Fully human chitinase-3 like-1 monoclonal antibody inhibits tumor growth, fibrosis, angiogenesis, and immune cell remodeling in lung, pancreatic, and colorectal cancers.

Considering the limited efficacy of current therapies in lung, colorectal, and pancreatic cancers, innovative combination treatments with diverse mechanisms of action are needed to improve patients' outcomes. Chitinase-3 like-1 protein (CHI3L1) emerges as a versatile factor with significant implications in various diseases, particularly cancers, fostering an immunosuppressive tumor microenvironment for cancer progression. Therefore, pre-clinical validation is imperative to fully realize its potential in cancer treatment. We developed phage display-derived fully human monoclonal CHI3L1 neutralizing antibodies (nAbs) and verified the nAbs-antigen binding affinity and specificity in lung, pancreatic and colorectal cancer cell lines. Tumor growth signals, proliferation and migration ability were all reduced by CHI3L1 nAbs in vitro. Orthotopic or subcutaneous tumor mice model and humanized mouse model were established for characterizing the anti-tumor properties of two CHI3L1 nAb leads. Importantly, CHI3L1 nAbs not only inhibited tumor growth but also mitigated fibrosis, angiogenesis, and restored immunostimulatory functions of immune cells in pancreatic, lung, and colorectal tumor mice models. Mechanistically, CHI3L1 nAbs directly suppressed the activation of pancreatic stellate cells and the transformation of macrophages into myofibroblasts, thereby attenuating fibrosis. These findings strongly support the therapeutic potential of CHI3L1 nAbs in overcoming clinical challenges, including the failure of gemcitabine in pancreatic cancer.

CARs derived from broadly neutralizing, human monoclonal antibodies identified by single B cell sorting target hepatitis B virus-positive cells.

To design new CARs targeting hepatitis B virus (HBV), we isolated human monoclonal antibodies recognizing the HBV envelope proteins from single B cells of a patient with a resolved infection. HBV-specific memory B cells were isolated by incubating peripheral blood mononuclear cells with biotinylated hepatitis B surface antigen (HBsAg), followed by single-cell flow cytometry-based sorting of live, CD19+ IgG+ HBsAg+ cells. Amplification and sequencing of immunoglobulin genes from single memory B cells identified variable heavy and light chain sequences. Corresponding immunoglobulin chains were cloned into IgG1 expression vectors and expressed in mammalian cells. Two antibodies named 4D06 and 4D08 were found to be highly specific for HBsAg, recognized a conformational and a linear epitope, respectively, and showed broad reactivity and neutralization capacity against all major HBV genotypes. 4D06 and 4D08 variable chain fragments were cloned into a 2nd generation CAR format with CD28 and CD3zeta intracellular signaling domains. The new CAR constructs displayed a high functional avidity when expressed on primary human T cells. CAR-grafted T cells proved to be polyfunctional regarding cytokine secretion and killed HBV-positive target cells. Interestingly, background activation of the 4D08-CAR recognizing a linear instead of a conformational epitope was consistently low. In a preclinical model of chronic HBV infection, murine T cells grafted with the 4D06 and the 4D08 CAR showed on target activity indicated by a transient increase in serum transaminases, and a lower number of HBV-positive hepatocytes in the mice treated. This study demonstrates an efficient and fast approach to identifying pathogen-specific monoclonal human antibodies from small donor cell numbers for the subsequent generation of new CARs.

Structural analyses of the GI.4 norovirus by cryo-electron microscopy and X-ray crystallography revealing binding sites for human monoclonal antibodies.

Noroviruses are major causative agents of acute nonbacterial gastroenteritis in humans. There are neither antiviral therapeutic agents nor vaccines for noroviruses at this time. To evaluate the potential usefulness of two previously isolated human monoclonal antibody fragments, CV-1A1 and CV-2F5, we first conducted a single-particle analysis to determine the cryo-electron microscopy structure of virus-like particles (VLPs) from the genogroup I genotype 4 (GI.4) Chiba strain uniformly coated with CV-1A1 fragments. The results revealed that the GI.4-specific CV-1A1 antibody bound to the P2 subdomain, in which amino acids are less conserved and variable. Interestingly, a part of the CV-1A1 intrudes into the histo-blood group antigen-binding site, suggesting that this antibody might exert neutralizing activity. Next, we determined the crystal structure of the protruding (P) domain of the capsid protein in the complex form with the CV-2F5 antibody fragment. Consistent with the cross-reactivity, the CV-2F5 bound to the P1 subdomain, which is rich in amino acids conserved among the GI strains, and moreover induced a disruption of Chiba VLPs. These results suggest that the broadly reactive CV-2F5 antibody can be used as both a universal detection reagent and an antiviral drug for GI noroviruses. IMPORTANCE: We conducted the structural analyses of the VP1 protein from the GI.4 Chiba norovirus to identify the binding sites of the previously isolated human monoclonal antibodies CV-1A1 and CV-2F5. The cryo-electron microscopy of the Chiba virus-like particles (VLPs) complexed with the Fv-clasp forms of GI.4-specific CV-1A1 revealed that this antibody binds to the highly variable P2 subdomain, suggesting that this antibody may have neutralizing ability against the GI.4 strains. X-ray crystallography revealed that the CV-2F5 antibody bound to the P1 subdomain, which is rich in conserved amino acids. This result is consistent with the ability of the CV-2F5 antibody to react with a wide variety of GI norovirus strains. It is also found that the CV-2F5 antibody caused a disruption of VLPs. Our findings, together with previous reports on the structures of VP1 proteins and VLPs, are expected to open a path for the structure-based development of antivirals and vaccines against norovirus disease.

Epitopes of an antibody that neutralizes a wide range of SARS-CoV-2 variants in a conserved subdomain 1 of the spike protein.

The evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has continued, enabling the virus to escape from host immunity by changing its spike antigen, while biased toward the receptor-binding domain and N-terminal domain. Here, we isolated a novel pan-SARS-CoV-2 neutralizing antibody (which we named MO11) for even the recent dominators XBB.1.16 and EG.5.1, from a convalescent patient who had received three doses of an original mRNA COVID-19 vaccination. A cryo-electron microscopy analysis of the spike-MO11 complex at 2.3 Å atomic resolution revealed that it recognizes a conserved epitope hidden behind a glycan shield at N331 on subdomain 1 (SD1), holding both the N- and C-terminal segments comprising SD1. Our identification of MO11 unveiled the functional importance of SD1 for the spike's function, and we discuss the potential availability of a novel common epitope among the SARS-CoV-2 variants.IMPORTANCENovel severe acute respiratory syndrome coronavirus 2 variants with immune evasion ability are still repeatedly emerging, nonetheless, a part of immunity developed in responding to the antigen of earlier variants retains efficacy against recent variants irrespective of the numerous mutations. In exploration for the broadly effective antibodies, we identified a cross-neutralizing antibody, named MO11, from the B cells of the convalescent patient. MO11 targets a novel epitope in subdomain 1 (SD1) and was effective against all emerging variants including XBB.1.16 and EG.5.1. The neutralizing activity covering from D614G to EG.5.1 variants was explained by the conservation of the epitope, and it revealed the importance of the subdomain on regulating the function of the antigen for viral infection. Demonstrated identification of the neutralizing antibody that recognizes a conserved epitope implies basal contribution of such group of antibodies for prophylaxis against COVID-19.

Crimean-Congo Hemorrhagic Fever Survivors Elicit Protective Non-Neutralizing Antibodies that Target 11 Overlapping Regions on Viral Glycoprotein GP38.

Crimean-Congo hemorrhagic fever virus can cause lethal disease in humans yet there are no approved medical countermeasures. Viral glycoprotein GP38, unique to Nairoviridae, is a target of protective antibodies, but extensive mapping of the human antibody response to GP38 has not been previously performed. Here, we isolated 188 GP38-specific antibodies from human survivors of infection. Competition experiments showed that these antibodies bind across five distinct antigenic sites, encompassing eleven overlapping regions. Additionally, we reveal structures of GP38 bound with nine of these antibodies targeting different antigenic sites. Although GP38-specific antibodies were non-neutralizing, several antibodies were found to have protection equal to or better than murine antibody 13G8 in two highly stringent rodent models of infection. Together, these data expand our understanding regarding this important viral protein and inform the development of broadly effective CCHFV antibody therapeutics.

Usefulness of Belimumab in Adult Patients With Systemic Lupus Erythematosus Evaluated Using Single Indexes: A Meta-Analysis and Systematic Review.

Background: Belimumab is the first antibody drug approved for systemic lupus erythematosus (SLE), and is a fully human monoclonal antibody that inhibits soluble B lymphocyte stimulator protein. In clinical trials, a composite index was used to assess efficacy of belimumab. However, clinical guidelines on SLE treatment currently use single efficacy indexes. Objective: The main objective of this study was to perform a meta-analysis to evaluate the efficacy of belimumab utilizing single indexes used in routine clinical practice, rather than the composite efficacy index used in clinical trials during the development phase. As a secondary endpoint, safety was also evaluated. Methods: Several databases were searched to identify reports published up to December 1, 2021 on randomized controlled trials examining the efficacy of belimumab in adult patients with SLE. From the clinical trial data, efficacy was evaluated using single indexes including the SLE Disease Activity Index (SLEDAI), British Isles Lupus Assessment Group Index, and Physician Global Assessment. Safety was also assessed. Data were synthesized and analyzed using Review Manager 5.4. This study protocol was registered in the UMIN Clinical Trials Registry (Registration number: UMIN000052846). Results: The search identified 12 reports that met the inclusion criteria. Five reports were included in efficacy evaluation and 9 in safety evaluation. The primary endpoint was SLEDAI. Significantly more belimumab-treated patients achieved a ≥4-point reduction in SLEDAI (relative risk 1.28; 95% confidence interval, 1.16-1.40; P < 0.00001) compared with placebo. Other efficacy endpoints were also improved significantly in the belimumab group. No difference in safety was found between belimumab and placebo. Conclusions: The present meta-analysis evaluating clinical trial data using various single indexes recommended by clinical guidelines for SLE verifies that addition of belimumab to standard of care is efficacious for moderate-to-severe SLE.

Protective human monoclonal antibodies target conserved sites of vulnerability on the underside of influenza virus neuraminidase.

Continuously evolving influenza viruses cause seasonal epidemics and pose global pandemic threats. Although viral neuraminidase (NA) is an effective drug and vaccine target, our understanding of the NA antigenic landscape still remains incomplete. Here, we describe NA-specific human antibodies that target the underside of the NA globular head domain, inhibit viral propagation of a wide range of human H3N2, swine-origin variant H3N2, and H2N2 viruses, and confer both pre- and post-exposure protection against lethal H3N2 infection in mice. Cryo-EM structures of two such antibodies in complex with NA reveal non-overlapping epitopes covering the underside of the NA head. These sites are highly conserved among N2 NAs yet inaccessible unless the NA head tilts or dissociates. Our findings help guide the development of effective countermeasures against ever-changing influenza viruses by identifying hidden conserved sites of vulnerability on the NA underside.

Antibody verification of HLA class I and class II eplets by human monoclonal HLA antibodies.

In solid organ transplantation, formation of de novo donor-specific HLA antibodies is induced by mismatched eplets on donor HLA molecules. While several studies have shown a strong correlation between the number of eplet mismatches and inferior outcomes, not every eplet mismatch is immunogenic. Eplets are theoretically defined entities, necessitating formal proof that they can be recognised and bound by antibodies. This antibody verification is pivotal to ensure that clinically relevant eplets are considered in studies on molecular matching. Recombinant human HLA-specific monoclonal antibodies (mAbs) were generated from HLA-reactive B cell clones isolated from HLA immunised individuals using recombinant HLA molecules. Subsequently, the reactivity patterns of the mAbs obtained from single antigen bead assay were analysed using HLA-EMMA software to identify single or configurations of solvent accessible amino acids uniquely present on the reactive HLA alleles and were mapped to eplets. Two HLA class I and seven HLA class II-specific human mAbs were generated from four individuals. Extensive mAb reactivity analysis, led to antibody verification of three HLA-DR-specific eplets, and conversion of five eplets (one HLA-A, one HLA-B, two HLA-DR, and one HLA-DP), from provisionally verified to truly antibody-verified. Finally, one HLA-DQ-specific eplet was upgraded from level A2 to level A1 verification evidence. The generation of recombinant human HLA-specific mAbs with different specificities contributes significantly to the antibody verification of eplets and therefore is instrumental for implementation of eplet matching in the clinical setting.

Neutralizing and enhancing monoclonal antibodies in SARS-CoV-2 convalescent patients: lessons from early variant infection and impact on shaping emerging variants.

Serological studies of COVID-19 convalescent patients have identified polyclonal lineage-specific and cross-reactive antibodies (Abs), with varying effector functions against virus variants. Individual specificities of anti-SARS-CoV-2 Abs and their impact on infectivity by other variants have been little investigated to date. Here, we dissected at a monoclonal level neutralizing and enhancing Abs elicited by early variants and how they affect infectivity of emerging variants. B cells from 13 convalescent patients originally infected by D614G or Alpha variants were immortalized to isolate 445 naturally-produced anti-SARS-CoV-2 Abs. Monoclonal antibodies (mAbs) were tested for their abilities to impact the cytopathic effect of D614G, Delta, and Omicron (BA.1) variants. Ninety-eight exhibited robust neutralization against at least one of the three variant types, while 309 showed minimal or no impact on infectivity. Thirty-eight mAbs enhanced infectivity of SARS-CoV-2. Infection with D614G/Alpha variants generated variant-specific (65 neutralizing Abs, 35 enhancing Abs) and cross-reactive (18 neutralizing Abs, 3 enhancing Abs) mAbs. Interestingly, among the neutralizing mAbs with cross-reactivity restricted to two of the three variants tested, none demonstrated specific neutralization of the Delta and Omicron variants. In contrast, cross-reactive mAbs enhancing infectivity (n = 3) were found exclusively specific to Delta and Omicron variants. Notably, two mAbs that amplified in vitro the cytopathic effect of the Delta variant also exhibited neutralization against Omicron. These findings shed light on functional diversity of cross-reactive Abs generated during SARS-CoV-2 infection and illustrate how the balance between neutralizing and enhancing Abs facilitate variant emergence.

Human monoclonal antibodies against Staphylococcus aureus A protein identified by high-throughput single-cell sequencing of phase I clinical volunteers' B cells.

Methicillin-resistant Staphylococcus aureus, poses a significant threat through infections in both community and hospital settings. To address this challenge, we conducted a phase I clinical trial study involving a recombinant Staphylococcus aureus vaccine. Utilizing peripheral blood lymphocytes from 64 subjects, we isolated antigen-specific memory B cells for subsequent single-cell sequencing. Among the 676 identified antigen-binding IgG1+ clones, we selected the top 10 antibody strains for construction within expression vectors. Successful expression and purification of these monoclonal antibodies led to the discovery of a highly expressed human antibody, designated as IgG-6. This antibody specifically targets the pentameric form of the Staphylococcus aureus protein A (SpA5). In vivo assessments revealed that IgG-6 provided prophylactic protection against MRSA252 infection. This study underscores the potential of human antibodies as an innovative strategy against Staphylococcus aureus infections, offering a promising avenue for further research and clinical development.

Efficacy of Bezlotoxumab Against Clostridioides difficile Infection: A Case-Series Study at a University Hospital in Japan and Literature Review.

Background Clostridioides difficile infection (CDI) recurrence is a public health concern as well as a health economic burden. Bezlotoxumab treatment is one way to prevent recurrence; however, its clinical results have not been reported in Japan. Therefore, we investigated the efficacy and safety of bezlotoxumab in patients with CDI at a university hospital in Japan and compared them with previously reported findings. Methodology We retrospectively examined all patients with some risk factors for recurrent CDI who received bezlotoxumab at the discretion of physicians at the Aichi Medical University Hospital, Aichi, Japan, between July 2018 and July 2022. The primary outcome was the three-month CDI recurrence rate. The secondary outcomes were an initial clinical cure and the six-month CDI recurrence rate. The safety of the administration was also assessed. Results A total of nine patients who received bezlotoxumab were included during the study period. The rate of CDI recurrence within three months was 28.5% (2/9). Two patients died due to other causes before their diarrhea improved. None of the patients experienced CDI recurrence between three and six months after the initial clinical cure of the baseline episode. Patients showed good tolerability to bezlotoxumab with no adverse effects. Two patients with a single episode of CDI recurrence before bezlotoxumab administration showed no recurrence. Conclusions In this Japanese case-series study, the efficacy of bezlotoxumab in preventing CDI recurrence in elderly patients with CDI and multiple underlying diseases was inferior to that reported in previous studies that analyzed real-world data. It is possible that bezlotoxumab may not be fully effective in elderly patients with CDI.

Active safety surveillance of rabies monoclonal antibody and rabies vaccine in patients with category III potential rabies exposure.

Background: A vero cell-based inactivated Rabies Vaccine (Rabivax-S) and Rabies Human Monoclonal Antibody (Rabishield) have been approved since 2016. A post-marketing surveillance was conducted in India from 2020 to 2021 to gather real world safety data on Rabivax-S and Rabishield. Methods: This was non-interventional active surveillance in patients with category III potential rabies exposure who were administered a post-exposure prophylaxis (PEP) regimen (Rabishield and Rabivax-S) by their healthcare providers (HCPs) as per the dosages and regimens mentioned in the package insert approved by the Indian regulators. The approved schedule for PEP was local infiltration of Rabishield on Day 0 and five doses of Rabivax-S on Day 0, 3, 7, 14, and 28 (Intramuscular route, IM) or four doses of Rabivax-S on Day 0, 3, 7, and 28 (Intradermal route, ID). The primary objective of this surveillance was to generate real-world evidence on the safety and tolerability of Rabishield and Rabivax-S. All patients enrolled in the surveillance were required to report any adverse events (AEs) occurring up to Day 31 after initiation of PEP (administration of Rabishield and the first dose of Rabivax-S) to their HCP. Findings: A total of 1000 patients with category III potential rabies exposure were enrolled across India. 991 patients received the PEP regimen with IM Rabivax-S while 9 received a PEP regimen with the ID regimen. While 32% of the patients were <12 years of age, 11.8% were ≥12 to <18 years of age and 56.2% were ≥18 years of age. The entire PEP regimen was completed by 97.3% of the enrolled patients. A total of 69 AEs were reported in 64 patients. Out of these, 49 AEs in 47 patients were assessed as causally related to the study products (26 with Rabishield and 23 with Rabivax-S). The majority of the AEs were mild and all recovered without any sequelae. One serious adverse event (SAE) of fracture of the hand was reported which was not related to either Rabishield or Rabivax-S. No case of rabies was reported. Interpretation: Rabishield and Rabivax-S have an excellent safety profile and are well tolerated. No participant developed rabies during 31 day follow up. Funding: The PMS was funded by Serum institute of India Private Limited which is the manufacturer of the study products.

A fully human monoclonal antibody possesses antibody-dependent cellular cytotoxicity (ADCC) activity against the H1 subtype of influenza A virus by targeting a conserved epitope at the HA1 protomer interface.

The DiversitabTM system produces target specific high titer fully human polyclonal IgG immunoglobulins from transchromosomic (Tc) bovines shown to be safe and effective against multiple virulent pathogens in animal studies and Phase 1, 2 and 3 human clinical trials. We describe the functional properties of a human monoclonal antibody (mAb), 38C2, identified from this platform, which recognizes recombinant H1 hemagglutinins (HAs) and induces appreciable antibody-dependent cellular cytotoxicity (ADCC) activity in vitro. Interestingly, 38C2 monoclonal antibody demonstrated no detectable neutralizing activity against H1N1 virus in both hemagglutination inhibition and virus neutralization assays. Nevertheless, this human monoclonal antibody induced appreciable ADCC against cells infected with multiple H1N1 strains. The HA-binding activity of 38C2 was also demonstrated in flow cytometry using Madin-Darby canine kidney cells infected with multiple influenza A H1N1 viruses. Through further investigation with the enzyme-linked immunosorbent assay involving the HA peptide array and 3-dimensional structural modeling, we demonstrated that 38C2 appears to target a conserved epitope located at the HA1 protomer interface of H1N1 influenza viruses. A novel mode of HA-binding and in vitro ADCC activity pave the way for further evaluation of 38C2 as a potential therapeutic agent to treat influenza virus infections in humans.

Thinking beyond Vaccination: Promising Add-On Strategies to Active Immunization and Vaccination in Pandemics-A Mini-Review.

There is little doubt that final victories over pandemics, such as COVID-19, are attributed to herd immunity, either through post-disease convalescence or active immunization of a high percentage of the world's population with vaccines, which demonstrate protection from infection and transmission and are available in large quantities at reasonable prices. However, it is assumable that humans with immune defects or immune suppression, e.g., as a consequence of allograft transplantation, cannot be immunized actively nor produce sufficient immune responses to prevent SARS-CoV-2 infections. These subjects desperately need other strategies, such as sophisticated protection measures and passive immunization. Hypertonic salt solutions attack vulnerable core areas of viruses; i.e., salt denatures surface proteins and thus prohibits virus penetration of somatic cells. It has to be ensured that somatic proteins are not affected by denaturation regarding this unspecific virus protection. Impregnating filtering facepieces with hypertonic salt solutions is a straightforward way to inactivate viruses and other potential pathogens. As a result of the contact of salt crystals on the filtering facepiece, these pathogens become denatured and inactivated almost quantitatively. Such a strategy could be easily applied to fight against the COVID-19 pandemic and other ones that may occur in the future. Another possible tool to fight the COVID-19 pandemic is passive immunization with antibodies against SARS-CoV-2, preferably from human origin. Such antibodies can be harvested from human patients' sera who have successfully survived their SARS-CoV-2 infection. The disadvantage of a rapid decrease in the immunoglobulin titer after the infection ends can be overcome by immortalizing antibody-producing B cells via fusion with, e.g., mouse myeloma cells. The resulting monoclonal antibodies are then of human origin and available in, at least theoretically, unlimited amounts. Finally, dry blood spots are a valuable tool for surveilling a population's immunity. The add-on strategies were selected as examples for immediate, medium and long-term assistance and therefore did not raise any claim to completeness.

Characterization of epitopes of human monoclonal antibodies against cytomegalovirus glycoprotein B for neutralization and antibody-dependent phagocytosis.

As congenital cytomegalovirus (CMV) infections are the leading non-genetic cause of sensorineural hearing loss and significant neurological disabilities in children, the development of CMV vaccines should be given the highest public health priority. Although MF59-adjuvanted glycoprotein B (gB) vaccine (gB/MF59) is safe and immunogenic, its efficacy in terms of protection from natural infection was around 50 % in clinical trials. Although gB/MF59 induced high antibody titers, anti-gB antibodies contributed little to the neutralization of infection. Recent studies have found that non-neutralizing functions, including antibody-dependent phagocytosis of virions and virus-infected cells, are likely to play important roles in pathogenesis and vaccine design. Previously, we isolated human monoclonal antibodies (MAbs) that reacted with the trimeric form of gB ectodomain and found that preferential epitopes for neutralization were present on Domains (Doms) I and II of gB, while there were abundant non-neutralizing antibodies targeting Dom IV. In this study, we analyzed the phagocytosis activities of these MAbs and found the following: 1) MAbs effective for phagocytosis of the virions targeted Doms I and II, 2) the MAbs effective for phagocytosis of the virions and those of virus-infected cells were generally distinct, and 3) the antibody-dependent phagocytosis showed little correlation with neutralizing activities. Taking account of the frequency and levels of neutralization and phagocytosis, incorporation of the epitopes on Doms I and II into developing vaccines is considered desirable for the prevention of viremia.

Characterization of the C6D7-RBD Human Monoclonal Antibody Specific to the SARS-CoV-2 S Protein Receptor-Binding Domain.

The new coronavirus infection COVID-19 is an acute viral disease that affects primarily the upper respiratory tract. The etiological agent of COVID-19 is the SARS-CoV-2 RNA virus (Coronaviridae family, Betacoronavirus genus, Sarbecovirus subgenus). We have developed a high-affinity human monoclonal antibody, called C6D7-RBD, which is specific to the S protein receptor-binding domain (RBD) from the SARS-CoV-2 Wuhan-Hu-1 strain and exhibits virus-neutralizing activity in a test with recombinant antigens: angiotensin-converting enzyme 2 (ACE2) and RBD.

Identification and Analysis of Monoclonal Antibodies with Neutralizing Activity against Diverse SARS-CoV-2 Variants.

We identified neutralizing monoclonal antibodies against severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) variants (including Omicron variants BA.5 and BA.2.75) from individuals who received two doses of mRNA vaccination after they had been infected with the D614G virus. We named them MO1, MO2, and MO3. Among them, MO1 showed particularly high neutralizing activity against authentic variants: D614G, Delta, BA.1, BA.1.1, BA.2, BA.2.75, and BA.5. Furthermore, MO1 suppressed BA.5 infection in hamsters. A structural analysis revealed that MO1 binds to the conserved epitope of seven variants, including Omicron variants BA.5 and BA.2.75, in the receptor-binding domain of the spike protein. MO1 targets an epitope conserved among Omicron variants BA.1, BA.2, and BA.5 in a unique binding mode. Our findings confirm that D614G-derived vaccination can induce neutralizing antibodies that recognize the epitopes conserved among the SARS-CoV-2 variants. IMPORTANCE Omicron variants of SARS-CoV-2 acquired escape ability from host immunity and authorized antibody therapeutics and thereby have been spreading worldwide. We reported that patients infected with an early SARS-CoV-2 variant, D614G, and who received subsequent two-dose mRNA vaccination have high neutralizing antibody titer against Omicron lineages. It was speculated that the patients have neutralizing antibodies broadly effective against SARS-CoV-2 variants by targeting common epitopes. Here, we explored human monoclonal antibodies from B cells of the patients. One of the monoclonal antibodies, named MO1, showed high potency against broad SARS-CoV-2 variants including BA.2.75 and BA.5 variants. The results prove that monoclonal antibodies that have common neutralizing epitopes among several Omicrons were produced in patients infected with D614G and who received mRNA vaccination.

Development of a recombinant hepatitis B immunoglobulin derived from B cells collected from healthy individuals administered with hepatitis B virus vaccines: A feasibility study.

BACKGROUND: In Japan, plasma with a high concentration of Hepatitis B Virus (HBV) antibodies for hepatitis B immunoglobulin (HBIG) is almost entirely imported. We aimed to produce recombinant HBIG by isolating immunoglobulin cDNAs against the HBV surface antigen (HBsAg). STUDY DESIGN AND METHODS: B cells expressing HBsAg antibodies were obtained from blood center personnel who had been administered HB vaccine booster and then isolated by either an Epstein-Barr virus hybridoma or an antigen-specific memory B cell sorting method. Each cDNA of the heavy and light chains of the target antibody was cloned into an IgG1 expression vector and transfected into Expi293F cells to produce a recombinant monoclonal antibody (mAb), which was screened by ELISA and in vitro HBV neutralizing assays. The cross-reactivity of the mAbs to normal human molecules was evaluated by ELISA and immunohistochemistry. RESULTS: Antibody cDNAs were cloned from 11 hybridoma cell lines and 204 HBsAg-bound memory B cells. Three of the resulting recombinant mAbs showed stronger neutralizing activity in vitro than the currently used HBIG. All three bind to the conformational epitope(s) of HBsAg but not to human DNA or cells. DISCUSSION: We successfully isolated HBV-neutralizing monoclonal antibodies from B cells collected from healthy plasma donors boosted against the HBV. To obtain an alternative source for HBIG, HBV-neutralizing monoclonal antibodies from B cells collected from healthy plasma donors boosted against the HBV may be useful.

Epitope analysis of human monoclonal antibodies from a patient with autoimmune factor XIII deficiency reveals their inhibitory mechanisms.

Autoimmune coagulation factor XIII (FXIII) deficiency (AiF13D) is a bleeding disorder caused by anti-FXIII autoantibodies. Recently, we generated human monoclonal antibodies (mAbs) from the peripheral blood of an AiF13D patient and classified them into three groups: FXIII-dissociation inhibitor, FXIII-assembly inhibitor, and non-neutralizing/inhibitory mAbs. However, the epitope region and molecular inhibitory mechanism of each mAb remain unknown. Here, we localized the epitope regions of the representative inhibitory mAbs A69K (dissociation inhibitor) and A78L (assembly inhibitor) to the ß-barrel-2 domain and boundary of ß-barrel-1&2 domains, respectively, of the FXIII-A subunit, by combining a binding assay using its synthesized peptides and a protease-protection assay. Our findings suggest that A69K inhibits the activation-related conformational changes and dissociation of FXIII and that A78L competitively inhibits FXIII-assembly.

CD46 targeted 212Pb alpha particle radioimmunotherapy for prostate cancer treatment.

We recently identified CD46 as a novel prostate cancer cell surface antigen that shows lineage independent expression in both adenocarcinoma and small cell neuroendocrine subtypes of metastatic castration resistant prostate cancer (mCRPC), discovered an internalizing human monoclonal antibody YS5 that binds to a tumor selective CD46 epitope, and developed a microtubule inhibitor-based antibody drug conjugate that is in a multi-center phase I trial for mCRPC (NCT03575819). Here we report the development of a novel CD46-targeted alpha therapy based on YS5. We conjugated 212Pb, an in vivo generator of alpha-emitting 212Bi and 212Po, to YS5 through the chelator TCMC to create the radioimmunoconjugate, 212Pb-TCMC-YS5. We characterized 212Pb-TCMC-YS5 in vitro and established a safe dose in vivo. We next studied therapeutic efficacy of a single dose of 212Pb-TCMC-YS5 using three prostate cancer small animal models: a subcutaneous mCRPC cell line-derived xenograft (CDX) model (subcu-CDX), an orthotopically grafted mCRPC CDX model (ortho-CDX), and a prostate cancer patient-derived xenograft model (PDX). In all three models, a single dose of 0.74 MBq (20 µCi) 212Pb-TCMC-YS5 was well tolerated and caused potent and sustained inhibition of established tumors, with significant increases of survival in treated animals. A lower dose (0.37 MBq or 10 µCi 212Pb-TCMC-YS5) was also studied on the PDX model, which also showed a significant effect on tumor growth inhibition and prolongation of animal survival. These results demonstrate that 212Pb-TCMC-YS5 has an excellent therapeutic window in preclinical models including PDXs, opening a direct path for clinical translation of this novel CD46-targeted alpha radioimmunotherapy for mCRPC treatment.