A Multivalent Vaccine Based on Ferritin Nanocage Elicits Potent Protective Immune Responses against SARS-CoV-2 Mutations.

The SARS-CoV-2 pandemic has created a global public crisis and heavily affected personal lives, healthcare systems, and global economies. Virus variants are continuously emerging, and, thus, the pandemic has been ongoing for over two years. Vaccines were rapidly developed based on the original SARS-CoV-2 (Wuhan-Hu-1) to build immunity against the coronavirus disease. However, they had a very low effect on the virus' variants due to their low cross-reactivity. In this study, a multivalent SARS-CoV-2 vaccine was developed using ferritin nanocages, which display the spike protein from the Wuhan-Hu-1, B.1.351, or B.1.429 SARS-CoV-2 on their surfaces. We show that the mixture of three SARS-CoV-2 spike-protein-displaying nanocages elicits CD4+ and CD8+ T cells and B-cell immunity successfully in vivo. Furthermore, they generate a more consistent antibody response against the B.1.351 and B.1.429 variants than a monovalent vaccine. This leads us to believe that the proposed ferritin-nanocage-based multivalent vaccine platform will provide strong protection against emerging SARS-CoV-2 variants of concern (VOCs).

Selection of Vaccinia Virus-Neutralizing Antibody from a Phage-Display Human-Antibody Library.

Although smallpox was eradicated in 1980, it is still considered a potential agent of biowarfare and bioterrorism. Smallpox has the potential for high mortality rates along with a major public health impact, eventually causing public panic and social disruption. Passive administration of neutralizing monoclonal antibodies (mAbs) is an effective intervention for various adverse reactions caused by vaccination and the unpredictable nature of emerging and bioterrorist-related infections. Currently, vaccinia immune globulin (VIG) is manufactured from vaccinia vaccine-boosted plasma; however, this production method is not ideal because of its limited availability, low specific activity, and risk of contamination with blood-borne infectious agents. To overcome the limitations of VIG production from human plasma, we isolated two human single chain variable fragments (scFvs) (SC34 and SC212) bound to vaccinia virus (VACV) from a scFv phage library constructed from the B cells of VACV vaccine-boosted volunteers. The scFvs were converted to human IgG1 (VC34 and VC212). These two anti-VACV mAbs were produced in Chinese Hamster Ovary (CHO) DG44 cells. The binding affinities of VC34 and VC212 were estimated by competition ELISA to IC50 values of 2 µg/mL (13.33 nM) and 22 µg/mL (146.67 nM), respectively. Only the VC212 mAb was proven to neutralize the VACV, as evidenced by the plaque reduction neutralization test (PRNT) result with a PRNT50 of ~0.16 mg/mL (~1.07 µM). This VC212 could serve as a valuable starting material for further development of VACV-neutralizing human immunoglobulin for a prophylactic measure against post-vaccination complications and for post-exposure treatment against smallpox.

Neutralization of hepatitis B virus (HBV) by human monoclonal antibody against HBV surface antigen (HBsAg) in chimpanzees.

The virus neutralizing efficacy of HB-C7A, a human monoclonal antibody raised against the surface antigen of hepatitis B virus (HBsAg), was proved using hepatitis B virus (HBV)-naïve chimpanzees. One control chimpanzee which received 100CID(50) of HBV, subtype adw, without HB-C7A antibody became infected by HBV as evidenced by the appearance of HBV DNA on week 10 and subsequent appearance of HBsAg, anti-HBc and anti-HBs in the serum. Two experimental chimpanzees were inoculated intravenously with same dose of HBV as the control chimpanzee, which was previously incubated with 0.1mg and 10mg of HB-C7A antibody prior to inoculation. HBV infection was not observed in the antibody-treated chimpanzees during 12 months of follow-up, exhibiting neither detectable HBsAg nor anti-HBc antibody. This work demonstrates the neutralization of HBV by HB-C7A monoclonal antibody and shows the possibility of prevention of HBV infection using this antibody in liver transplantation and exposure to HBV.

Human monoclonal antibody against Hepatitis B virus surface antigen (HBsAg).

Hepatitis B virus (HBV) is one of the main pathogens responsible for hepatitis and hepatocellular carcinoma. Human plasma-derived Hepatitis B immune globulin (HBIG) is being used for prophylactic and liver transplantation currently. However, it may be necessary to replace a HBIG with a recombinant one because of limited availability of human plasma with high anti-HBsAg antibody titer and possible contamination of human pathogens. A Chinese hamster ovary (CHO) cell line, HB-C7A, was established which produces a fully human IgG1 that binds HBsAg. The HB-C7A exhibits approximately 2600 units/mg of antibody. The affinity (K(a)) of HB-C7A is 1.1 x 10(8) M(-1) by Biacore analysis and estimated 6.7-fold higher than that of Hepabig (a plasma-derived HBIG from Green Cross Corp., Yongin, Korea) by competition ELISA. The HB-C7A recognizes the conformational "a" determinant of HBsAg and binds HBV particle more efficiently than the Hepabig. The HB-C7A binds to HBV-infected human liver tissue but does not bind to normal human tissues. This HB-C7A has several advantages compared to plasma-derived Hepabig such as activity, safety and availability.

ER2, a novel human anti-EGFR monoclonal antibody inhibit tumor activity in non-small cell lung cancer models.

OBJECTIVES: The epidermal growth factor receptor (EGFR) abnormalities including amplification, mutation, and overexpression are frequent in non-small cell lung cancer (NSCLC). We investigated in vitro and in vivo antitumor activity of ER2, a novel human anti-EGFR monoclonal antibody, in NSCLC. METHODS: A panel of NSCLC cell lines (A549, H460, H322, H358, H1299, HCC827, PC9, H1975, and PC9-GR) was used to evaluate in vitro antitumor activity of ER2 and cetuximab. The inhibitory effects of ER2 and cetuximab on downstream signaling were assessed by western blot. Secreted VEGF was measured by Human VEGF Quantikine ELISA kit. Antitumor effects of ER2 and cetuximab as single agents and in combination with cisplatin were evaluated in H322, HCC827 and A549 xenograft models. RESULTS: ER2 efficiently inhibits EGFR and its downstream signaling molecules including Akt and Erk1/2 in NSCLC cell lines with wild-type or mutant EGFR. ER2 inhibited cell viability of H322, HCC827 and A549 cells in a dose-dependent manner by inducing cell cycle arrest and apoptosis. Also, ER2 suppressed EGF-stimulated VEGF production as efficiently as cetuximab in H322, HCC827 and A549 cells. Moreover, ER2 alone and in combination with cisplatin showed a significant anti-tumor efficacy in xenograft mouse models. CONCLUSION: Taken together, ER2 has significant anti-tumor activity in in vitro and in vivo NSCLC models, suggesting a rationale for clinical development of ER2 in NSCLC.

GC1118, an Anti-EGFR Antibody with a Distinct Binding Epitope and Superior Inhibitory Activity against High-Affinity EGFR Ligands.

The EGFR-targeted monoclonal antibodies are a valid therapeutic strategy for patients with metastatic colorectal cancer (mCRC). However, only a small subset of mCRC patients has therapeutic benefits and there are high demands for EGFR therapeutics with a broader patient pool and more potent efficacy. In this study, we report GC1118 exhibiting a different character in terms of binding epitope, affinity, mode of action, and efficacy from other anti-EGFR antibodies. Structural analysis of the EGFR-GC1118 crystal complex revealed that GC1118 recognizes linear, discrete N-terminal epitopes of domain III of EGFR, critical for EGF binding but not overlapping with those of other EGFR-targeted antibodies. GC1118 exhibited superior inhibitory activity against high-affinity EGFR ligands in terms of EGFR binding, triggering EGFR signaling, and proliferation compared with cetuximab and panitumumab. EGFR signaling driven by low-affinity ligands, on the contrary, was well inhibited by all the antibodies tested. GC1118 demonstrated robust antitumor activity in tumor xenografts with elevated expression of high-affinity ligands in vivo, whereas cetuximab did not. Considering the significant role of high-affinity EGFR ligands in modulating tumor microenvironment and inducing resistance to various cancer therapeutics, our study suggests a potential therapeutic advantage of GC1118 in terms of efficacy and a range of benefited patient pool. Mol Cancer Ther; 15(2); 251-63. ©2015 AACR.

Affinity Maturation of an Epidermal Growth Factor Receptor Targeting Human Monoclonal Antibody ER414 by CDR Mutation.

It is well established that blocking the interaction of EGFR with growth factors leads to the arrest of tumor growth, resulting in tumor cell death. ER414 is a human monoclonal antibody (mAb) derived by guided selection of the mouse mAb A13. The ER414 exhibited a ~17-fold lower affinity and, as a result, lower efficacy of inhibition of the EGF-mediated tyrosine phosphorylation of EGFR when compared with mAb A13 and cetuximab. We performed a stepwise in vitro affinity maturation to improve the affinity of ER414. We obtained a 3D model of ER414 to identify the amino acids in the CDRs that needed to be mutated. Clones were selected from the phage library with randomized amino acids in the CDRs and substitution of amino acids in the HCDR3 and LCDR1 of ER414 led to improved affinity. A clone, H3-14, with a ~20-fold increased affinity, was selected from the HCDR3 randomized library. Then three clones, ER2, ER78 and ER79, were selected from the LCDR1 randomized library based on the H3-14 but did not show further increased affinities compared to that of H3-14. Of the three, ER2 was chosen for further characterization due to its better expression than others. We successfully performed affinity maturation of ER414 and obtained antibodies with a similar affinity as cetuximab. And antibody from an affinity maturation inhibits the EGF-mediated tyrosine phosphorylation of EGFR in a manner similar to cetuximab.

Conversion of a murine monoclonal antibody A13 targeting epidermal growth factor receptor to a human monoclonal antibody by guided selection.

Epidermal growth factor receptor (EGFR) is an attractive target for tumor therapy because it is overexpressed in the majority of solid tumors and the increase in receptor expression levels has been linked with a poor clinical prognosis. Also it is well established that blocking the interaction of EGFR and the growth factors could lead to the arrest of tumor growth and possibly result in tumor cell death. A13 is a murine monoclonal antibody (mAb) that specifically binds to various sets of EGFR-expressing tumor cells and inhibits EGF-induced EGFR phosphorylation. We isolated human immunoglobulin genes by guided selection based on the mAb A13. Four different human single chain Fvs (scFvs) were isolated from from hybrid scFv libraries containing a human VH repertoire with the VL of mAb A13 and a human VL repertoire with the VH of mAb A13. All the 4 scFvs bound to EGFR-expressing A431 cells. One scFv (SC414) with the highest affinity was converted to IgG1 (ER414). The ER414 exhibited ∼17 fold lower affinity compared to the A13 mAb. In addition the ER414 inhibited an EGF-induced tyrosine phosphorylation of EGFR with much lower efficacy compared to the A13 mAb and Cetuximab (Merck KgaA, Germany). We identified that the epitope of A13 mAb is retained in ER414. This approach will provide an efficient way of converting a murine mAb to a human mAb.

A novel anti-EGFR monoclonal antibody inhibiting tumor cell growth by recognizing different epitopes from cetuximab.

The epidermal growth factor receptor (EGFR) overexpressed in many epithelial tumors is an attractive target for tumor therapy since numerous blocking agents of EGFR signaling have proven their anti-tumor activity. Here we report a novel monoclonal antibody (mAb), A13, which was generated from mice immunized with human cervical carcinoma A431 cells. In addition to binding to soluble EGFR with affinity of K(D) approximately 5.8nM, mAb A13 specifically bound to a variety of tumor cells and human placenta tissues expressing EGFR. A13 efficiently inhibited both EGF-dependant EGFR tyrosine phosphorylation in cervical and breast tumor cells and also in vitro colony formation of EGFR-overexpressing lung tumors. Competition and sandwich ELISAs, competitive surface plasmon resonance, and domain-level epitope mapping analyses demonstrated that mAb A13 competitively bound to the domain III (amino acids 302-503) of EGFR with EGF, but recognized distinct epitopes from those of cetuximab (Erbitux). Our results demonstrated that anti-EGFR mAb A13 interfered with EGFR proliferation signaling by blocking EGF binding to EGFR with different epitopes from those of cetuximab, suggesting that combination therapies of mAb A13 with cetuximab may prove beneficial for anti-tumor therapy.