2 or 9NHAc-GD2 with the other four mice not exhibiting any binding, confirming that Qβ is a superior carrier (Figure 3b).

Figure 3.

Figure 3.

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(a) Stacked bar graphs depicting the IgG signals at a serum dilution of 1:1,000 for each mouse (n = 5) immunized with Qβ−9NHAc-GD2 14 on the full array. Controls have been omitted for clarity. Antibodies to blood group A4 (BG-A4) are often seen in mice in the absence of immunization. (b) A heat map comparison of the glyco-microarray profiles of sera from KLH-GD2, Qβ-GD2, and Qβ−9NHAc-GD2 immunized mice in binding with various gangliosides, glycolipid glycans, other sialylated glycans, and GalNAc terminal glycans. For each group, the results from each individual mouse (n = 5) were shown. Data are IgG signals for sera diluted 1:1,000. The list of the library components is given in Table S6 with the full microarray results in Table S7.

Analysis of microarray binding results revealed an interesting distinction in epitope profiles between IgG antibodies generated by Qβ−9NHAc-GD2 14 and Qβ-GD2 13. Sera from 4 of the 5 mice immunized with Qβ−9NHAc-GD2 14 only reacted strongly with 9NHAc-GD2 (Figure 3b). It is particularly noteworthy that the signal intensities for GD2 on the microarray were several orders of magnitude weaker (Figure 3b), highlighting the high specificity of the antibodies induced by Qβ−9NHAc-GD2 14. This bodes well for future clinical translation to avoid the potential side effects associated with the recognition of GD2 on normal cells by antibodies. Interestingly, the sera elicited by Qβ-GD2 13 exhibited binding to both GD2 and 9NHAc-GD2 antigens with less differentiation of GD2 vs NHAc-GD2, and the binding to 9NHAc-GD2 was generally weaker than Qβ−9NHAc-GD2 14 induced antibodies. The anti-Qβ-GD2 13 sera had much weaker binding to GD2 tetrasaccharide 18 and GD3 tetrasaccharide 19 (Scheme 6) as compared to GD2 pentasaccharide 2. In contrast, ganglioside GM2 20, GT2 pentasaccharide 21, and GQ2 pentasaccharide 22 (Scheme 6) were recognized well by anti-Qβ-GD2 13 sera. As GM2, GD2, GT2, and GQ2 share the same reducing end GM2 tetrasaccharide structure, these results suggest that the epitopes recognized by antibodies elicited by Qβ-GD2 13 reside in the GM2 tetrasaccharide with the glucose and the GalNAc playing critical roles for antibody recognition. The lack of strong binding of GM2, GT2, and GQ2 by antibodies from Qβ−9NHAc-GD2 14 immunized mice indicates that the non-reducing end 9NHAc-Neu5Ac structure of 9NHAc-GD2 is the major epitope for these antibodies.

Scheme 6.

Scheme 6.

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The structures of GD2 tetrasaccharide 18, GD3 tetrasaccharide 19, GM2 tetrasaccharide 20, GT2 pentasaccharide 21, and GQ2 pentasaccharide 22.

Qβ−9NHAc-GD2 conjugate 14 elicited antibodies with stronger binding and higher cytotoxicity to tumor cells compared to those induced by Qβ-GD2 conjugate 13

We evaluated next the abilities of post-immune sera to recognize GD2/9OAc-GD2 expressed in the native environment, i.e., on the surface of tumor cells. EL4 tumor cells were incubated with sera from mice immunized with the conjugates 13 and 14, and IgG antibody bindings to cancer cells were analyzed using flow cytometry. As shown in Figure 4, significant enhancements in cellular binding to cancer cells were observed with 13 and 14 induced sera compared to those from control mice immunized with Qβ only, suggesting the successful induction of anti-cancer antibodies. Importantly, sera induced by Qβ−9NHAc-GD2 14 were found to bind EL4 cells stronger compared to that elicited by Qβ-GD2 13, suggesting the generation of higher levels of anti-cancer antibodies by conjugate 14.

Figure 4.

Figure 4.

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Flow cytometry analysis showed Qβ−9NHAc-GD2 14 elicited IgG antibodies with significantly stronger binding to EL4 tumor cells compared with Qβ-GD2 13 elicited antibodies. The assay was tested with 1:20 dilution of the corresponding sera. Each symbol represents one mouse (n = 5–9 mice for each group). The p values were determined through a two-tailed unpaired Student’s t-test using GraphPad Prism. * p < 0.05, ** p < 0.01.

The abilities of the post-immune sera to kill the tumor cells were measured next. Upon incubation of EL4 tumor cells (Figure 5) with sera and rabbit complement, significantly higher percentages of tumor cells were killed by Qβ−9NHAc-GD2 conjugate 14 immunized mouse sera as compared to cells treated with Qβ-GD2 conjugate 13 and Qβ immunized sera. These results are consistent with the ELISA and flow cytometry results of high levels of antibodies produced by Qβ−9NHAc-GD2 14.

Figure 5.

Figure 5.

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Sera from Qβ−9NHAc-GD2 conjugate 14 exhibited significantly higher CDC towards tumor cells compared with those of the conjugate 13 and Qβ. CDC towards EL4 cells was determined by MTS assay. Each symbol represents one mouse (n = 5 mice for each group). The p values were determined through a two-tailed t test using GraphPad Prism. * p < 0.05, *** p < 0.001.

Evaluation of immunogenicity and potential side effects of Qβ−9NHAc-GD2 conjugate 14 in canines

As a critical step towards future translation, we evaluated the Qβ−9NHAc-GD2 conjugate 14 in canines. Canines enable the evaluation of allodynia and serve as a large animal model complementing the mouse studies. Canines (n = 3) were immunized with Qβ−9NHAc-GD2 conjugate 14/MPLA biweekly four times with the age matched control animals (n = 2) immunized with Qβ/MPLA by the same schedule. Sera were drawn from the animals on day 49 after the initial immunization. As shown in Figure 6, Qβ−9NHAc-GD2 conjugate 14 elicited significantly higher levels of anti-9NHAc-GD2 IgG antibodies compared to the Qβ control suggesting that Qβ−9NHAc-GD2 14 is immunogenic in canines. Allodynia analysis was performed on the animals, which showed there were no significant changes in pain tolerance following immunization (Table S1). Furthermore, hematology analysis and serum chemistry analysis of immunized animals demonstrated no significant adverse effects associated with immunization (Tables S2–S5). These results indicate that Qβ-NHAc-GD2 14 is immunogenic and safe in a large animal model, which paves the way for future translation to humans.

Figure 6.

Figure 6.

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Titers of anti-9NHAc-GD2 IgG antibodies from Qβ−9NHAc-GD2 conjugate 14 in canines. Each symbol represents one canine (n = 2–3 canines for each group). The statistical significance was determined through a two-tailed t-test using GraphPad Prism. ** p < 0.01.

Conclusions

Due to its important role in tumor development, high levels of expression on cancer cell surfaces, and clinical successes of anti-GD2 mAb, GD2 is an attractive antigen for cancer vaccine development.[3–6] However, its low immunogenicity and the neurological pain observed upon administration of anti-GD2 mAb are some of the significant obstacles to overcome for successful anti-cancer vaccines. 9-OAc-GD2 is an appealing alternative to GD2 as the expression of 9-OAc GD2 is restricted to tumor cells.[19] Since the OAc moiety in sialosides is known to be unstable,[20] which is also corroborated in our studies where the antigen lost the OAc during 9-OAc-GD2 synthesis, it is challenging to apply 9-OAc GD2 as the antigen. To solve this problem, we explored the 9-NHAc-GD2 as the surrogate for vaccine design.

While chemical synthesis was challenging, the 9-NHAc-GD2 3 and GD2 1 bearing a reducing end amine group for conjugation were successfully synthesized via a chemoenzymatic strategy. To boost the antibody responses, 9-NHAc-GD2 and GD2 were conjugated with a powerful protein carrier, bacteriophage Qβ. To benchmark the performance, GD2 was also conjugated to KLH since a KLH-GD2 construct has been evaluated in phase II clinical trials of cancer patients. Head-to-head immunization studies of Qβ- and KLH-GD2 conjugates showed that the Qβ construct elicited significantly stronger anti-GD2 IgG antibody responses compared to the corresponding benchmark KLH conjugate, demonstrating the superiority of Qβ as a carrier for anti-cancer vaccine development.

Mice immunized with Qβ−9NHAc-GD2 were able to generate significantly higher levels of IgG antibodies as compared to those with Qβ-GD2 suggesting the NHAc substitution can enhance its antigenicity. The antibody responses induced were long lasting and boostable. Furthermore, the antibodies produced by Qβ−9NHAc-GD2 were able to bind stronger with GD2 positive tumor cells and were much more cytotoxic against the tumor cells. Glycan microarray analysis revealed interesting epitope profiles in mice immunized with Qβ−9NHAc-GD2 vs those with Qβ-GD2. The antibodies produced by Qβ−9NHAc-GD2 were highly selective for 9NHAc-GD2 binding and the main epitopes residing in the non-reducing end 9NHAc-Neu5Ac, while Qβ-GD2 induced antibodies recognized reducing end part of the GD2 structure including the reducing end glucose unit.

To facilitate future translation of the vaccine candidate, canines were immunized with Qβ−9NHAc-GD2. IgG antibodies were generated from the immunization suggesting Qβ−9NHAc-GD2 is immunogenic in canines. There were no significant changes in pain tolerance, serum chemistry, or hematology, highlighting the safety of Qβ−9NHAc-GD2 immunization. This was the first time a Qβ glycan conjugate has been evaluated in a canine model, indicating its high potential for translation to human patients.

O-Acetylation is a common modification of naturally existing glycans, including those on tumor cells and microbial glycans. Our results of 9NHAc-GD2 suggest that the NHAc may be a suitable surrogate of the hydrolytically unstable OAc. This can open up a new direction for vaccine design targeting O-acetylated carbohydrate antigens.

Supplementary Material

Supporting info

Acknowledgments

We are grateful to the National Cancer Institute, National Institutes of Health (Grant R01 CA225105 and the Intramural Research Program), Michigan State University Foundation, the National Science Foundation (1913654), and the National Natural Science Foundation of China (22007058, 21961142016, 21877073) for financial support of our work. We thank the Consortium for Functional Glycomics (GM62116; the Scripps Research Institute), T. Tolbert (University of Kansas), L.-X. Wang (University of Maryland), J. Barchi (National Cancer Institute), T. Lowary (University of Alberta), Omicron Biochemicals Inc., GlycoHub, and Glycan Therapeutics for generously contributing glycans for the array.

Footnotes

Xuefei Huang is the founder of Iaso Therapeutics Inc., which is dedicated to the development of next generation of vaccines, including GD2 based anti-cancer vaccines. Herbert Kavunja is currently an employee of Iaso Therapeutics.

Supporting Information: The Supporting Information includes detailed experimental procedures, characterization data and spectra, supplementary figures, and supplementary schemes.

References:

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Supplementary Materials

Supporting info
NIHMS1739548-supplement-Supporting_info.pdf (8.9MB, pdf)

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