Murine Monoclonal Antibody Research Articles

HTA58 Cost-Utility Analysis of Palivizumab to Prevent Respiratory Syncytial Virus Infections in the Dominican Republic

Respiratory Syncytial Virus (RSV) is the most common cause of lower respiratory tract infections in infants and young children worldwide. Palivizumab is a humanized murine monoclonal antibody administered monthly as an intramuscular injection and significantly reduces the overall hospitalization rate due to RSV. This study evaluated the cost-utility ratio of palivizumab to prevent RSV in the Dominican Republic.

Oregovomab and non-platinum chemotherapy in PARP inhibitor-resistant ovarian, fallopian tube, or primary peritoneal cancer patients not candidates for platinum retreatment: A multicenter, two-cohort, single-arm phase 2 trial (OPERA/KGOG 3065/APGOT-OV6).

TPS5621 Background: As PAPR inhibitors (PARPis) are becoming the standard of care option for epithelial ovarian cancer (EOC), PARPi-resistant patients are increasing. Moreover, non-platinum single agent chemotherapy in these patients has been found to elicit poor survival outcomes. Oregovomab, an investigational murine monoclonal antibody directed against CA-125, has shown promising efficacy in the previous phase II study of combinational front-line adjuvant chemotherapy. The aim of the present study is to investigate the efficacy of oregovomab + non-platinum-based chemotherapy in PARPi-resistant EOC patients not suitable for platinum-based therapy. Methods: OEPRA is a multi-centre, investigator-initiated, two-cohort, single-arm phase II trial recruiting PARPi-resistant EOC patients from five sites in South Korea. The trial includes patients with recurrent EOC who have experienced disease progression despite treatment with PARPis and who have either received bevacizumab or are not eligible for bevacizumab treatment. Mucinous histology type is excluded. Patients who have received one to three prior lines of chemotherapy are to be assigned to Cohort 1 (oregovamab 2 mg [C1,2,3,5,7 for five doses] + pegylated liposomal doxorubicin [PLD] 40 mg/m2 q4w, n = 28), while patients who have received more than three prior lines of chemotherapy are to be assigned to Cohort 2 (oregovamab 2 mg [C1,2,3,5,7 for five doses] + weekly paclitaxel 80 mg/m2 [D1,8,15 q4w], n = 28). A total of 56 patients will be recruited and treated with oregovomab + PLD / weekly paclitaxel until disease progression, unacceptable toxicity, or withdrawal of patient consent. The primary endpoint is objective response rate by RECIST 1.1. Accrual is expected to be completed in 2023, followed by presentation of results in 2024. The study is registered with Clinicaltrials.gov (NCT05407584). Clinical trial information: NCT05407584 .

Neutralizing and protective murine monoclonal antibodies to the hemagglutinin of influenza H5 clades 2.3.2.1 and 2.3.4.4.

Highly pathogenic avian H5 influenza viruses have spread and diversified genetically and antigenically into multiple clades and subclades. Most isolates of currently circulating H5 viruses are in clade 2.3.2.1 or 2.3.4.4. Panels of murine monoclonal antibodies (mAbs) were generated to the influenza hemagglutinin (HA) of H5 viruses from the clade 2.3.2.1 H5N1 vaccine virus A/duck/Bangladesh/19097/2013 and the clade 2.3.4.4 H5N8 vaccine virus A/gyrfalcon/Washington/41088-6/2014. Antibodies were selected and characterized for binding, neutralization, epitope recognition, cross-reactivity with other H5 viruses, and the ability to provide protection in passive transfer experiments. All mAbs bound homologous HA in an ELISA format; mAbs 5C2 and 6H6 were broadly binding for other H5 HAs. Potently neutralizing mAbs were identified in each panel, and all neutralizing mAbs provided protection in passive transfer experiments in mice challenged with a homologous clade influenza virus. Cross-reacting mAb 5C2 neutralized a wide variety of clade 2.3.2.1 viruses, as well as H5 viruses from other clades, and also provided protection against heterologous H5 clade influenza virus challenge. Epitope analysis indicated that the majority of mAbs recognized epitopes in the globular head of the HA. The mAb 5C2 appeared to recognize an epitope below the globular head but above the stalk region of HA. The results suggested that these H5 mAbs would be useful for virus and vaccine characterization. The results confirmed the functional cross-reactivity of mAb 5C2, which appears to bind a novel epitope, and suggest the therapeutic potential for H5 infections in humans with further development.

Diverse Murine Vaccinations Reveal Distinct Antibody Classes to Target Fusion Peptide and Variation in Peptide Length to Improve HIV Neutralization.

While neutralizing antibodies that target the HIV-1 fusion peptide have been elicited in mice by vaccination, antibodies reported thus far have been from only a single antibody class that could neutralize ~30% of HIV-1 strains. To explore the ability of the murine immune system to generate cross-clade neutralizing antibodies and to investigate how higher breadth and potency might be achieved, we tested 17 prime-boost regimens that utilized diverse fusion peptide-carrier conjugates and HIV-1 envelope trimers with different fusion peptides. We observed priming in mice with fusion peptide-carrier conjugates of variable peptide length to elicit higher neutralizing responses, a result we confirmed in guinea pigs. From vaccinated mice, we isolated 21 antibodies, belonging to 4 distinct classes of fusion peptide-directed antibodies capable of cross-clade neutralization. Top antibodies from each class collectively neutralized over 50% of a 208-strain panel. Structural analyses - both X-ray and cryo-EM - revealed each antibody class to recognize a distinct conformation of fusion peptide and to have a binding pocket capable of accommodating diverse fusion peptides. Murine vaccinations can thus elicit diverse neutralizing antibodies, and altering peptide length during prime can improve the elicitation of cross-clade responses targeting the fusion peptide site of HIV-1 vulnerability. IMPORTANCE The HIV-1 fusion peptide has been identified as a site for elicitation of broadly neutralizing antibodies, with prior studies demonstrating that priming with fusion peptide-based immunogens and boosting with soluble envelope (Env) trimers can elicit cross-clade HIV-1-neutralizing responses. To improve the neutralizing breadth and potency of fusion peptide-directed responses, we evaluated vaccine regimens that incorporated diverse fusion peptide-conjugates and Env trimers with variation in fusion peptide length and sequence. We found that variation in peptide length during prime elicits enhanced neutralizing responses in mice and guinea pigs. We identified vaccine-elicited murine monoclonal antibodies from distinct classes capable of cross-clade neutralization and of diverse fusion peptide recognition. Our findings lend insight into improved immunogens and regimens for HIV-1 vaccine development.

Humanization and characterization of a murine monoclonal neutralizing antibody against human adenovirus 7

Human adenovirus type 7 (HAdV7) is commonly associated with febrile acute respiratory disease (ARD) outbreaks. We have reported that 10G12, a mouse monoclonal antibody (mAb) specifically recognizing and neutralizing HAdV7, is a promising candidate for humanization. In this study, we engineered the six variants of 10G12 with increased degree of humanization and investigated their biological activity. The humanized monoclonal antibody (mAb) 10G12-M2 was shown to retain the parental antibody's high binding affinity, specificity and potent efficacy of viral suppression. The mAb 10G12-M2 recognized a conformational neutralization epitope of the hexon protein. Complex structure-based molecular docking simulation showed that the hexon protein formed several interactions with 10G12-M2, including hydrogen bonds and salt bridges interaction. Physicochemical properties analysis of 10G12-M2 demonstrated that it is stable and desirable lead candidate. In general, 10G12-M2 had excellent biological activity after humanization combined with the potential for use in prophylactic or therapeutic applications against HAdV7.

Establishment and Functional Characterization of Murine Monoclonal Antibodies Recognizing Neuritin

Neuritin represents a neurotrophic factor that is not only important in neuronal development and plasticity but also impacts endothelial angiogenesis, cell migration, tumor growth and the production of antibodies by B cells. We established monoclonal mouse anti-mouse neuritin antibodies by immunizing knock-out mice with two different neuritin-derived peptides. Because neuritin is well conserved between species, these new monoclonal antibodies recognize the neuritin of a wide variety of species, including human. Moreover, they not only recognize specifically surface-bound neuritin expressed by murine follicular regulatory T cells but also the block binding of recombinant neuritin to germinal center B cells. This suggests that these newly generated tools will be of great use in studying neuritin expression and function.

Abstract 3590: Targeted detection of ovarian cancer using functionalized iron oxide nanoparticles

Abstract SuperParaMagnetic Relaxometry (SPMR) is a highly sensitive detection technology that can differentiate the magnetic signature of nanoparticles (NP) bound to tumor cells from unbound nanoparticles. Nanoparticles that reach and bind to the target cells are measurable by superconducting quantum interference device (SQUID) magnetometers (MRX instrument developed in house), while unbound nanoparticles such as those freely circulating in the bloodstream are not detected and bone and normal tissue do not produce any magnetic signal. We have developed a protocol to produce high precision 25nm+1nm (<7% dispersion) Fe3O4 nanoparticle core. These core nanoparticles are coated by a polymer shell functionalized with carboxylate groups. Antibody are then conjugated on the surface providing molecular targeting capabilities and PEG is also attached to the surface to reduce opsonization. In previous studies, we have demonstrated that when conjugated with anti-HER2 antibody such as Herceptin, these nanoparticles exhibited great specificity and selectivity towards HER2+ tumor cells in vitro and in vivo. In current studies, we expanded our nanoparticles applications to other type of cancers, such as ovarian cancer. The CA125 is a tissue-specific antigen expressed in ovarian cancer. It is associated with greater than 80% of epithelial ovarian neoplasms. OC125, a murine monoclonal antibody, reacts with glycosylation-dependent antigens present exclusively in the cleaved portion of the molecule. OC125 antibody is conjugated to our nanoparticles using the same strategy developed for Herceptin nanoparticles. Each nanoparticle contains one to three OC125 antibody molecules covalently attached to the surface based on ELISA analysis. Our results have shown that OC125-NP can distinguish CA125+ and CA125- cell lines, OVCAR3 and HeyA8 respectively. Positive signal can be competed out by pre-incubation with free OC125 antibody and negative cell line produce undetectable SPMR signal, demonstrating good sensitivity, specificity and selectivity. Antigen glypican-1 (GPC1) is a proteoglycan located on cell surface composed of a membrane-associated protein core anchored to the cytoplasmic membrane. GPC1 may play a functional role in the control of cell division and growth regulation. The expression of GPC1 has been found to be elevated in many cancer cells, including ovarian. Applying the same conjugation strategy developed for Herceptin NP, humanized anti-GPC1 antibody is conjugated to the nanoparticles with minor modification. GPC1-NP generate appreciable signal using SKOV3 cell line (GPC1+ ovarian cell lines) and the signal can be competed out by the presence of excess free GPC1 antibody. Together, these results suggest that in additional to breast cancer application, our antibody functionalized nanoparticle system can be developed for other targeted cancer detection, such as ovarian cancer. Citation Format: Marie Zhang, Eric Smith-Nguyen. Targeted detection of ovarian cancer using functionalized iron oxide nanoparticles. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3590.

Abstract 4068: Ganglioside SSEA4 in Ewing sarcoma: A marker of tumor cells with highly aggressive features and a potential immune target

Abstract Cancers can aberrantly express carbohydrate antigens restricted to immature cells during prenatal human development, and these may be exploited for selective immune targeting. We studied expression and functional associations of the globo-series ganglioside stage-specific embryonic antigen 4 (SSEA4), a cell surface marker of human embryonic stem cells, in Ewing sarcoma (EwS). Flow cytometry revealed SSEA4 expression on the cell surface of each of 13 EwS cell lines, with moderate to high expression densities in 6 of 13 (46%) cell lines. Of 31 primary EwS tumor biopsies, 21 (68%) were SSEA4 positive by immunofluorescence staining. No associations of SSEA4 positivity with molecular or clinical disease parameters were found. Comparisons of paired tumor cell subpopulations with high and low/negative SSEA4 expression selected by cell sorting from 8 EwS cell lines revealed increased cell proliferation and colony formation of SSEA4high tumor cells, along with higher doxocrubicin chemoresistance and a higher propensity to migrate in transwell assays. SSEA4 negative cells selected from bulk populations of EwS cells regained SSEA4 expression during continued in vitro culture as well as after in vivo xenografting into immunodeficient mice. SSEA4high expression in EwS cells was not dependent on expression levels of the EWSR1-FLI1 fusion transcript or its direct targets, nor associated with markers of epithelial/mesenchymal plasticity. To retarget human T cells to SSEA4, we generated a second-generation, 41BBζ CAR using the antigen-binding domains of a murine anti-SSEA4 monoclonal antibody. SSEA4-specific CAR T cells specifically interacted with SSEA4 positive EwS cells in a strictly antigen-dependent manner, resulting in effective tumor cell lysis in vitro. We conclude that targeting of SSEA4 with CAR T cells or alternative immune therapeutics could be an attractive strategy to eliminate tumor cell subsets with high propensity to drive disease progression in EwS and therefore deserves further evaluation towards clinical translation. Citation Format: Silke Jamitzky, Bianca Altvater, Carolin Krekeler, Laura Hoen, Caroline Brandes, Lisa Richter, Julia Ebbinghaus, Laurin Ochs, Nicole Farwick, Katja Urban, Dennis Görlich, Ian Johnston, Rita Pfeifer, Claudia Rossig, Wolfgang Hartmann, Sareetha Kailayangiri. Ganglioside SSEA4 in Ewing sarcoma: A marker of tumor cells with highly aggressive features and a potential immune target. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4068.

Intact Transition Epitope Mapping—Serological Inspection by Epitope EXtraction (ITEM—SIX)

Precision medicine requests accurate serological inspections to precisely stratify patients for targeted treatment. Intact transition epitope mapping analysis proved surrogate seroconversion of a model organism's serum when spiked with a monoclonal murine anti-Ovalbumin antibody (mAb) with epitope resolution. Isolation of the IgG fraction from blood serum applied two consecutive protein precipitation steps followed by ultrafiltration and resulted in an ESI-MS analysis-ready IgG preparation. For epitope mapping by epitope extraction, the Ovalbumin antigen was digested with trypsin. After desalting, the peptide mixture was added to the ESI-MS-ready IgG preparation from mAb-spiked serum and the solution was incubated to form an immune complex between the Ovalbumin-derived epitope peptide and the anti-Ovalbumin mAb. Then, the entire mixture of proteins and peptides was directly electrosprayed. Sorting of ions in the mass spectrometer's gas phase, dissociation of the immune complex ions by collision-induced dissociation, and recording of the epitope peptide ion that had been released from the immune complex proved the presence of the anti-Ovalbumin mAb in serum. Mass determination of the complex-released epitope peptide ion with isotope resolution is highly accurate, guaranteeing high specificity of this novel analysis approach, which is termed Intact Transition Epitope Mapping-Serological Inspections by Epitope EXtraction (ITEM-SIX).

Monoclonal antibody targeting a novel linear epitope on nucleoprotein confers pan-reactivity to influenza A virus.

Nucleoprotein (NP) functions crucially in the replicative cycle of influenza A virus (IAV) via forming the ribonucleoprotein complex together with PB2, PB1, and PA proteins. As its high conservation, NP ranks one of the hot targets for design of universal diagnostic reagents and antiviral drugs for IAV. Here, we report an anti-NP murine monoclonal antibody (mAb) 5F10 prepared from traditional lymphocyte hybridoma technique with the immunogen of a clade 2.3.4.4 H5N1 subtype avian influenza virus. The specificity of mAb 5F10 to NP protein was confirmed by immunofluorescence assay and western blotting, and the mAb 5F10 could be used in immunoprecipitation and immunohistochemistry assays. Importantly, mAb 5F10 possessed broad-spectrum reactivity against H1~H11 subtypes of avian influenza viruses, including various HA clades of H5Nx subtype. In addition, mAb 5F10 also showed good affinity with H1N1 and H3N2 subtype influenza viruses of swine and human origin. Furthermore, the recognized antigenic epitope of mAb 5F10 was identified to consist of the conserved amino acid motif 81EHPSA85 in the second flexible loop region of NP protein through screening the phage display peptide library. Collectively, the mAb 5F10 which recognizes the novel universal NP linear B-cell epitope of IAV with diverse origins and subtypes will be a powerful tool for NP protein-based structural, functional, and mechanistic studies, as well as the development of detection methods and universal vaccines for IAV. KEY POINTS: • A broad-spectrum mAb against various subtypes and sources of IAV was developed • The mAb possessed good reactivity in IFA, western blot, IP, and IHC assays • The mAb targeted a novel conserved linear B-cell epitope involving 81EHPSA85 on NP protein.

Antibody-based protection against respiratory syncytial virus in mice and their offspring through vectored immunoprophylaxis.

Respiratory syncytial virus (RSV) causes acute lower respiratory tract infections, with potential lower respiratory tract infections, which can be particularly problematic in infants and the elderly. There are no approved vaccines for RSV. The current standard of care for high-risk individuals is monthly administration of palivizumab, a humanized murine monoclonal antibody (mAb) targeting the RSV fusion protein. Adeno-associated virus (AAV)-mediated expression of mAbs has previously led to sustained expression of therapeutic concentrations of mAbs in several animal models, representing an alternative to repetitive passive administration. Intramuscular (IM) administration of AAV6.2FF expressing RSV antibodies, palivizumab or hRSV90, resulted in high concentrations of human (h)IgG1 mAbs in the serum and at various mucosal surfaces, while intranasal administration limited hIgG expression to the respiratory tract. IM administration of AAV6.2FF-hRSV90 or AAV6.2FF-palivizumab in a murine model provided sterilizing immunity against challenge with RSV A2. Evidence of maternal passive transfer of vectorized hRSV90 was detected in both murine and ovine models, with circulating mAbs providing sterilizing immunity in mouse progeny. Finally, addition of a "kill switch" comprised of LoxP sites flanking the mAb genes resulted in diminished serum hIgG after AAV-DJ-mediated delivery of Cre recombinase to the same muscle group that was originally transduced with the AAV-mAb vector. The ability of this AAV-mAb system to mediate robust, sustained mAb expression for maternal transfer to progeny in murine and ovine models emphasizes the potential of this platform for use as an alternative prophylactic vaccine for protection against neonatal infections, particularly in high-risk infants.

Preparation and characterization of a neutralizing murine monoclonal antibody against tetanus toxin

After Clostridium tetani infects the human body, it propagates under anaerobic conditions and produces tetanus neurotoxin (TeNT). TeNT can affect the central nervous system, inhibit the release of neurotransmitters, and result in respiratory failure, which are the root causes of death in tetanus patients. Identifying monoclonal antibodies (mAbs) targeting TeNT with neutralizing activity is urgently needed for the prevention and treatment of tetanus infection. In this study, through immunizing BALB/c mice with tetanus toxoid (TT), we obtained six positive hybridoma cell lines (1A7, 2C7, 3A7, 3H4, 4C1, and 4E12). Antibody isotyping showed that the antibodies are all of the IgG1/κ subclass. Ascites fluid was prepared by allogeneic ascites induction and the antibodies were purified through protein G affinity chromatography columns. Purities of the produced murine mAbs were all greater than 95%. All six antibodies bound to linear epitopes, among which 3A7 bound to the TeNT/L domain and the other five antibodies bound to the TeNT/Hc domain. Moreover, the affinity constants of these six antibodies against the antigen were all in the nanomolar range, and the affinity of 4E12 antibody reached the picomolar range. Results from toxin-neutralization assays in mice showed that 2C7 antibody delayed animal death, while 1A7, 3A7, 3H4, and 4E12 antibodies conferred partial protection. Additionally, 4C1 antibody offered complete protection, as 200 μg of 4C1 antibody fully protected against toxin challenge with 10 LD50 of TeNT and had a window period of 1 h. Antibody epitope grouping results revealed that the binding epitopes of 4C1 antibody were different from those of the other five antibodies. When 4C1 antibody was used in combination with another antibody, the neutralizing activities of antibodies were all evidently enhanced. Specifically, 4C1 combined with 3A7 antibody led to the greatest improvement in neutralizing activities, and 20 μg antibodies total (10 + 10 μg) fully protected against toxin challenge with 10 LD50. When 4E12, 3A7, and 4C1 antibodies were used in combination, 18 μg antibodies total (6 + 6 + 6 μg) completely neutralized 10 LD50 toxin. The present study derived murine mAbs with neutralizing activities and laid the foundation for follow-up therapeutic drug development for TeNT poisoning as well as establishment of TeNT detection methods.

Progress and perspectives of rabbit monoclonal antibodies

Since the advent of murine hybridomas, the emergence of a variety of monoclonal antibody (mAb) technologies has enabled the wide applications of murine monoclonal antibodies in medicine, life science, agronomy, and food science. Compared with murine monoclonal antibodies, rabbit monoclonal antibodies (RabmAbs) exhibit higher affinity, presenting with increased detection sensitivity and greater specificity for the particular structure of epitopes. This paper reviews the history, preparation techniques, advantages and disadvantages, current applications, and future perspectives for RabmAbs.

Оценка эффективности различных адъювантов при получении мышиных моноклональных антител к рецептор-связывающему домену S-белка SARS-CоV-2

Оценка эффективности различных адъювантов при получении мышиных моноклональных антител к рецептор-связывающему домену S-белка SARS-CоV-2

Development, Preparation and Evaluation of a Murine Monoclonal Antibodies Based Interferon-Gamma Enzyme-Linked Immunosorbent Assay (ELISA) for Rapid and Accurate Diagnosis of Bovine Tuberculosis

Development, Preparation and Evaluation of a Murine Monoclonal Antibodies Based Interferon-Gamma Enzyme-Linked Immunosorbent Assay (ELISA) for Rapid and Accurate Diagnosis of Bovine Tuberculosis

A Monoclonal Antibody Targeting C-Terminal Domain of Transmissible Gastroenteritis Virus Spike Protein.

The structure and function of the C-terminus domain (CTD) of porcine transmissible gastroenteritis virus (TGEV) spike protein remain largely unknown, thereby a specific monoclonal antibody (MAb) allows us to fully understand this domain. In this study, we developed a murine MAb against CTD of TGEV spike protein, as evidenced by the results of indirect fluorescent assay, Western blotting, and fluorescence-activated cell sorter. Further study showed that the MAb is able to exclusively recognize a 12-residue peptide (FKNVSDGVIYSV) derived from CTD of TGEV spike protein. This MAb can be used to elucidate the potential function of CTD of TGEV spike in virus attachment and entry, and warrants further intensive investigation.

A group of infection-enhancing and focus size-reducing monoclonal antibodies recognized an ‘a and c’ strands epitope in the pr domain of Dengue Virus prM

Partial cleavage of a dengue virus envelope protein, prM, by furin results in a mixture of extracellular particles with variable levels of maturation and infectivity. Partially mature particles can infect leukocytes via interaction between the prM-anti-prM antibody complex with Fcγ receptors. Known prM epitopes involved in antibody-mediated infection are localized to the pr domain. In this study, a group of murine anti-prM monoclonal antibodies with strong infection-enhancing activity was found to reduce the focus size of subsets of multiple dengue serotypes that they could enhance. By employing sets of overlapping peptides, four antibodies recognizing 2-mercaptoethanol-insensitive epitopes were mapped to a common tetrapeptide located distantly in the b-c loop and furin binding site. Substitution mutations of each, or both, of the tetrapeptides in virus-like particles, however, failed to reduce binding. Further mapping experiments were performed using immature virus-like particles with abolished furin binding site to minimize the differential influence of various pr substitutions on pr-M cleavage. Reduction of antibody binding was detected when single alanine substitutions were introduced into the ‘a’ strand and ‘c' strand of pr domain. These findings suggest that the pr ‘a and c' strands region is the major binding site of these unusual focus size-reducing anti-prM antibodies.

Targeted alpha therapy with the 224Ra/212Pb-TCMC-TP-3 dual alpha solution in a multicellular tumor spheroid model of osteosarcoma.

Osteosarcoma patients with overt metastases at primary diagnosis have a 5-year survival rate of less than 20%. TP-3 is a murine IgG2b monoclonal antibody with high affinity for an epitope residing on the p80 osteosarcoma cell surface membrane antigen. The tumor-associated antigen p80 is overexpressed in osteosarcomas, and has very low normal tissue expression. We propose a novel dual alpha targeting solution containing two radionuclides from the same decay chain, including the bone-seeking 224Ra, and cancer cell-surface seeking 212Pb-TCMC-TP-3 for the treatment of osteoblastic bone cancers, circulating cancer cells and micrometastases. In this in vitro study, the cytotoxic effects of 212Pb-TCMC-TP-3 (single alpha solution) and 224Ra/212Pb-TCMC-TP-3 (dual alpha solution) were investigated in a multicellular spheroid model mimicking micrometastatic disease in osteosarcoma. OHS spheroids with diameters of 253 ± 98 μm treated with 4.5, 2.7, and 3.3 kBq/ml of 212Pb-TCMC-TP-3 for 1, 4, and 24 h, respectively, were disintegrated within 3 weeks. The 212Pb-TCMC-TP-3 induced a 7-fold delay in spheroid doubling time compared to a 28-times higher dose with the non-specific 212Pb-TCMC-rituximab. The 224Ra/212Pb-TCMC-TP-3 completely disintegrated spheroids with diameters of 218-476 μm within 3 and 2 weeks after 4 and 24 h incubation with 5 kBq/ml, respectively. Treatment with 1 kBq/ml of 224Ra/212Pb-TCMC-TP-3 for 24 h caused an 11.4-fold reduction in spheroid viability compared with unconjugated 224Ra/212Pb. The single and dual alpha solutions with TP-3 showed cytotoxicity in spheroids of clinically relevant size, which warrant further testing of the dual alpha solution using in vivo osteosarcoma models.

Evaluation of panel of neutralising murine monoclonal antibodies and a humanised bispecific antibody against influenza A(H1N1)pdm09 virus infection in a mouse model

The influenza A (H1N1) pdm09 virus attracted public attention because of its high prevalence. The annual global morbidity and mortality rates of influenza remain high despite the application of influenza vaccines and antiviral drugs, which indicates the urgent need to identify a more effective strategy for controlling and treating A(H1N1) pdm09 influenza infection. To produce a highly effective therapeutic with broad specificity for A(H1N1) pdm09 influenza viruses, we generated 15 murine monoclonal antibodies (mAbs) via hybridoma technology: 11 mAbs demonstrated 20–100% therapeutic protection in a mouse model of A(H1N1) pdm09 infection at a single dose of 10 mg/kg. A humanised bispecific antibody (Bis-Hu11-1) generated based on the mAbs 3D2 and 3D11, combining the specificities of the two mAbs, was also effective in preventing and treating A(H1N1) pdm09 infection in a mouse model. Bis-Hu11-1 demonstrated hemagglutination inhibition (HI) activity against the escape mutants generated by its parental mAbs that resulted in the obvious reduction in the HI activity of the parental mAbs. In summary, we generated a panel of neutralising mAbs against A(H1N1) pdm09 influenza virus. This study presents a promising method for developing neutralising antibodies that potentially target a series of antigenically diverse influenza viruses.

Phase 1 study of intraventricular 131I-omburtamab targeting B7H3 (CD276)-expressing CNS malignancies

BackgroundThe prognosis for metastatic and recurrent tumors of the central nervous system (CNS) remains dismal, and the need for newer therapeutic targets and modalities is critical. The cell surface glycoprotein B7H3 is expressed on a range of solid tumors with a restricted expression on normal tissues. We hypothesized that compartmental radioimmunotherapy (cRIT) with the anti-B7H3 murine monoclonal antibody omburtamab injected intraventricularly could safely target CNS malignancies.Patients and methodsWe conducted a phase I trial of intraventricular 131I-omburtamab using a standard 3 + 3 design. Eligibility criteria included adequate cerebrospinal fluid (CSF) flow, no major organ toxicity, and for patients > dose level 6, availability of autologous stem cells. Patients initially received 74 MBq radioiodinated omburtamab to evaluate dosimetry and biodistribution followed by therapeutic 131I-omburtamab dose-escalated from 370 to 2960 MBq. Patients were monitored clinically and biochemically for toxicity graded using CTCAEv 3.0. Dosimetry was evaluated using serial CSF and blood sampling, and serial PET or gamma-camera scans. Patients could receive a second cycle in the absence of grade 3/4 non-hematologic toxicity or progressive disease.ResultsThirty-eight patients received 100 radioiodinated omburtamab injections. Diagnoses included metastatic neuroblastoma (n = 16) and other B7H3-expressing solid tumors (n = 22). Thirty-five patients received at least 1 cycle of treatment with both dosimetry and therapy doses. Acute toxicities included < grade 4 self-limited headache, vomiting or fever, and biochemical abnormalities. Grade 3/4 thrombocytopenia was the most common hematologic toxicity. Recommended phase 2 dose was 1850 MBq/injection. The median radiation dose to the CSF and blood by sampling was 1.01 and 0.04 mGy/MBq, respectively, showing a consistently high therapeutic advantage for CSF. Major organ exposure was well below maximum tolerated levels. In patients developing antidrug antibodies, blood clearance, and therefore therapeutic index, was significantly increased. In patients receiving cRIT for neuroblastoma, survival was markedly increased (median PFS 7.5 years) compared to historical data.ConclusionscRIT with 131I-omburtamab is safe, has favorable dosimetry and may have a therapeutic benefit as adjuvant therapy for B7-H3-expressing leptomeningeal metastases.Trial registration: clinicaltrials.gov NCT00089245, August 5, 2004.