Huaizu Guo

Targeted Delivery of Tumor Antigens to Activated Dendritic Cells via CD11c Molecules Induces Potent Antitumor Immunity in Mice

Purpose: CD11c is an antigen receptor predominantly expressed on dendritic cells (DC), to which antigen targeting has been shown to induce robust antigen-specific immune responses. To facilitate targeted delivery of tumor antigens to DCs, we generated fusion proteins consisting of the extracellular domain of human HER or its rat homologue neu, fused to the single-chain fragment variable specific for CD11c (scFvCD11c-HER2/neu). Experimental Design: Induction of cellular and humoral immune responses and antitumoral activity of the fusion proteins admixed with DC-activating CpG oligonucleotides (scFvCD11c-HER2/neuCpG) were tested in transplantable HER2/neu-expressing murine tumor models and in transgenic BALB-neuT mice developing spontaneous neu-driven mammary carcinomas. Results: Vaccination of BALB/c mice with scFvCD11c-HER2CpG protected mice from subsequent challenge with HER2-positive, but not HER2-negative, murine breast tumor cells, accompanied by induction of strong HER2-specific T-cell and antibody responses. In a therapeutic setting, injection of scFvCD11c-HER2CpG caused rejection of established HER2-positive tumors. Importantly, antitumoral activity of such a fusion protein vaccine could be reproduced in immunotolerant BALB-neuT mice, where scFvCD11c-neuCpG vaccination significantly protected against a subsequent challenge with neu-expressing murine breast tumor cells and markedly delayed the onset of spontaneous mammary carcinomas. Conclusions: CD11c-targeted protein vaccines for in vivo delivery of tumor antigens to DCs induce potent immune responses and antitumoral activities and provide a rationale for further development of this approach for cancer immunotherapy.

Comparison of the inhibition mechanisms of Adalimumab and Infliximab in treating tumor necrosis factor α-associated diseases from a molecular view.

Background: The epitope and the TNFα inhabitation mechanism of Adalimumab remain unclear. Results: The crystal structure of the TNFα in complex with Adalimumab is reported at a resolution of 3.1 Å. Conclusion: The epitope of Adalimumab provided information that Adalimumab may have clinical advantage compared with Infliximab. Significance: These data reveal the Adalimumabs mechanism of TNFα inhibition and its advantages compared with other TNF inhibitors in clinical practice. TNFα-targeting therapy with the use of the drugs Etanercept, Infliximab, and Adalimumab is used in the clinical treatment of various inflammatory and immune diseases. Although all of these reagents function to disrupt the interaction between TNFα and its receptors, clinical investigations showed the advantages of Adalimumab treatment compared with Etanercept and Infliximab. However, the underlying molecular mechanism of action of Adalimumab remains unclear. In our previous work, we presented structural data on how Infliximab binds with the E-F loop of TNFα and functions as a TNFα receptor-binding blocker. To further elucidate the variations between TNFα inhibitors, we solved the crystal structure of TNFα in complex with Adalimumab Fab. The structural observation and the mutagenesis analysis provided direct evidence for identifying the Adalimumab epitope on TNFα and revealed the mechanism of Adalimumab inhibition of TNFα by occupying the TNFα receptor-binding site. The larger antigen-antibody interface in TNFα Adalimumab also provided information at a molecular level for further understanding the clinical advantages of Adalimumab therapy compared with Infliximab.

Structural Basis for Treating Tumor Necrosis Factor α (TNFα)-associated Diseases with the Therapeutic Antibody Infliximab

Background: Although infliximab has high efficacy in treating TNFα-associated diseases, the epitope on TNFα remains unclear. Results: The crystal structure of the TNFα in complex with the infliximab Fab is reported at a resolution of 2.6 Å. Conclusion: TNFα E-F loop plays a crucial role in the interaction. Significance: The structure may lead to understanding the mechanism of mAb anti-TNFα. Monoclonal antibody (mAb) drugs have been widely used for treating tumor necrosis factor α (TNFα)-related diseases for over 10 years. Although their action has been hypothesized to depend in part on their ability to bind precursor cell surface TNFα, the precise mechanism and the epitope bound on TNFα remain unclear. In the present work, we report the crystal structure of the infliximab Fab fragment in complex with TNFα at a resolution of 2.6 Å. The key features of the TNFα E-F loop region in this complex distinguish the interaction between infliximab and TNFα from other TNF-receptor structures, revealing the mechanism of TNFα inhibition by overlapping with the TNFα-receptor interface and indicating the crucial role of the E-F loop in the action of this therapeutic antibody. This structure also indicates the formation of an aggregated network for the activation of complement-dependent cytolysis and antibody-dependent cell-mediated cytotoxicity, which result in development of granulomatous infections through TNFα blockage. These results provide the first experimental model for the interaction of TNFα with therapeutic antibodies and offer useful information for antibody optimization by understanding the precise molecular mechanism of TNFα inhibition.

Methylation of Tip30 Promoter Is Associated with Poor Prognosis in Human Hepatocellular Carcinoma

Purpose: To investigate Tip30 promoter methylation status in human hepatocellular carcinoma (HCC) and the correlation with clinicopathologic features and prognosis. Experimental Design: The methylation status of CpG islands in Tip30 promoter was examined in 15 HCC cell lines as well as 59 paired HCC and adjacent nontumor tissues. The associations between Tip30 methylation status and the survival of patients were analyzed. Results:Tip30 promoter was hypermethylated in 6 of 10 HCC cell lines with reduced Tip30 mRNA. DNA methyltransferase inhibitor, 5-aza-2′-deoxycytidine, greatly enhanced TIP30 expression and sensitized HCC cells to cytotoxic drug-induced cell death. The promoter region of Tip30 was identified and the main promoter activity was located in the -135 to -45 region sited within a CpG island. The minimal promoter element contained four Sp1 binding sites, which were hypermethylated in HCC cell-derived promoters. Moreover, analyses of Tip30 promoter methylation status in 59 paired HCC tissues showed that 47% of the cases were hypermethylated. Recurrence rate (95% versus 67%; P = 0.011) and mortality (82% versus 53%; P = 0.033) were significantly higher in patients with methylated Tip30. Disease-free survival was significantly higher in patients with unmethylated Tip30 (33.3% versus 4.5%; P = 0.036). Conclusions: Our results show that epigenetic silencing of Tip30 gene expression by CpG island DNA hypermethylation is associated with poor prognosis in patients with HCC.

Identification of a novel functional domain of ricin responsible for its potent toxicity

Ribosome-inactivating proteins (RIPs) are toxic N-glycosidases that depurinate the universally conserved α-sarcin loop of large rRNAs. They have received attention in biological and biomedical research because of their unique biological activities toward animals and human cells as cell-killing agents. A better understanding of the depurination mechanism of RIPs could allow us to develop potent neutralizing antibodies and to design efficient immunotoxins for clinical use. Among these RIPs, ricin exhibited remarkable efficacy in depurination activity and highly conserved tertiary structure with other RIPs. It can be considered as a prototype to investigate the depurination mechanism of RIPs. In the present study, we successfully identified a novel functional domain responsible for controlling the depurination activity of ricin, which is located far from the enzymatic active site reported previously. Our study indicated that ricin A-chain mAbs binding to this domain (an α-helix comprising the residues 99–106) exhibited an unusual potent neutralizing ability against ricin in vivo. To further investigate the potential role of the α-helix in regulating the catalytic activity of ricin, ricin A-chain variants with different flexibility of the α-helix were rationally designed. Our data clearly demonstrated that the flexibility of the α-helix is responsible for controlling the depurination activity of ricin and determining the extent of protein synthesis inhibition, suggesting that the conserved α-helix might be considered as a potential target for the prevention and treatment of RIP poisoning.

Increased Expression of iASPP, Regulated by Hepatitis B Virus X Protein-Mediated NF-κB Activation, in Hepatocellular Carcinoma

BACKGROUND & AIMS iASPP is an inhibitory member of the ankyrin-repeat-, SH3-domain- and proline-rich-region-containing protein (ASPP) family; iASPP expression is up-regulated in different human tumor types. We explored the molecular mechanism increased expression of iASPP and its role in hepatocellular carcinoma (HCC). METHODS iASPP expression levels in human liver samples and cell lines were determined by polymerase chain reaction, immunoblot, and immunohistochemical analyses. Luciferase reporter, chromatin immunoprecipitation, and electrophoretic mobility shift assays were used to measure transcriptional activation by nuclear factor-κB (NF-κB). Effects on tumor growth were characterized with MTS, soft agar colony formation, and flow cytometry analyses. Tumorigenicity of cells was studied in nude mice. RESULTS Compared with normal liver cells or tissues, iASPP was expressed at significantly higher levels in HCC cell lines (9/14) and liver samples from patients with HCC, cirrhosis, or hepatitis B virus infection. Increased expression of iASPP was significantly associated with time to recurrence and survival time of patients with HCC. NF-κB activation increased the expression of iASPP through p65/p50 binding to a putative NF-κB-binding site in the iASPP promoter; hepatitis B virus X gene product might up-regulate expression of iASPP. Transgenic expression of iASPP promoted tumor cell proliferation and resistance to chemotherapeutic drugs in vitro and in vivo. CONCLUSIONS iASPP is up-regulated in HCC; it is a direct transcription target of NF-κB. Increased iASPP expression contributes to tumor progression by proliferative and antiapoptotic effects. iASPP might be developed as an HCC therapeutic target or to sensitize cancer cells to chemotherapeutic drugs; it might also be used as a prognostic factor.

Structural basis of immunosuppression by the therapeutic antibody daclizumab

Interleukin-2 (IL)-2 signaling plays a pivotal role in the activation of immune responses, and drugs that block this pathway have been shown to be effective for the immunosuppression in patients with organ transplantation to alleviate/eliminate allograft rejection. The first humanized monoclonal antibody (mAb) daclizumab falls into this category and shows high specificity and affinity against a key component of the IL-2 receptor complex, namely IL-2Rα. To reveal the molecular mechanism of the inhibition of the IL-2 signaling pathway by daclizumab, we determined the crystal structures of the daclizumab Fab in free form and in complex with the IL-2Rα ectodomain at 2.6 and 2.8 Å resolution, respectively. The daclizumab Fab adopts a similar conformation in the presence or absence of the IL-2Rα ectodomain. The antigen-binding site of daclizumab is mainly composed of five complementarity determining regions (CDRs) that form a large positively charged surface depression and two flanking patches that are generally hydrophobic. The conformational epitope consists of several discontinuous segments of the IL-2Rα ectodomain, a large portion of which overlaps with the regions that interact with IL-2, suggesting that the binding of daclizumab to IL-2Rα would prevent the IL-2 binding to IL-2Rα and the subsequent formation of the IL-2/IL-2Rαβγc complex, and therefore block the IL-2 signaling pathway. These results also have implications for the design and development of improved mAb drugs targeting IL-2Rα.

Structural Basis for the Blockage of IL-2 Signaling by Therapeutic Antibody Basiliximab

IL-2 signaling plays a central role in the initiation and activation of immune responses. Correspondingly, blockage of this pathway leads to inhibition of the immune system and would provide some therapeutic benefits. Basiliximab (Simulect), a therapeutic mAb drug with specificity against IL-2Rα of T cells, was approved by U.S. Food and Drug Administration in 1998. It has been proven to be effective in the suppression of the IL-2 pathway and hence has been widely used to prevent allograft rejection in organ transplantation, especially in kidney transplants. In this study, we report the crystal structure of the basiliximab Fab in complex with the ectodomain of IL-2Rα at 2.9 Å resolution. In the complex structure, the Fab interacts with IL-2Rα with extensive hydrophobic and hydrophilic interactions, accounting for a high binding affinity of 0.14 nM. The Ag binding site of basiliximab consists of all six CDR loops that form a large binding interface with a central shallow hydrophobic groove surrounded by four hydrophilic patches. The discontinuous epitope is composed of several segments from the D1 domain and a minor segment from the D2 domain that overlap with most of the regions responsible for the interactions with IL-2. Thus, basiliximab binding can completely block the interactions of IL-2 with IL-2Rα and hence inhibit the activation of the IL-2 signal pathway. The structural results also provide important implications for the development of improved and new IL-2Rα–targeted mAb drugs.

Versatile characterization of glycosylation modification in CTLA4-Ig fusion proteins by liquid chromatography-mass spectrometry

CTLA4-Ig is a highly glycosylated therapeutic fusion protein that contains multiple N- and O-glycosylation sites. Glycosylation plays a vital role in protein solubility, stability, serum half-life, activity, and immunogenicity. For a CTLA4-Ig biosimilar development program, comparative analytical data, especially the glycosylation data, can influence decisions about the type and amount of animal and clinical data needed to establish biosimilarity. Because of the limited clinical experience with biosimilars before approval, a comprehensive level of knowledge about the biosimilar candidates is needed to achieve subsequent development. Liquid chromatography-mass spectrometry (LC–MS) is a versatile technique for characterizing N- and O-glycosylation modification of recombinant therapeutic proteins, including 3 levels: intact protein analysis, peptide mapping analysis, and released glycans analysis. In this report, an in-depth characterization of glycosylation of a candidate biosimilar was carried out using a systematic approach: N- and O-linked glycans were identified and electron-transfer dissociation was then used to pinpoint the 4 occupied O-glycosylation sites for the first time. As the results show, the approach provides a set of routine tools that combine accurate intact mass measurement, peptide mapping, and released glycan profiling. This approach can be used to comprehensively research a candidate biosimilar Fc-fusion protein and provides a basis for future studies addressing the similarity of CTLA4-Ig biosimilars.

The Protein-Protein Interface Evolution Acts in a Similar Way to Antibody Affinity Maturation

Understanding the evolutionary mechanism that acts at the interfaces of protein-protein complexes is a fundamental issue with high interest for delineating the macromolecular complexes and networks responsible for regulation and complexity in biological systems. To investigate whether the evolution of protein-protein interface acts in a similar way as antibody affinity maturation, we incorporated evolutionary information derived from antibody affinity maturation with common simulation techniques to evaluate prediction success rates of the computational method in affinity improvement in four different systems: antibody-receptor, antibody-peptide, receptor-membrane ligand, and receptor-soluble ligand. It was interesting to find that the same evolutionary information could improve the prediction success rates in all the four protein-protein complexes with an exceptional high accuracy (>57%). One of the most striking findings in our present study is that not only in the antibody-combining site but in other protein-protein interfaces almost all of the affinity-enhancing mutations are located at the germline hotspot sequences (RGYW or WA), indicating that DNA hot spot mechanisms may be widely used in the evolution of protein-protein interfaces. Our data suggest that the evolution of distinct protein-protein interfaces may use the same basic strategy under selection pressure to maintain interactions. Additionally, our data indicate that classical simulation techniques incorporating the evolutionary information derived from in vivo antibody affinity maturation can be utilized as a powerful tool to improve the binding affinity of protein-protein complex with a high accuracy.