Jianbing Zhang

3′,5′-Cyclic diguanylic acid elicits mucosal immunity against bacterial infection

3,5-Cyclic diguanylic acid (cdiGMP) is emerging as a universal bacterial second messenger in regulating bacterial growth on surfaces. It has been recently shown that cdiGMP stimulates innate immunity and enhances antigen-specific humoral and cellular immune responses. We herein report that intranasal (i.n.) administration with cdiGMP induces an acute but transient inflammatory response and activation of dendritic cells in the lungs. Moreover, i.n. immunization of mice with pneumococcal surface adhesion A (PsaA) in conjunction with cdiGMP elicited strong antigen-specific serum immunoglobulin G (IgG) and secretory IgA antibody responses at multiple mucosal surfaces. More importantly, the immunized mice showed significantly reduced nasopharyngeal Streptococcus pneumoniae colonization. These results, for the first time, provide direct evidence for the induction of protection against mucosal bacterial infections by cdiGMP as an adjuvant.

Recent advances in the development of novel mucosal adjuvants and antigen delivery systems.

Mucosal infections and associated diseases remain a major socio-economic burden to society. Since parenteral immunizations fail to induce efficient protective immunity at mucosal surfaces, mucosal immunization is a logical approach to prevent and treat mucosally-initiated infections. All currently approved human mucosal vaccines are based on attenuated or killed whole pathogen cells but this strategy does pose safety concerns. Therefore, substantial effort is being invested to develop safe and effective mucosal adjuvants and delivery systems for mucosal vaccines. Encouragingly, some of these have progressed to advanced preclinical and clinical studies. This review discusses the promising preclinical research and the potential applications of several novel mucosal adjuvants and delivery systems: an archaeal lipid mucosal vaccine adjuvant and delivery (AMVAD) system, 3’,5’-cyclic diguanylic acid (c-di-GMP) and detoxified bacterial AB5 toxins. The potential and challenges in targeting M cells for mucosal vaccination are also discussed.

Pentabody-mediated antigen delivery induces antigen-specific mucosal immune response

An efficient immunization system is essential for the development of mucosal vaccine. Cholera toxin (CT) and Escherichia coli heat labile toxin (LT) are among the strongest adjuvants tested in experimental animals but their use in humans has been hindered by their toxicity. On the other hand, the role of their non-toxic B-subunits, CTB or LTB, in enhancing mucosal immune response is not clear. We propose here a novel strategy for the induction of mucosal immune responses. Single domain antibodies (sdAbs) against a model antigen bovine serum albumin (BSA) were raised from the antibody repertoire of a llama immunized with BSA, pentamerized by fusing the sdAbs to CTB, generating the so-called pentabodies. These pentabodies were used to deliver the antigen by mixing the two components and administering the mixture to mice intranasally. One construct was equivalent to CT in helping induce mucosal immune response. It was also found that this ability was probably due to its high affinity to BSA, providing some insight into the controversial role of CTB in mucosal immunization: at least for BSA, the model antigen BSA employed in this study, CTB has to be tightly linked to the antigen to have adjuvant/immune-enhancing effect.

Isolation of functional single domain antibody by whole cell immunization: implications for cancer treatment.

Carcinoembryonic antigen related cell adhesion molecule (CEACAM) 6 is over-expressed in different types of cancer cells. In addition, it has also been implicated in some infectious diseases. Targeting this molecule by an antibody might have applications in diverse tumor models. Single domain antibody (sdAb) is becoming very useful format in antibody engineering as potential tools for treating acute and chronic disease conditions such as cancer for both diagnostic as well as therapeutic application. Generally, sdAbs with good affinity are isolated from an immune library. Discovery of a new target antigen would require a new immunization with purified antigen which is not always easy. In this study, we have isolated, by phage display, an sdAb against CEACAM6 with an affinity of 5 nM from a llama immunized with cancer cells. The antibody has good biophysical properties, and it binds to the cells expressing the target antigen. Furthermore, it reduces cancer cells proliferation in vitro and shows an excellent tumor targeting in vivo. This sdAb could be useful in diagnosis as well as therapy of CEACAM6 expressing tumors. Finally, we envisage it would be feasible to isolate good sdAbs against other interesting tumor associated antigens from this library. Therefore, this immunization method could be a general strategy for isolating sdAbs against any surface antigen without immunizing the animal with the antigen of interest each time.

Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Single Domain Antibodies Are Potent Inhibitors of Low Density Lipoprotein Receptor Degradation.

Single domain antibodies (sdAbs) correspond to the antigen-binding domains of camelid antibodies. They have the same antigen-binding properties and specificity as monoclonal antibodies (mAbs) but are easier and cheaper to produce. We report here the development of sdAbs targeting human PCSK9 (proprotein convertase subtilisin/kexin type 9) as an alternative to anti-PCSK9 mAbs. After immunizing a llama with human PCSK9, we selected four sdAbs that bind PCSK9 with a high affinity and produced them as fusion proteins with a mouse Fc. All four sdAb-Fcs recognize the C-terminal Cys-His-rich domain of PCSK9. We performed multiple cellular assays and demonstrated that the selected sdAbs efficiently blocked PCSK9-mediated low density lipoprotein receptor (LDLR) degradation in cell lines, in human hepatocytes, and in mouse primary hepatocytes. We further showed that the sdAb-Fcs do not affect binding of PCSK9 to the LDLR but rather block its induced cellular LDLR degradation. Pcsk9 knock-out mice expressing a human bacterial artificial chromosome (BAC) transgene were generated, resulting in plasma levels of ∼300 ng/ml human PCSK9. Mice were singly or doubly injected with the best sdAb-Fc and analyzed at day 4 or 11, respectively. After 4 days, mice exhibited a 32 and 44% decrease in the levels of total cholesterol and apolipoprotein B and ∼1.8-fold higher liver LDLR protein levels. At 11 days, the equivalent values were 24 and 46% and ∼2.3-fold higher LDLR proteins. These data constitute a proof-of-principle for the future usage of sdAbs as PCSK9-targeting drugs that can efficiently reduce LDL-cholesterol, and as tools to study the Cys-His-rich domain-dependent sorting the PCSK9-LDLR complex to lysosomes.

PCSK9 Prosegment Chimera as Novel Inhibitors of LDLR Degradation

The proprotein convertase PCSK9, a target for the treatment of hypercholesterolemia, is a negative regulator of the LDL receptor (LDLR) leading to its degradation in endosomes/lysosomes and up-regulation of plasma LDL-cholesterol levels. The proprotein convertases, a family of nine secretory serine proteases, are first synthesized as inactive zymogens. Except for PCSK9, all other convertases are activated following the autocatalytic excision of their inhibitory N-terminal prosegment. PCSK9 is unique since the mature enzyme exhibits a cleaved prosegment complexed with the catalytic subunit and has no protease activity towards other substrates. Similar to other convertases, we hypothesized that the in trans presence of the PCSK9 prosegment would interfere with PCSK9s activity on the LDLR. Since the prosegment cannot be secreted alone, we engineered a chimeric protein using the Fc-region of human IgG1 fused to the PCSK9 prosegment. The expression of such Fcpro-fusion protein in HEK293 and HepG2 cells resulted in a secreted protein that binds PCSK9 and markedly inhibits its activity on the LDLR. This was observed by either intracellular co-expression of PCSK9 and Fcpro or by an extracellular in vitro co-incubation of Fcpro with PCSK9. Structure-function studies revealed that the inhibitory function of Fcpro does not require the acidic N-terminal stretch (residues 31–58) nor the C-terminal Gln152 of the prosegment. Fcpro likely interacts with the prosegment and/or catalytic subunit of the prosegment≡PCSK9 complex thereby allosterically modulating its function. Our data suggest a novel strategic approach for the design and isolation of PCSK9 inhibitors.

Crystal Structure of a Human Single Domain Antibody Dimer Formed through V(H)-V(H) Non-Covalent Interactions.

Single-domain antibodies (sdAbs) derived from human VH are considered to be less soluble and prone to aggregate which makes it difficult to determine the crystal structures. In this study, we isolated and characterized two anti-human epidermal growth factor receptor-2 (HER2) sdAbs, Gr3 and Gr6, from a synthetic human VH phage display library. Size exclusion chromatography and surface plasmon resonance analyses demonstrated that Gr3 is a monomer, but that Gr6 is a strict dimer. To understand this different molecular behavior, we solved the crystal structure of Gr6 to 1.6 Å resolution. The crystal structure revealed that the homodimer assembly of Gr6 closely mimics the VH-VL heterodimer of immunoglobulin variable domains and the dimerization interface is dominated by hydrophobic interactions.

A Novel Affinity Tag, ABTAG, and Its Application to the Affinity Screening of Single-Domain Antibodies Selected by Phage Display

ABTAG is a camelid single-domain antibody (sdAb) that binds to bovine serum albumin (BSA) with low picomolar affinity. In surface plasmon resonance (SPR) analyses using BSA surfaces, bound ABTAG can be completely dissociated from the BSA surfaces at low pH, over multiple cycles, without any reduction in the capacity of the BSA surfaces to bind ABTAG. A moderate throughput, SPR-based, antibody screening assay exploiting the unique features of ABTAG is described. Anti-carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) sdAbs were isolated from a phage-displayed sdAb library derived from the heavy chain antibody repertoire of a llama immunized with CEACAM6. Following one or two rounds of panning, enriched clones were expressed as ABTAG fusions in microtiter plate cultures. The sdAb-ABTAG fusions from culture supernatants were captured on BSA surfaces and CEACAM6 antigen was then bound to the captured molecules. The SPR screening method gives a read-out of relative expression levels of the fusion proteins and kinetic and affinity constants for CEACAM6 binding by the captured molecules. The library was also panned and screened by conventional methods and positive clones were subcloned and expressed for SPR analysis. Compared to conventional panning and screening, the SPR-based ABTAG method yielded a considerably higher diversity of binders, some with affinities that were three orders of magnitude higher affinity than those identified by conventional panning.