Protective Epitope Discovery and Design of MUC1-based Vaccine for Effective Tumor Protections in Immunotolerant Mice.

Human mucin-1 (MUC1) is a highly attractive antigen for the development of anticancer vaccines. However, in human clinical trials of multiple MUC1 based vaccines, despite the generation of anti-MUC1 antibodies, the antibodies often failed to exhibit much binding to tumor presumably due to the challenges in inducing protective immune responses in the immunotolerant environment. To design effective MUC1 based vaccines functioning in immunotolerant hosts, vaccine constructs were first synthesized by covalently linking the powerful bacteriophage Qß carrier with MUC1 glycopeptides containing 20-22 amino acid residues covering one full length of the tandem repeat region of MUC1. However, IgG antibodies elicited by these first generation constructs in tolerant human MUC1 transgenic (Tg) mice did not bind tumor cells strongly. To overcome this, a peptide array has been synthesized. By profiling binding selectivities of antibodies, the long MUC1 glycopeptide was found to contain immunodominant but nonprotective epitopes. Critical insights were obtained into the identity of the key protective epitope. Redesign of the vaccine focusing on the protective epitope led to a new Qß-MUC1 construct, which was capable of inducing higher levels of anti-MUC1 IgG antibodies in MUC1.Tg mice to react strongly with and kill a wide range of tumor cells compared to the construct containing the gold standard protein carrier, i.e., keyhole limpet hemocyanin. Vaccination with this new Qß-MUC1 conjugate led to significant protection of MUC1.Tg mice in both metastatic and solid tumor models. The antibodies exhibited remarkable selectivities toward human breast cancer tissues, suggesting its high translational potential.

Delivery of foreign cytotoxic T lymphocyte epitopes to tumor tissues for effective antitumor immunotherapy against pre-established solid tumors in mice.

Cytotoxic T lymphocyte (CTL) can have remarkable abilities to kill tumor cells. However, the establishment of successful CTL-based anticancer therapy has met with many challenges. Within tumor cells, there exist subpopulations with low or no expression of the targeted antigen (termed as antigen-loss variants). In addition, tumor cells can downregulate the levels of major histocompatibility complex class I (MHC-I) molecules on cell surface due to immune pressure. As a result, some tumor cells can escape the immune pressure bestowed by CTLs, resulting in treatment failure. To address these difficulties, a new approach is developed to deliver foreign high-affinity CTL epitopes to tumor tissues utilizing pH-responsive "smart" microparticles (MPs). These MPs could encapsulate CTL peptide epitope, release the peptide under acidic condition encountered in tumor tissues and enhance CTL activation. Mice bearing pre-established tumor as "antigen-loss variant" solid tumor models were administered intratumorally with MPs containing the CTL peptide, which showed 100% survival following the treatment. In contrast, all control mice died from tumor. Significant protection from tumor-induced death was also observed with systemic administration of CTL peptide-MPs. The therapeutic efficacy can be attributed to enhanced delivery of the epitope to tumor tissues, presentation of the epitope by tumor cells as well as tumor stromal cells and/or generation of epitope-specific CTLs by the peptide-containing MPs. These findings offer a promising new direction for treating established solid tumor using CTL therapy.

Chemical Synthesis of GM2 Glycans, Bioconjugation with Bacteriophage Qß, and the Induction of Anticancer Antibodies.

The development of carbohydrate-based antitumor vaccines is an attractive approach towards tumor prevention and treatment. Herein, we focused on the ganglioside GM2 tumor-associated carbohydrate antigen (TACA), which is overexpressed in a wide range of tumor cells. GM2 was synthesized chemically and conjugated with a virus-like particle derived from bacteriophage Qß. Although the copper-catalyzed azide-alkyne cycloaddition reaction efficiently introduced 237 copies of GM2 per Qß, this construct failed to induce significant amounts of anti-GM2 antibodies compared to the Qß control. In contrast, GM2 immobilized on Qß through a thiourea linker elicited high titers of IgG antibodies that recognized GM2-positive tumor cells and effectively induced cell lysis through complement-mediated cytotoxicity. Thus, bacteriophage Qß is a suitable platform to boost antibody responses towards GM2, a representative member of an important class of TACA: the ganglioside.

Nonclinical immunogenicity risk assessment for knobs-into-holes bispecific IgG1 antibodies.

Bispecific antibodies, including bispecific IgG, are emerging as an important new class of antibody therapeutics. As a result, we, as well as others, have developed engineering strategies designed to facilitate the efficient production of bispecific IgG for clinical development. For example, we have extensively used knobs-into-holes (KIH) mutations to facilitate the heterodimerization of antibody heavy chains and more recently Fab mutations to promote cognate heavy/light chain pairing for efficient in vivo assembly of bispecific IgG in single host cells. A panel of related monospecific and bispecific IgG1 antibodies was constructed and assessed for immunogenicity risk by comparison with benchmark antibodies with known low (Avastin and Herceptin) or high (bococizumab and ATR-107) clinical incidence of anti-drug antibodies. Assay methods used include dendritic cell internalization, T cell proliferation, and T cell epitope identification by in silico prediction and MHC-associated peptide proteomics. Data from each method were considered independently and then together for an overall integrated immunogenicity risk assessment. In toto, these data suggest that the KIH mutations and in vitro assembly of half antibodies do not represent a major risk for immunogenicity of bispecific IgG1, nor do the Fab mutations used for efficient in vivo assembly of bispecifics in single host cells. Comparable or slightly higher immunogenicity risk assessment data were obtained for research-grade preparations of trastuzumab and bevacizumab versus Herceptin and Avastin, respectively. These data provide experimental support for the common practice of using research-grade preparations of IgG1 as surrogates for immunogenicity risk assessment of their corresponding pharmaceutical counterparts.

A carbohydrate-based approach for the total synthesis of (-)-dinemasone B, (+)-4a-epi-dinemasone B, (-)-7-epi-dinemasone B, and (+)-4a,7-Di-epi-dinemasone B.

(-)-Dinemasone B was isolated by Krohn and co-workers from a culture of the endophytic fungus Dinemasporium strigosum and has shown promising antimicrobial activity. Described herein is the first total synthesis of (-)-dinemasone B, (+)-4a-epi-dinemasone B, (-)-7-epi-dinemasone B, and (+)-4a,7-di-epi-dinemasone B. Their absolute configurations were also determined. The developed synthesis features a stereoselective reduction of C-glycosidic ketone, lactonization, and E-olefination of aldehyde starting from D-glucose.

Development of a Cell-based Neutralizing Antibody Assay for Zinpentraxin Alfa: Challenges and Mitigation Strategies.

Therapeutic protein drugs can potentially induce immune responses in patients and result in the production of anti-drug antibodies (ADAs), including a subset of ADAs called neutralizing antibodies (NAbs) that might cause loss of efficacy by inhibiting clinical activities of the drug. Herein, we describe the unique challenges encountered during the development of a fit-for-purpose cell-based NAb assay for a new protein modality, zinpentraxin alfa, including our strategies for assay design to overcome various matrix interferences and improve assay drug tolerance. We demonstrated that a typical biotin-drug extraction with acid dissociation (BEAD) approach alone was not sufficient to eliminate matrix interferences in this assay. Instead, the combination of the BEAD and ZebaTM spin size exclusion plate (SEP) was required to achieve the desirable assay performance. We also demonstrated that appropriate acidic buffers were critical in sample pretreatment to improve assay drug tolerance, which not only dissociated the drug/NAb immune complex but also effectively and irreversibly denatured the free drug. The final assay performed well with confirmed assay robustness and suitability for the clinical applications.

Tobacco mosaic virus as a new carrier for tumor associated carbohydrate antigens.

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.

Structure Guided Design of Bacteriophage Qß Mutants as Next Generation Carriers for Conjugate Vaccines.

Vaccines are critical tools to treat and prevent diseases. For an effective conjugate vaccine, the carrier is crucial, but few carriers are available for clinical applications. In addition, a drawback of current protein carriers is that high levels of antibodies against the carrier are induced by the conjugate vaccine, which are known to interfere with the immune responses against the target antigen. To overcome these challenges, we obtained the near atomic resolution crystal structure of an emerging protein carrier, i.e., the bacteriophage Qß virus like particle. On the basis of the detailed structural information, novel mutants of bacteriophage Qß (mQß) have been designed, which upon conjugation with tumor associated carbohydrate antigens (TACAs), a class of important tumor antigens, elicited powerful anti-TACA IgG responses and yet produced lower levels of anticarrier antibodies as compared to those from the wild type Qß-TACA conjugates. In a therapeutic model against an aggressive breast cancer in mice, 100% unimmunized mice succumbed to tumors in just 12 days even with chemotherapy. In contrast, 80% of mice immunized with the mQß-TACA conjugate were completely free from tumors. Besides TACAs, to aid in the development of vaccines to protect against COVID-19, the mQß based conjugate vaccine has been shown to induce high levels of IgG antibodies against peptide antigens from the SARS-CoV-2 virus, demonstrating its generality. Thus, mQß is a promising next-generation carrier platform for conjugate vaccines, and structure-based rational design is a powerful strategy to develop new vaccine carriers.

Divergent heparin oligosaccharide synthesis with preinstalled sulfate esters.

Traditional chemical synthesis of heparin oligosaccharides first involves assembly of the full length oligosaccharide backbone followed by sulfation. Herein, we report an alternative strategy in which the O-sulfate was introduced onto glycosyl building blocks as a trichloroethyl ester prior to assembly of the full length oligosaccharide. This allowed divergent preparation of both sulfated and non-sulfated building blocks from common advanced intermediates. The O-sulfate esters were found to be stable during glycosylation as well as typical synthetic manipulations encountered during heparin oligosaccharide synthesis. Furthermore, the presence of sulfate esters in both glycosyl donors and acceptors did not adversely affect the glycosylation yields, which enabled us to assemble multiple heparin oligosaccharides with preinstalled 6-O-sulfates.

Active cobalt catalyst for the cleavage of benzyl ether.

An improved method for the deprotection of benzyl ethers using a catalytic amount of Co(2)(CO)(8) in the presence of Me(2)PhSiH and CO (1 atm) is described. The deprotection reaction is compatible with double bond or sulfur-containing substrates. The method also tolerates other functional groups, such as Ac, Piv, and Bz, and shows potential selectivity in perbenzylated monosaccharides.

MicroRNA-217 acts as a tumor suppressor and correlates with the chemoresistance of cervical carcinoma to cisplatin.

BACKGROUND: MicroRNA-217 (miR-217) has been demonstrated to participate in the tumorigenesis and progression of various types of cancers. Nevertheless, the role of miR-217 in cervical carcinoma still remains not fully elucidated. This current work sought to investigate the role of miR-217 in the growth, migration, and invasion of cervical carcinoma and detect the role of miR-217 in the chemosensitivity of cervical carcinoma cell to cisplatin. MATERIALS AND METHODS: The levels of miR-217 in 65 pairs of cervical carcinoma tissues and matched normal tissues were detected using quantitative real-time-PCR assay. The roles of miR-217 on the growth, apoptosis, migration, and invasion of cervical cancer SiHa and Ca-Ski cells were analyzed using Cell Counting Kit-8, flow cytometry, wound healing, and Transwell invasion assays, respectively. The target of miR-217 was identified using the online analysis tool TargetScan (http://www.targetscan.org/vert_72/) and was verified by luciferase reporter and immunoblotting assays. The xenograft tumor model was constructed to explore the impact of miR-217 on the growth of cervical carcinoma cell in vivo. RESULTS: The level of miR-217 was remarkably lower in cervical carcinoma tissues than that in noncancerous tissues. Overregulation of miR-217 markedly suppressed the aggressiveness of cervical cancer cell and induced cell apoptosis through regulating V-Ki-Ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS). Finally, upregulation of miR-217 enhanced the chemosensitivity of both SiHa and Ca-Ski cervical cancer cells toward cisplatin. CONCLUSION: Altogether, upregulation of miR-217 inhibits the aggressiveness phenotypes of cervical carcinoma cell via regulating KRAS gene and increases the sensitivity of cervical cancer cell to cisplatin.

Antitumor Humoral and T Cell Responses by Mucin-1 Conjugates of Bacteriophage Qß in Wild-type Mice.

Mucin-1 (MUC1) is one of the top ranked tumor associated antigens. In order to generate effective anti-MUC1 immune responses as potential anticancer vaccines, MUC1 peptides and glycopeptides have been covalently conjugated to bacteriophage Qß. Immunization of mice with these constructs led to highly potent antibody responses with IgG titers over one million, which are among the highest anti-MUC1 IgG titers reported to date. Furthermore, the high IgG antibody levels persisted for more than six months. The constructs also elicited MUC1 specific cytotoxic T cells, which can selectively kill MUC1 positive tumor cells. The unique abilities of Qß-MUC1 conjugates to powerfully induce both antibody and cytotoxic T cell immunity targeting tumor cells bode well for future translation of the constructs as anticancer vaccines.

Design and synthesis of novel multivalent mannosides targeting the mannose receptor.

According to the characteristics of C-type lectin-like domains in the mannose receptor (MR), a novel design of multivalent mannosides targeting the MR was accomplished. Beginning with a divalent mannoside as the sugar unit, a series of multivalent mannosides with variations in both valence and space were synthesized in a convergent approach. The synthetic multivalent mannosides are to be explored to study MR-sugar binding events.

Valency and density matter: Deciphering impacts of immunogen structures on immune responses against a tumor associated carbohydrate antigen using synthetic glycopolymers.

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.

PD-1 Suppresses Development of Humoral Responses That Protect against Tn-Bearing Tumors.

Tn is a carbohydrate antigen uniquely exposed on tumor mucins and, thus, an ideal target for immunotherapy. However, it has been difficult to elicit protective antibody responses against Tn antigen and other tumor-associated carbohydrate antigens. Our study demonstrates this can be attributed to PD-1 immuno-inhibition. Our data show a major role for PD-1 in suppressing mucin- and Tn-specific B-cell activation, expansion, and antibody production important for protection against Tn-bearing tumor cells. These Tn/mucin-specific B cells belong to the innate-like B-1b cell subset typically responsible for T cell-independent antibody responses. Interestingly, PD-1-mediated regulation is B cell-intrinsic and CD4+ cells play a key role in supporting Tn/mucin-specific B-cell antibody production in the context of PD-1 deficiency. Mucin-reactive antibodies produced in the absence of PD-1 inhibition largely belong to the IgM subclass and elicit potent antitumor effects via a complement-dependent mechanism. The identification of this role for PD-1 in regulating B cell-dependent antitumor immunity to Tn antigen highlights an opportunity to develop new therapeutic strategies targeting tumor-associated carbohydrate antigens. Cancer Immunol Res; 4(12); 1027-37. ©2016 AACR.

Lipopeptide-Coated Iron Oxide Nanoparticles as Potential Glycoconjugate-Based Synthetic Anticancer Vaccines.

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.

Significant Impact of Immunogen Design on the Diversity of Antibodies Generated by Carbohydrate-Based Anticancer Vaccine.

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.

Carbohydrate antigen delivery by water soluble copolymers as potential anti-cancer vaccines.

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.

Boosting immunity to small tumor-associated carbohydrates with bacteriophage qß capsids.

The development of an effective immunotherapy is an attractive strategy toward cancer treatment. Tumor associated carbohydrate antigens (TACAs) are overexpressed on a variety of cancer cell surfaces, which present tempting targets for anticancer vaccine development. However, such carbohydrates are often poorly immunogenic. To overcome this challenge, we show here that the display of a very weak TACA, the monomeric Tn antigen, on bacteriophage Qß virus-like particles elicits powerful humoral responses to the carbohydrate. The effects of adjuvants, antigen display pattern, and vaccine dose on the strength and subclasses of antibody responses were established. The local density of antigen rather than the total amount of antigen administered was found to be crucial for induction of high Tn-specific IgG titers. The ability to display antigens in an organized and high density manner is a key advantage of virus-like particles such as Qß as vaccine carriers. Glycan microarray analysis showed that the antibodies generated were highly selective toward Tn antigens. Furthermore, Qß elicited much higher levels of IgG antibodies than other types of virus-like particles, and the IgG antibodies produced reacted strongly with the native Tn antigens on human leukemia cells. Thus, Qß presents a highly attractive platform for the development of carbohydrate-based anticancer vaccines.