Significant Impact of Immunogen Design on the Diversity of Antibodies Generated by Carbohydrate-Based Anticancer Vaccine.
ABSTRACT: Development of an effective vaccine targeting tumor associated carbohydrate antigens (TACAs) is an appealing approach toward tumor immunotherapy. While much emphasis has been typically placed on generating high antibody titers against the immunizing antigen, the impact of immunogen design on the diversity of TACA-specific antibodies elicited has been overlooked. Herein, we report that the immunogen structure can significantly impact the breadth and the magnitude of humoral responses. Vaccine constructs that induced diverse TACA-binding antibodies provided much stronger recognition of a variety of Tn positive tumor cells. Optimization of the breadth of the antibody response led to a vaccine construct that demonstrated long lasting efficacy in a mouse tumor model. After challenged with the highly aggressive TA3Ha cells, mice immunized with the new construct exhibited a statistically significant improvement in survival relative to controls (0% vs 50% survival; p < 0.0001). Furthermore, the surviving mice developed long-term immunity against TA3Ha. Thus, both the magnitude and the breadth of antibody reactivity should be considered when designing TACA-based antitumor vaccines.
Project description:Tumor associated carbohydrate antigens (TACAs) are overexpressed on tumor cells, which renders them attractive targets for anti-cancer vaccines. To overcome the poor immunogenicity of TACAs, we designed a polymer platform for antigen presentation by co-delivering TACA and helper T (Th) cell epitope on the same chain. The block copolymer was synthesized by cyanoxyl-mediated free radical polymerization followed by conjugation with a TACA Tn antigen and a mouse Th-cell peptide epitope derived from polio virus (PV) to afford the vaccine construct. The glycopolymer vaccine elicited an anti-Tn immune response with significant titers of IgG antibodies, which recognized Tn-expressing tumor cells.
Project description:Tumor-associated carbohydrate antigens (TACAs) support cell survival that could be interrupted by anti-TACA antibodies. Among TACAs that mediate cell survival signals are the neolactoseries antigen Lewis Y (LeY) and the ganglioside GD2. To induce sustained immunity against both LeY and GD2, we developed a carbohydrate mimicking peptide (CMP) as a surrogate pan-immunogen that mimics both. This CMP, referred to as P10s, is the N-terminal half of a peptide vaccine named P10s-PADRE, the C-terminal half of which (PADRE) is a Pan-T-cell epitope. A Phase I dose-escalation trial of P10s-PADRE plus adjuvant MONTANIDE™ ISA 51 VG was conducted in subjects with metastatic breast cancer to test 300 and 500 ?g/injection in two cohorts of 3 subjects each. Doses of the P10s-PADRE vaccine were administered to research participants subcutaneously on weeks 1, 2, 3, 7 and 19. Antibody responses to P10s, GD2, and LeY were measured by ELISA. The P10s-PADRE vaccine induced antibodies specifically reactive with P10s, LeY and GD2 in all 6 subjects. Serum antibodies displayed Caspase-3-dependent apoptotic functionality against LeY or GD2 expressing breast cancer cell lines. Immunization with the P10s-PADRE vaccine was well-tolerated and induced functional antibodies, and the data suggest potential clinical benefit.
Project description:For successful carbohydrate based anti-cancer vaccines, it is critical that B cells are activated to secret antibodies targeting the tumor associated carbohydrate antigens (TACAs). Despite the availability of many TACA based constructs, systematic understanding of the effects of structural features on anti-glycan antibody responses is lacking. In this study, a series of defined synthetic glyco-polymers bearing a representative TACA, i.e., the Thomsen-nouveau (Tn) antigen, have been prepared to probe the induction of early B cell activation and antibody production via a T cell independent mechanism. Valency and density of the antigen in the polymers turned out to be critical. An average of greater than 6 Tn per chain was needed to induce antibody production. Glycopolymers with 40 antigens per chain and backbone molecular weight of 450 kDa gave the strongest stimulation to B cells in vitro, which correlated well with its in vivo activity. Deviations from the desired valency and density led to decreased antibody production or even antigen specific B cell non-responsiveness. These findings provide important insights on how to modulate anti-TACA immune responses facilitating the development of TACA based anti-cancer vaccines using glycopolymers.
Project description:PURPOSE:Keyhole limpet hemocyanin (KLH) is used as a vaccine adjuvant, as a carrier protein for small haptens, and as a treatment for bladder cancer. Immunization with KLH produces antibodies to tumor-associated carbohydrate antigens (TACAs) in animals, and these antibodies have been postulated as the basis of efficacy for bladder cancer treatment. The purpose of this study was to evaluate antibody responses to KLH in humans. EXPERIMENTAL DESIGN:A carbohydrate microarray was used to profile antibody responses in 14 individuals immunized with KLH plus alum adjuvant. RESULTS:Eight out of fourteen individuals produced antibodies to at least one TACA. Increases to Lewis X, Lewis Y, GA1di, GM3, and sialyl Lewis A were observed in certain individuals, but, in general, antibody profiles were highly variable. Pre-immunization antibody levels to a subset of array antigens had a statistically significant correlation with the magnitude of the antibody response to KLH. CONCLUSIONS AND CLINICAL RELEVANCE:Antibodies to TACAs can be produced in humans, but antibody profiles differ considerably from person to person, which may contribute to variable clinical responses with KLH. Pre-treatment antibody levels to certain antigens may be useful for predicting which patients will respond favorably to KLH.
Project description:Although iron oxide magnetic nanoparticles (NPs) have been widely utilized in molecular imaging and drug delivery studies, they have not been evaluated as carriers for glycoconjugate-based anticancer vaccines. Tumor-associated carbohydrate antigens (TACAs) are attractive targets for the development of anticancer vaccines. Due to the weak immunogenicity of these antigens, it is highly challenging to elicit strong anti-TACA immune responses. With their high biocompatibilities and large surface areas, magnetic NPs were synthesized for TACA delivery. The magnetic NPs were coated with phospholipid-functionalized TACA glycopeptides through hydrophobic-hydrophobic interactions without the need for any covalent linkages. Multiple copies of glycopeptides were presented on NPs, potentially leading to enhanced interactions with antibody-secreting B cells through multivalent binding. Mice immunized with the NPs generated strong antibody responses, and the glycopeptide structures important for high antibody titers were identified. The antibodies produced were capable of recognizing both mouse and human tumor cells expressing the glycopeptide, resulting in tumor cell death through complement-mediated cytotoxicities. These results demonstrate that magnetic NPs can be a new and simple platform for multivalently displaying TACA and boosting anti-TACA immune responses without the need for a typical protein carrier.
Project description:Seasonal epidemics caused by influenza A (H1 and H3 subtypes) and B viruses are a major global health threat. The traditional, trivalent influenza vaccines have limited efficacy because of rapid antigenic evolution of the circulating viruses. This antigenic variability mediates viral escape from the host immune responses, necessitating annual vaccine updates. Influenza vaccines elicit a protective antibody response, primarily targeting the viral surface glycoprotein hemagglutinin (HA). However, the predominant humoral response is against the hypervariable head domain of HA, thereby restricting the breadth of protection. In contrast, the conserved, subdominant stem domain of HA is a potential "universal" vaccine candidate. We designed an HA stem-fragment immunogen from the 1968 pandemic H3N2 strain (A/Hong Kong/1/68) guided by a comprehensive H3 HA sequence conservation analysis. The biophysical properties of the designed immunogen were further improved by C-terminal fusion of a trimerization motif, "isoleucine-zipper", or "foldon". These immunogens elicited cross-reactive, antiviral antibodies and conferred partial protection against a lethal, homologous HK68 virus challenge in vivo. Furthermore, bacterial expression of these immunogens is economical and facilitates rapid scale-up.
Project description:Tumor-associated carbohydrate antigens (TACAs) are being actively studied as targets for antitumor vaccine development. One serious challenge was the low immunogenecity of these antigens. Herein, we report the results of using the tobacco mosaic virus (TMV) capsid as a promising carrier of a weakly immunogenic TACA, the monomeric Tn antigen. The copper(I) catalyzed azide-alkyne cycloaddition reaction was highly efficient in covalently linking Tn onto the TMV capsid without resorting to a large excess of the Tn antigen. The location of Tn attachment turned out to be important. Tn introduced at the N terminus of TMV was immunosilent, while that attached to tyrosine 139 elicited strong immune responses. Both Tn specific IgG and IgM antibodies were generated as determined by enzyme-linked immunosorbent assay and a glycan microarray screening study. The production of high titers of IgG antibodies suggested that the TMV platform contained the requisite epitopes for helper T cells and was able to induce antibody isotype switching. The antibodies exhibited strong reactivities toward Tn antigen displayed in its native environment, i.e., cancer cell surface, thus highlighting the potential of TMV as a promising TACA carrier.
Project description:Tumor associated carbohydrate antigens (TACAs) are a class of attractive antigens for the development of anti-cancer immunotherapy. Besides monoclonal antibodies and vaccines, chimeric antigen receptor (CAR) T cells and bispecific antibodies (BsAbs) targeting TACA are exciting directions to harness the power of the immune system to fight cancer. In this review, we focus on two TACAs, i.e., the GD2 ganglioside and the mucin-1 (MUC1) protein. The latest advances in CAR T cells and bispecific antibodies targeting these two antigens are presented. The roles of co-stimulatory molecules, structures of the sequences for antigen binding, methods for CAR and antibody construction, as well as strategies to enhance solid tumor penetration and reduce T cell exhaustion and death are discussed. Furthermore, approaches to reduce "on target, off tumor" side effects are introduced. With further development, CAR T cells and BsAbs targeting GD2 and MUC1 can become powerful agents to effectively treat solid tumor.
Project description:Tumor-associated carbohydrate antigens (TACA) result from the aberrant glycosylation that is seen with transformation to a tumor cell. The carbohydrate antigens that have been found to be tumor-associated include the mucin related Tn, Sialyl Tn, and Thomsen-Friedenreich antigens, the blood group Lewis related Lewis(Y), Sialyl Lewis(X) and Sialyl Lewis(A), and Lewis(X) (also known as stage-specific embryonic antigen-1, SSEA-1), the glycosphingolipids Globo H and stage-specific embryonic antigen-3 (SSEA-3), the sialic acid containing glycosphingolipids, the gangliosides GD2, GD3, GM2, fucosyl GM1, and Neu5GcGM3, and polysialic acid. Recent developments have furthered our understanding of the T-independent type II response that is seen in response to carbohydrate antigens. The selection of a vaccine target antigen is based on not only the presence of the antigen in a variety of tumor tissues but also on the role this antigen plays in tumor growth and metastasis. These roles for TACAs are being elucidated. Newly acquired knowledge in understanding the T-independent immune response and in understanding the key roles that carbohydrates play in metastasis are being applied in attempts to develop an effective vaccine response to TACAs. The role of each of the above mentioned carbohydrate antigens in cancer growth and metastasis and vaccine attempts using these antigens will be described.
Project description:Comparative immune response profiling is important for selecting next-generation vaccines. We comprehensively evaluated the antibody responses from a panel of nine respiratory vaccines against Ebola virus (EBOV) derived from human and avian paramyxoviruses expressing EBOV glycoprotein (GP). Most vaccines were protective in guinea pigs but yielded antibody repertoires that differed in proportion targeting key antigenic regions, avidity, neutralizing antibody specificities, and linear epitope preferences. Competition studies with monoclonal antibodies from human survivors revealed that some epitopes in GP targeted for neutralization were vector dependent, while EBOV-neutralizing titers correlated with the response magnitude toward the receptor-binding domain and GP1/GP2 interface epitopes. While an immunogen determines the breadth of antibody response, distinct vaccine vectors can induce qualitatively different responses, affecting protective efficacy. These data suggest that immune correlates of vaccine protection cannot be generalized for all vaccines against the same pathogen, even if they use the exact same immunogen.