Individual Monoclonal Antibodies Research Articles

IL1RAP-specific T cell engager depletes acute myeloid leukemia stem cells

BackgroundThe interleukin-1 receptor accessory protein (IL1RAP) is highly expressed on acute myeloid leukemia (AML) bulk blasts and leukemic stem cells (LSCs), but not on normal hematopoietic stem cells (HSCs), providing an opportunity to target and eliminate the disease, while sparing normal hematopoiesis. Herein, we report the activity of BIF002, a novel anti-IL1RAP/CD3 T cell engager (TCE) in AML.MethodsAntibodies to IL1RAP were isolated from CD138+ B cells collected from the immunized mice by optoelectric positioning and single cell sequencing. Individual mouse monoclonal antibodies (mAbs) were produced and characterized, from which we generated BIF002, an anti-human IL1RAP/CD3 TCE using Fab arm exchange. Mutations in human IgG1 Fc were introduced to reduce FcγR binding. The antileukemic activity of BIF002 was characterized in vitro and in vivo using multiple cell lines and patient derived AML samples.ResultsIL1RAP was found to be highly expressed on most human AML cell lines and primary blasts, including CD34+ LSC-enriched subpopulation from patients with both de novo and relapsed/refractory (R/R) leukemia, but not on normal HSCs. In co-culture of T cells from healthy donors and IL1RAPhigh AML cell lines and primary blasts, BIF002 induced dose- and effector-to-target (E:T) ratio-dependent T cell activation and leukemic cell lysis at subnanomolar concentrations. BIF002 administered intravenously along with human T cells led to depletion of leukemic cells, and significantly prolonged survival of IL1RAPhigh MOLM13 or AML patient-derived xenografts with no off-target side effects, compared to controls. Of note, BiF002 effectively redirects T cells to eliminate LSCs, as evidenced by the absence of disease initiation in secondary recipients of bone marrow (BM) from BIF002+T cells-treated donors (median survival not reached; all survived > 200 days) compared with recipients of BM from vehicle- (median survival: 26 days; p = 0.0004) or isotype control antibody+T cells-treated donors (26 days; p = 0.0002).ConclusionsThe novel anti-IL1RAP/CD3 TCE, BIF002, eradicates LSCs and significantly prolongs survival of AML xenografts, representing a promising, novel treatment for AML.

Rapid isolation of anti-idiotype aptamers for quantification of human monoclonal antibodies against SARS-CoV-2 spike protein

Therapeutic antibodies that block viral entry have already proven to be important, first line drugs for treatments of viral infections. In the case of SARS-CoV-2, combinations of multiple therapeutic antibodies may need to be rapidly identified and formulated in a way that blocks each new, predominant variant of the virus. For efficient introduction of any new antibody combination into patients, it is important to be able to monitor patient-specific pharmacokinetics of individual antibodies, which would include the time course of their specific capacity to block the viral spike proteins. Here, we present three examples of microfluidic-based rapid isolation of companion reagents useful for establishing combination antibody therapies. These reagents are specific three-dimensional imprints of variable regions of individual human monoclonal antibodies against the -spike protein of SARS-CoV-2 virus in the form of oligonucleotide-based ligands (aptamers). We implement these anti-idiotypic aptamers as bioreceptors in graphene-based field-effect transistor sensors to accomplish label free, rapid, and sensitive detection of matching antibodies within minutes. Through this work we have demonstrated the general applicability of anti-idiotype aptamers as capture reagents in quantification of active forms of monoclonal antibodies in complex biological mixtures.

Febrile temperatures modulate the formation of immune complexes relevant for autoimmune diseases

BackgroundAutoimmune disorders encompass a diverse subset of diseases whose common symptoms include, among others, fever. Fever of unknown origins, once an infectious or tumor agent have been ruled out as possible causes, may originate with an autoimmune disease. ObjectiveTo determine the role of febrile temperatures on the stability of antigens pertinent to autoimmunity, and on the immune complexes they form with commercial therapeutic monoclonal antibodies. MethodsUsing molecular dynamics simulations, the binding between four antigens belonging to a set of autoimmune diseases and their individual monoclonal antibodies was investigated under different febrile temperatures. ResultsIt was determined that at febrile temperatures, monoclonal antibodies used in the therapy of autoimmune diseases bind with higher binding free energy to pertinent antigens, once the autoimmune condition has been established and treatment is warranted. ConclusionPerforming molecular dynamics simulations at fever temperatures may be important for delineating the role antibodies may play in other diseases, including in cancers and infections.

Humoral immunity to SARS-CoV-2 elicited by combination COVID-19 vaccination regimens.

The SARS-CoV-2 pandemic prompted a global vaccination effort and the development of numerous COVID-19 vaccines at an unprecedented scale and pace. As a result, current COVID-19 vaccination regimens comprise diverse vaccine modalities, immunogen combinations, and dosing intervals. Here, we compare vaccine-specific antibody and memory B cell responses following two-dose mRNA, single-dose Ad26.COV.2S, and two-dose ChAdOx1, or combination ChAdOx1/mRNA vaccination. Plasma-neutralizing activity, as well as the magnitude, clonal composition, and antibody maturation of the RBD-specific memory B cell compartments, showed substantial differences between the vaccination regimens. While individual monoclonal antibodies derived from memory B cells exhibited similar binding affinities and neutralizing potency against Wuhan-Hu-1 SARS-CoV-2, there were significant differences in epitope specificity and neutralizing breadth against viral variants of concern. Although the ChAdOx1 vaccine was inferior to mRNA and Ad26.COV.2S in several respects, biochemical and structural analyses revealed enrichment in a subgroup of memory B cell neutralizing antibodies with distinct RBD-binding properties resulting in remarkable potency and breadth.

Induced antigen-binding polyreactivity in human serum IgA

Previous studies have shown that polyreactive antibodies play an important role in the frontline defense against the dissemination of pathogens in the pre-immune host. Interestingly, antigen-binding polyreactivity can not only be inherent, but also acquired post-translationally. The ability of individual monoclonal IgG and IgE antibodies to acquire polyreactivity following contact with various agents that destabilize protein structure (urea, low pH) or have a pro-oxidative potential (heme, ferrous ions) has been studied in detail. However, to the best of our knowledge this property of human IgA has previously been described only cursorily. In the present study pooled human serum IgA and two human monoclonal IgA antibodies were exposed to buffers with acidic pH, to free heme or to ferrous ions, and the antigen-binding behavior of the native and modified IgA to viral and bacterial antigens were compared using immunoblot and ELISA. We observed a dose-dependent increase in reactivity to several bacterial extracts and to pure viral antigens. This newly described property of IgA may have therapeutic potential as has already been shown for pooled IgG with induced polyreactivity.

Antibody-Mediated Immunosuppression Can Result from RBC Antigen Loss Independent of Fcγ Receptors in Mice

Background: Prophylaxis against red blood cell (RBC) alloimmunization can be achieved by Rh immune globulin (RhIg), a polyclonal anti-RhD antibody preparation used to prevent de novo anti-RhD alloantibody formation, a process commonly termed antibody-mediated immunosuppression (AMIS). As the availability of RhIg donors can be limited and the only available immunoprophylaxis is against RhD, there is a critical need to develop monoclonal antibody alternatives to RhD and immunoprophylaxis strategies in general to other RBC alloantigens. As different monoclonal antibodies to the RhD antigen can have differential immunological outcomes,understanding the mechanism regarding how monoclonal antibodies induce AMIS can facilitate the development and accurate assessment of monoclonal antibodies against RhD and other RBC alloantigens for appropriate therapeutic activity. Recent studies suggest that AMIS may occur through alterations in the target antigen, otherwise known as antigen modulation. However, several studies suggest that AMIS may occur independent of antigen modulation. In particular, AMIS to RBCs that transgenically express the HOD (hen egg lysozyme (HEL), ovalbumin and Duffy) antigen have been shown to occur independent of the activating Fcγ receptors thought to be required for modulation of the HEL antigen. Therefore, we sought to determine whether anti-HEL monoclonal antibodies can induce antigen modulation and whether AMIS to HOD RBCs occurs via monoclonal antibody-induced alterations to the target antigen.Methods: Using RBCs transgenically express the HOD antigen, we transferred HOD RBCs into C57BL/6 wild-type (WT) or FcγR-chain knockout recipients (FcγR KO) in the presence or absence of anti-HEL monoclonal antibodies individually or in combination. Serum was collected at days 3, 5, 7, 14, 21 and 28 post-transfusion. Antibody development in WT or FcγR KO recipients was examined by flow crossmatch, where serum was incubated with HOD RBCs. Following incubation, RBCs were washed and incubated with fluorescently-tagged secondary antibody and analyzed using flow cytometry. To determine the impact of antibody engagement, RBCs were sampled at 10 minutes and 2 hours, along with days 1, 2, 3 and 5 post-transfusion. The level of detectable HEL antigen, complement deposition, HOD RBC survival and bound antibody to the RBC surface was measured by flow cytometry.Results: A combination of anti-HEL antibodies suppressed anti-HOD IgM formation more effectively than monoclonal anti-HEL antibodies alone, which correlated with the rate of decrease in bound antibody and detectable HEL on HOD RBCs over time. Unexpectedly, combination or individual anti-HEL monoclonal antibodies not only induced AMIS, but also likewise induced antigen modulation in FcγR KO recipients. Loss of detectable antigen did not appear to reflect anti-HEL induced complement fixation and subsequent complement-mediated masking of the HEL antigen, as no complement could be detected on HOD RBCs when evaluated in parallel. Furthermore, regardless of anti-HEL antibody exposure, in combination or individually, anti-HEL antibodies failed to induce significant RBC clearance in WT or FcγR KO recipients. Pre-exposure of HOD RBCs to anti-HEL antibodies reduced antigen levels and likewise suppressed anti-HOD antibody formation following HOD RBC exposure.Conclusion: In summary, these results suggest that antibody-mediated antigen modulation may reflect a mechanism of AMIS that can occur independent of activating FcγRs and may provide a surrogate to identify antibodies capable of inducing AMIS against different RBC alloantigens. DisclosuresNo relevant conflicts of interest to declare.

Antibody resistance of SARS-CoV-2 variants B.1.351 and B.1.1.7.

The COVID-19 pandemic has had widespread effects across the globe, and its causative agent, SARS-CoV-2, continues to spread. Effective interventions need to be developed to end this pandemic. Single and combination therapies with monoclonal antibodies have received emergency use authorization1-3, and more treatments are under development4-7. Furthermore, multiple vaccine constructs have shown promise8, including two that have an approximately 95% protective efficacy against COVID-199,10. However, these interventions were directed against the initial SARS-CoV-2 virus that emerged in 2019. The recent detection of SARS-CoV-2 variants B.1.1.7 in the UK11 and B.1.351 in South Africa12 is of concern because of their purported ease of transmission and extensive mutations in the spike protein. Here we show that B.1.1.7 is refractory to neutralization by most monoclonal antibodies against the N-terminal domain of the spike protein and is relatively resistant to a few monoclonal antibodies against the receptor-binding domain. It is not more resistant to plasma from individuals who have recovered from COVID-19 or sera from individuals who have been vaccinated against SARS-CoV-2. The B.1.351 variant is not only refractory to neutralization by most monoclonal antibodies against the N-terminal domain but also by multiple individual monoclonal antibodies against the receptor-binding motif of the receptor-binding domain, which is mostly due to a mutation causing an E484K substitution. Moreover, compared to wild-type SARS-CoV-2, B.1.351 is markedly more resistant to neutralization by convalescent plasma (9.4-fold) and sera from individuals who have been vaccinated (10.3-12.4-fold). B.1.351 and emergent variants13,14 with similar mutations in the spike protein present new challenges for monoclonal antibody therapies and threaten the protective efficacy of current vaccines.

A hydrophobic interaction chromatography method suitable for quantitating individual monoclonal antibodies contained in co-formulated drug products

A co-formulated monoclonal antibody (mAb) product containing two or more antibodies offers several therapeutic advantages. However, quantitating the individual antibodies in a co-formulated product is challenging due to the similar biochemical and biophysical properties of mAbs. To identify a method suitable to support the development of a co-formulated drug product with three mAbs, a hydrophobic interaction chromatography method was developed, utilizing a Dionex ProPac HIC-10 column, 100 mM phosphate buffer (pH 7.0), and an ammonium sulfate gradient. Compared to other methods that were evaluated, the HIC method showed the best separation, as well as accurate quantitation of the three mAbs in the co-formulated drug product. The calibration curves were linear over column loads of 225 μg to 900 μg (R2 > 0.99) and the accuracy was between 91% and 106%. Intra-day and inter-day precisions (RSD) were less than or equal to 0.6 % and 1.7%, respectively. The method was used to quantitate individual mAb concentrations in the co-formulated drug product and to monitor any changes in concentration during stability studies.

High microbiota reactivity of adult human intestinal IgA requires somatic mutations.

The gut is home to the body's largest population of plasma cells. In healthy individuals, IgA is the dominating isotype, whereas patients with inflammatory bowel disease also produce high concentrations of IgG. In the gut lumen, secretory IgA binds pathogens and toxins but also the microbiota. However, the antigen specificity of IgA and IgG for the microbiota and underlying mechanisms of antibody binding to bacteria are largely unknown. Here we show that microbiota binding is a defining property of human intestinal antibodies in both healthy and inflamed gut. Some bacterial taxa were commonly targeted by different monoclonal antibodies, whereas others selectively bound single antibodies. Interestingly, individual human monoclonal antibodies from both healthy and inflamed intestines bound phylogenetically unrelated bacterial species. This microbiota cross-species reactivity did not correlate with antibody polyreactivity but was crucially dependent on the accumulation of somatic mutations. Therefore, our data suggest that a system of affinity-matured, microbiota cross-species-reactive IgA is a common aspect of SIgA-microbiota interactions in the gut.

Affinities of human NMDA receptor autoantibodies: implications for disease mechanisms and clinical diagnostics

Anti-N-methyl-d-aspartate receptor (NMDAR) encephalitis is a common autoimmune encephalitis presenting with psychosis, dyskinesias, autonomic dysfunction and seizures. The underlying autoantibodies against the NR1 subunit are directly pathogenic by disrupting synaptic NMDAR currents. However, antibody titers correlate only partially with the clinical outcome, suggesting the relevance of other factors such as antibody affinity. We thus determined the binding curves of human monoclonal autoantibodies and patients’ cerebrospinal fluid (CSF) against NR1-expressing HEK293 cells using flow cytometry. Antibody affinity was highly variable with binding constants (half-maximal concentration, c50) ranging from 1 to 74 µg/ml for monoclonal antibodies. Comparing values of individual monoclonal antibodies with human CSF samples suggested that the CSF signal is predominantly represented by higher-affinity antibodies, potentially in a concentration range of NR1 antibodies between 0.1 and 5 µg/ml, roughly reflecting 1–10% of total CSF IgG in NMDAR encephalitis. Binding curves further depended on the CSF composition which must be considered when interpreting established clinical routine assays. Normalization of measurements using reference samples allowed high reproducibility. Accurate and reproducible measurement of NR1 antibody binding suggested that biophysical properties of the antibody might contribute to disease severity. Normalization of the data can be an elegant way to allow comparable inter-laboratory quantification of CSF NR1 antibody titers in autoimmune encephalitis patients, a prerequisite for use as surrogate markers in clinical trials. Based on our calculations, low-affinity antibodies can easily remain undetected in routine cell-based assays, indicating that their relation to clinical symptoms should be analyzed in future studies.

Development of an analytical method to assess the occupational health risk of therapeutic monoclonal antibodies using LC-HRMS.

Monoclonal antibodies are a group of commonly used therapeutics, whose occupational health risk is still discussed controversially. The long-term low-dose exposure side effects are insufficiently evaluated; hence, discussions are often based on a theoretical level or extrapolating side effects from therapeutic dosages. While some research groups recommend applying the precautionary principle for monoclonal antibodies, others consider the exposure risk too low for measures taken towards occupational health and safety. However, both groups agree that airborne monoclonal antibodies have the biggest risk potential. Therefore, we developed a peptide-based analytical method for occupational exposure monitoring of airborne monoclonal antibodies. The method will allow collecting data about the occupational exposure to monoclonal antibodies. Thus, the mean daily intake for personnel in pharmacies and the pharmaceutical industry can be determined for the first time and will help to substantiate the risk assessment by relevant data. The introduced monitoring method includes air sampling, sample preparation and detection by liquid chromatography coupled with high-resolution mass spectrometry of individual monoclonal antibodies as well as sum parameter. For method development and validation, a chimeric (rituximab), humanised (trastuzumab) and a fully humanised (daratumumab) monoclonal antibody are used. A limit of detection between 1μg per sample for daratumumab and 25μg per sample for the collective peptide is achieved. Graphical abstract Demonstration of the analytical workflow, from the release of monoclonal antibodies to the detection as single substances as well as sum parameter.

CD335 (NKp46)+ T-Cell Recruitment to the Bovine Upper Respiratory Tract during a Primary Bovine Herpesvirus-1 Infection.

Bovine natural killer (NK) cells were originally defined by the NK activation receptor CD335 [natural killer cell p46-related protein (NKp46)], but following the discovery of NKp46 expression on human T-cells, the definition of conventional bovine NK cells was modified to CD335+CD3− cells. Recently, a bovine T-cell population co-expressing CD335 was identified and these non-conventional T-cells were shown to produce interferon (IFN)-γ and share functional properties with both conventional NK cells and T-cells. It is not known, however, if CD335+ bovine T-cells are recruited to mucosal surfaces and what chemokines play a role in recruiting this unique T-cell subpopulation. In this study, bovine herpesvirus-1 (BHV-1), which is closely related to herpes simplex virus-1, was used to investigate bovine lymphocyte cell populations recruited to the upper respiratory tract following a primary respiratory infection. Immunohistochemical staining with individual monoclonal antibodies revealed significant (P < 0.05) recruitment of CD335+, CD3+, and CD8+ lymphocyte populations to the nasal turbinates on day 5 following primary BHV-1 infection. Dual-color immunofluorescence revealed that cells recruited to nasal turbinates were primarily T-cells that co-expressed both CD335 and CD8. This non-conventional T-cell population represented 77.5% of CD355+ cells and 89.5% of CD8+ cells recruited to nasal turbinates on day 5 post-BHV-1 infection. However, due to diffuse IFN-γ staining of nasal turbinate tissue, it was not possible to directly link increased IFN-γ production following BHV-1 infection with the recruitment of non-conventional T-cells. Transcriptional analysis revealed CCL4, CCL5, and CXCL9 gene expression was significantly (P < 0.05) upregulated in nasal turbinate tissue following BHV-1 infection. Therefore, no single chemokine was associated with recruitment of non-conventional T-cells. In conclusion, the specific recruitment of CD335+ and CD8+ non-conventional T-cells to viral-infected tissue suggests that these cells may play an important role in either the clearance of a primary BHV-1 infection or regulating host responses during viral infection. The early recruitment of non-conventional T-cells following a primary viral infection may enable the host to recognize viral-infected cells through NKp46 while retaining the possibility of establishing T-cell immune memory.

An Adaptive Phase 3 Trial for Evaluating Two Monoclonal Antibodies and the Combination for Prevention of Recurrence of C. difficile Infection

ABSTRACTPre-clinical and epidemiology studies suggest that antibodies directed at C. difficile toxins protect against recurrence of C. difficile infection (CDI). Human monoclonal antibodies (MAb) targeting toxin A and toxin B were developed and evaluated in Phase 2 trials as adjunctive therapy in CDI patients. Results showed that the MAb to toxin A alone was not sufficient to protect against recurrent disease, but that the combination of the two MAbs reduced CDI recurrence relative to placebo. Because human data for the individual MAbs were considered insufficient to select a regimen for evaluation in confirmatory trials, an adaptive Phase 3 program was developed to assess efficacy and safety of the two individual MAbs, as well as the combination product.An adaptive Phase 3 study was designed with a planned interim analysis to: (1) assess futility and (2) drop individual MAb therapy arms if inferior to the combination therapy arm. A four-step multiplicity strategy provides control of Type 1 error across multiple comparisons at two analysis times. Design performance was further assessed using simulations. Results demonstrate that Type 1 error is controlled at planned levels, and that the design provides sufficient power to select an effective treatment regimen.

Polyclonal anti-Candida antibody improves phagocytosis and overall outcome in zebrafish model of disseminated candidiasis

Fungal infections are a major cause of animal and plant morbidity and mortality worldwide. Effective biological therapeutics could complement current antifungal drugs, but understanding of their in vivo mechanisms has been hampered by technical barriers to intravital imaging of host-pathogen interactions. Here we characterize the fungal infection of zebrafish as a model to understand the mechanism-of-action for biological antifungal therapeutics through intravital imaging of these transparent animals. We find that non-specific human IgG enhances phagocytosis by zebrafish phagocytes in vivo. Polyclonal anti-Candida antibodies enhance containment of fungi in vivo and promote survival. Analysis suggests that early phagocytic containment is a strong prognostic indicator for overall survival. Although polyclonal anti-Candida antibodies protect against disease, this is not necessarily the case for individual monoclonal anti-Candida antibodies. Thus, the zebrafish appears to provide a useful model host for testing if a biological therapeutic promotes phagocytosis in vivo and enhances protection against candidemia.

Abstract 873: A novel MET-EGFR bispecific antibody LY3164530 shows advantage over combining MET and EGFR antibodies in tumor inhibition and overcome resistance

Abstract The epidermal growth factor receptor (EGFR) and the mesenchymal-epithelial transition factor (MET) are receptor tyrosine kinases that each plays a key role in cancer signaling. Co-expression and activation of MET and EGFR are found in a number of tumor types, including non-small cell lung, colorectal, gastric, and head and neck cancers (Nanjo et al. 2013). Blocking one receptor tends to up-regulate the other, leading to resistance to single-agent treatment (Engelman et al. 2007). Amplification of MET and/or high levels of HGF expression has been observed in non-small cell lung cancer (NSCLC) patients with intrinsic or acquired resistance to tyrosine kinase inhibitors of EGFR (Engelman et al. 2007; Yano et al. 2011). Conversely, MET-amplified lung cancer cells exposed to MET-inhibiting agents for a prolonged period develop resistance via the EGFR pathway (McDermott et al. 2010). The crosstalk between the MET and EGFR pathways suggests that dual inhibition of these targets may lead to improved outcomes for patients with MET- and EGFR-positive cancers, and that simultaneous inhibition may overcome or delay resistance compared to the blockade of just a single pathway. LY3164530 is an engineered mAb-scFv bispecific antibody that consists of an immunoglobulin G4 (IgG4) antibody to MET and a single-chain variable fragment (scFv) to EGFR fused to the N-terminus of each heavy chain (HC). LY3164530 binds to extracellular domains of MET and EGFR with high affinity and inhibits signaling via both MET and EGFR receptors by blocking ligand binding and internalizing and degrading both receptors. In tumor cells, it binds and co-immunoprecipitates both receptors. LY3164530 has increased avidity binding to MET in cells expressing higher level of EGFR. This increased avidity binding leads to better neutralization of HGF compared to parental MET antibody in these cells. Surprisingly, LY3164530 has superior activity in internalizing/degrading EGFR (wild type and mutant forms) in vitro and in vivo relative to the combination of LY2875358 (i.e., emibetuzumab) and cetuximab in cells expressing high MET and EGFR. In addition, LY3164530 has superior activity in overcoming HGF-mediated resistance to erlotinib, gefitinib, lapatinib, or vemurafenib as compared to the combination of individual monoclonal antibodies targeting these receptors in cell-based assays. In vivo, administration of LY3164530 results in dose-dependent antitumor activity in multiple cell line-derived NSCLC and gastric xenografts. The antitumor activity of LY3164530 is equivalent, and in some cases superior to the combination of emibetuzumab and cetuximab in NSCLC and gastric tumor models. The Phase 1 study with LY3164530 is on-going (NCT02221882). Citation Format: Ling Liu, Wei Zeng, Marcio Chedid, Yi Zeng, Sheng-hung Tschang, Yu Tian, Ying Tang, Jirong Lu. A novel MET-EGFR bispecific antibody LY3164530 shows advantage over combining MET and EGFR antibodies in tumor inhibition and overcome resistance. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 873.

Functional Activity of Antibodies Directed towards Flagellin Proteins of Non-Typhoidal Salmonella.

Non-typhoidal Salmonella (NTS) serovars Typhimurium and Enteritidis are major causes of invasive bacterial infections in children under 5 years old in sub-Saharan Africa, with case fatality rates of ~20%. There are no licensed NTS vaccines for humans. Vaccines that induce antibodies against a Salmonella Typhi surface antigen, Vi polysaccharide, significantly protect humans against typhoid fever, establishing that immune responses to Salmonella surface antigens can be protective. Flagella proteins, abundant surface antigens in Salmonella serovars that cause human disease, are also powerful immunogens, but the functional capacity of elicited anti-flagellar antibodies and their role in facilitating bacterial clearance has been unclear. We examined the ability of anti-flagellar antibodies to mediate microbial killing by immune system components in-vitro and assessed their role in protecting mice against invasive Salmonella infection. Polyclonal (hyperimmune sera) and monoclonal antibodies raised against phase 1 flagellin proteins of S. Enteritidis and S. Typhimurium facilitated bacterial uptake and killing of the homologous serovar pathogen by phagocytes. Polyclonal anti-flagellar antibodies accompanied by complement also achieved direct bacterial killing. Serum bactericidal activity was restricted to Salmonella serovars expressing the same flagellin used as immunogen. Notably, individual anti-flagellin monoclonal antibodies with complement were not bactericidal, but this biological activity was restored when different monoclonal anti-flagellin antibodies were combined. Passive transfer immunization with a monoclonal IgG antibody specific for phase 1 flagellin from S. Typhimurium protected mice against lethal challenge with a representative African invasive S. Typhimurium strain. These findings have relevance for the use of flagellin proteins in NTS vaccines, and confirm the role of anti-flagellin antibodies as mediators of protective immunity.

A First-in-Human Study To Assess the Safety and Pharmacokinetics of Monoclonal Antibodies against Human Cytomegalovirus in Healthy Volunteers.

Human cytomegalovirus (HCMV) can cause significant disease in immunocompromised patients and treatment options are limited by toxicities. CSJ148 is a combination of two anti-HCMV human monoclonal antibodies (LJP538 and LJP539) that bind to and inhibit the function of viral HCMV glycoprotein B (gB) and the pentameric complex, consisting of glycoproteins gH, gL, UL128, UL130, and UL131. Here, we evaluated the safety, tolerability, and pharmacokinetics of a single intravenous dose of LJP538 or LJP539 or their combination in healthy volunteers. Adverse events and laboratory abnormalities occurred sporadically with similar incidence between antibody and placebo groups and without any apparent relationship to dose. No subject who received antibody developed a hypersensitivity, infusion-related reaction or anti-drug antibodies. After intravenous administration, both LJP538 and LJP539 demonstrated typical human IgG1 pharmacokinetic properties, with slow clearances, limited volumes of distribution, and long terminal half-lives. The pharmacokinetic parameters were linear and dose proportional for both antibodies across the 50-fold range of doses evaluated in the study. There was no apparent impact on pharmacokinetics when the antibodies were administered alone or in combination. CSJ148 and the individual monoclonal antibodies were safe and well tolerated, with pharmacokinetics as expected for human immunoglobulin.

A bi-functional antibody-receptor domain fusion protein simultaneously targeting IGF-IR and VEGF for degradation

Bi-specific antibodies (BsAbs), which can simultaneously block 2 tumor targets, have emerged as promising therapeutic alternatives to combinations of individual monoclonal antibodies. Here, we describe the engineering and development of a novel, human bi-functional antibody-receptor domain fusion molecule with ligand capture (bi-AbCap) through the fusion of the domain 2 of human vascular endothelial growth factor receptor 1 (VEGFR1) to an antibody directed against insulin-like growth factor – type I receptor (IGF-IR). The bi-AbCap possesses excellent stability and developability, and is the result of minimal engineering. Beyond potent neutralizing activities against IGF-IR and VEGF, the bi-AbCap is capable of cross-linking VEGF to IGF-IR, leading to co-internalization and degradation of both targets by tumor cells. In multiple mouse xenograft tumor models, the bi-AbCap improves anti-tumor activity over individual monotherapies. More importantly, it exhibits superior inhibition of tumor growth, compared with the combination of anti-IGF-IR and anti-VEGF therapies, via powerful blockade of both direct tumor cell growth and tumor angiogenesis. The unique “capture-for-degradation” mechanism of the bi-AbCap is informative for the design of next-generation bi-functional anti-cancer therapies directed against independent signaling pathways. The bi-AbCap design represents an alternative approach to the creation of dual-targeting antibody fusion molecules by taking advantage of natural receptor-ligand interactions.

Application of D2-40/S100 and CD34/S100 detection in the diagnosis of blood and lymphatic vessel invasion of cutaneous malignant melanoma

Objective To develop an immunohistochemical assay for the diagnosis of cutaneous malignant melanoma (CMM) micrometastasis via blood and lymphatic vessels, and to evaluate its clinical significance. Methods Fifty-three patients (32 males and 21 females) histopathologically diagnosed as CMM were enrolled in this study. The patients were aged (61.2±8.4) years (range, 52- 72 years). Tissue specimens were obtained from the central area of tumor in each case , and also from removed lymph nodes in some cases. The average duration of follow-up was (65.00±5.68) months. During the follow-up, 17 patients died of the recurrence or metastasis of CMM, and 6 patients were lost to follow-up. The expressions of D2- 40, S100 and CD34 antigens in 53 tissue specimens were examined by immunohistochemical staining with three individual monoclonal antibodies, or by an immunohistochemical method using 2 two-antibody cocktails (D2-40/S100 and CD34/S100) and double-color chromogens in single tissue sections. Results Of the 53 patients, 30.19% (16/53) were positive for hematoxylin-eosin (HE) staining combined with immunohistochemical staining with individual monoclonal antibodies, and 49.06% (26/53) for the immunohistochemical method using two-antibody cocktails and double-color chromogens. Statistical differences were found in the positive rate between the two methods (χ2=3.94, P<0.05). Compared with patients without blood/lymph vessel tumor emboli, those with blood/lymph vessel tumor emboli showed higher lymph node metastasis rate (80.77%(21/26) vs. 37.04% (10/27), χ2=10.43, P<0.001), but lower five-year survival rate (42.31% (11/26) vs. 70.37%(19/27), χ2=4.25, P<0.05). Conclusions The immunohistochemical method with two-antibody cocktails is superior to HE staining combined with immunohistochemical staining with individual monoclonal antibodies in the detection of blood/lymph vessel tumor emboli. And blood/lymph vessel tumor emboli may be an important prognostic factor in patients with CMM. Key words: Melanoma; Lymphatic metastasis; S100 proteins; Antigens, CD34; D2-40; Immunohistochemical method with antibody cocktails

Blood Vascular Changes Associated with Chronic Active Multiple Sclerosis Plaques: Detection by Anti-Human Brain Vascular Endothelia-Specific Monoclonal Antibodies

Recent studies suggest that brain angiogenesis is an important component of multiple sclerosis (MS) lesions and may be a potential target for investigating the pathogenesis and treatment of the disease. In this preliminary study, a panel of blood vessel-specific mouse monoclonal antibodies raised against normal human whole temporal lobe homogenates was used to study changes in vascular immunoreactivity and morphological features in cerebral tissue obtained from two clinically and neuropath logically confirmed cases of chronic active multiple sclerosis. Immunoperoxidase and immunofluorescence staining techniques revealed more frequent detection of intensely labelled blood vessels within active multiple sclerosis plaques compared to the adjacent white matter and normal control brain sections. Strongly reactive vascular endothelial sprouts as detected by epifluorescence immunohistochemistry were observed in whole vessel mounts isolated by partial homogenization and sieving of cerebral tissue containing active plaques, but not in vessels isolated from normal control cerebral tissue. These vascular changes suggest increased angiogenesis and endothelial proliferation within the active multiple sclerosis plaques and may be part of the inflammatory response by the central nervous system to this disease. This study also suggests that monoclonal antibodies raised specifically against whole homogenates of human brain could potentially be useful tools for investigations of antigenic heterogeneity and morphologic characterization of vascular endothelial changes associated with multiple sclerosis lesions in the human model system. Plans are under way to further investigate the exact biochemical nature of the specific antigens being recognized by the individual monoclonal antibodies.