Early Parenting Interactions and First-Time Mothers' Postnatal Depression and Parental Competence.

Objectives: Schema Therapy, an approach that integrates cognitive-behavioural and attachment principles, helps us understand the impact of early interactions with caregivers on adult mental health. These early interactions can be assessed through Schema Therapy-informed tools; however, these tools have yet to be used with a postnatal population, which represents a period of vulnerability for new mothers. Therefore, the present study aimed to evaluate the impact of positive and negative early parenting interactions on a first-time mother's mental health and her sense of competence during the postnatal period, using recently revised and newly developed Schema Therapy-informed tools. Design: This is a cross-sectional study. Method: First-time mothers (N = 220) participated in an online survey within 12 months post-birth. Participants completed the Positive Parenting Schema Inventory (PPSI), Young Parenting Inventory-Revised (YPI-R2), Edinburgh Postnatal Depression Scale (EPDS), and Parenting Sense of Competence (PSOC) scale. The data were analysed using hierarchical multiple regression and mediational analysis. Results: Negative early interactions with mothers and fathers led to greater postnatal depressive symptomology, while positive early interactions with mothers led to fewer postnatal depressive symptoms. Mediation analyses revealed that postnatal depressive symptoms mediated early parenting interactions and participants' sense of parenting competence as a new mother. Conclusions: The protective effects of positive early interactions with caregivers can help first-time mothers' postnatal emotional adjustment and their sense of competence through diminished postnatal depressive symptoms. However, the enduring effects of negative early interactions with caregivers can contribute to a first-time mother's risk of developing postnatal depression and negatively affect her sense of parental competence.

Investing in late-stage clinical trials and manufacturing of product candidates for five major infectious diseases: a modelling study of the benefits and costs of investment in three middle-income countries.

BACKGROUND: Investing in late-stage clinical trials, trial sites, and production capacity for new health products could improve access to vaccines, therapeutics, and infectious disease diagnostics in middle-income countries. This study assesses the case for such investment in three of these countries: India, Kenya, and South Africa. METHODS: We applied investment case modelling and assessed how many cases, deaths, and disability-adjusted life years (DALYs) could be averted from the development and manufacturing of new technologies (therapeutics and vaccines) in these countries from 2021 to 2036, for five diseases-HIV, tuberculosis, malaria, pneumonia, and diarrhoeal diseases. We also estimated the economic benefits that might accrue from making these investments and we developed benefit-cost ratios for each of the three middle-income countries. Our modelling applies two investment case perspectives: a societal perspective with all costs and benefits measured at the societal level, and a country perspective to estimate how much health and economic benefit accrues to each middle-income country for every dollar invested in clinical trials and manufacturing by the middle-income country government. For each perspective, we modelled two scenarios: one that considers only domestic health and economic benefits; and one that includes regional health and economic benefits. In the regional scenarios, we assumed that new products developed and manufactured in India would benefit eight countries in south Asia, whereas new products developed and manufactured in Kenya would benefit all 21 countries in the Common Market for Eastern and Southern Africa (COMESA). We also assumed that all 16 countries in the Southern African Development Community (SADC) would benefit from products developed and manufactured in South Africa. FINDINGS: From 2021 to 2036, product development and manufacturing in Kenya could avert 4·44 million deaths and 206·27 million DALYs in the COMESA region. In South Africa, it could prevent 5·19 million deaths and 253·83 million DALYs in the SADC region. In India, it could avert 9·76 million deaths and 374·42 million DALYs in south Asia. Economic returns would be especially high if new tools were produced for regional markets rather than for domestic markets only. Under a societal perspective, regional returns outweigh investments by a factor of 20·51 in Kenya, 33·27 in South Africa, and 66·56 in India. Under a country perspective, the regional benefit-cost ratios amount to 60·71 in India, 8·78 in Kenya, and 11·88 in South Africa. INTERPRETATION: Our study supports the creation of regional hubs for clinical trials and product manufacturing compared with narrow national efforts. FUNDING: Bill & Melinda Gates Foundation.

A phase I/II study to evaluate safety, tolerability and immunogenicity of Hillchol®, an inactivated single Hikojima strain based oral cholera vaccine, in a sequentially age descending population in Bangladesh.

BACKGROUND: The World Health Organization (WHO) recommends the use of oral cholera vaccines (OCVs) as part of an integrated control program, both in highly endemic settings and during cholera epidemics. The available and internationally recommended WHO-prequalified OCVs (Dukoral, Shanchol, Euvichol) contain multiple heat and formalin-killed V. cholerae strains of Inaba and Ogawa serotypes. MSD Wellcome Trust Hilleman Laboratories Pvt. Ltd. in technical collaboration with University of Gothenburg, Sweden has developed a new single strain OCV, Hillchol. This vaccine consists of formaldehyde-inactivated whole cell El Tor V. cholerae O1 bacteria engineered into the Hikojima serotype for stable expression of both the Ogawa (AB) and Inaba (AC) LPS antigens on the bacterial surface. We evaluated the safety and immunogenicity of this novel and potentially much less expensive OCV in comparison with Shanchol. METHODS: We conducted a randomized, non-inferiority, age-descending clinical trial of OCV (Hillchol vs. Shanchol) in the Mirpur area of Dhaka city from July 2016 to May 2017. This study was carried out in three different age cohorts (1-<5, 5-17 and ≥18 years old). Two doses of vaccine were given at 14 days intervals to 560 healthy participants. FINDINGS: No serious adverse events were reported. There were no significant differences in the rates of adverse events between the test vaccine (Hillchol) and the comparator (Shanchol) group. Serum vibriocidal antibody responses in all age groups combined were comparable for all the O1 Ogawa (59% vs. 67%; 90% CI of difference: -14.55, -0.84) and Inaba (70% vs. 71%; 90% CI of difference: -7.24, 5.77) serotypes, showing that the Hillchol vaccine was non-inferior to Shanchol. This new vaccine was also non-inferior to Shanchol in the different age strata. CONCLUSION: The safety and immunogenicity profile of the new OCV Hillchol is comparable to Shanchol in persons residing in a cholera-endemic setting. ClinicalTrials.gov number: NCT02823899.

Development of Hillchol®, a low-cost inactivated single strain Hikojima oral cholera vaccine.

Cholera remains an important global health problem with up to 4 million cases and 140,000 deaths annually. Oral cholera vaccines (OCVs) are now a cornerstone of the WHOs "Ending Cholera - A Global Roadmap to 2030" global program for the eventual elimination of cholera. There are currently three WHO prequalified OCVs available, Dukoral®, Shanchol® and Euvichol-Plus®. These vaccines are effective but due to a multiple strain composition and two different methods of inactivation, are complex and costly to manufacture. We describe here the characterization and industrial scale development of Hillchol®; a novel, likely affordable single-component OCV for low and middle-income countries. Hillchol® consists of formalin-inactivated bacteria of a stable recombinant Vibrio cholerae O1 El Tor Hikojima serotype strain expressing approximately 50% each of Ogawa and Inaba O1 LPS antigens. The novel OCV can be manufactured on an industrial scale at a low cost. Hillchol® was well tolerated in animal toxicology studies and shown to have non-inferior oral immunogenicity in mice for both intestinal-mucosal and serological immune responses when compared with a WHO-prequalified OCV. The optimized production of this single component OCV will reduce cost of OCV production and thus substantially increase vaccine availability. Based on these results, Hillchol® has been produced at a GMP facility and used successfully for clinical phase I/II studies.

A randomized Phase I/II study to evaluate safety and reactogenicity of a heat-stable rotavirus vaccine in healthy adults followed by evaluation of the safety, reactogenicity, and immunogenicity in infants.

Objectives: To assess the safety and reactogenicity of single oral dose of heat-stable rotavirus vaccine (HSRV) in healthy adults aged 18-45 years followed by assessment of safety, reactogenicity, and immunogenicity of three doses of HSRV in healthy infants aged 6-8 weeks at enrollment.Trial Design: Single-center randomized controlled, sequential, blinded (adults) and open-label (infants).Setting: Single site at International Center for Diarrheal Disease Research, Bangladesh (icddr,b).Participants: Fifty eligible adults randomized in 1:1 ratio (HSRV: Placebo) followed by 50 eligible infants randomized in 1:1 ratio (HSRV: Comparator (RotaTeq®, pentavalent human-bovine (WC3) reassortant live-attenuated, rotavirus vaccine)).Intervention: Adults received either a single dose of HSRV or placebo and followed for 14 days. Infants received three doses of either HSRV or comparator with a follow-up for 28 days after each dose.Main Outcome Measures: Solicited and unsolicited adverse events (AEs) along with any serious adverse events (SAEs) were part of the safety and reactogenicity assessment in adults and infants whereas serum anti-rotavirus IgA response rates were part of immunogenicity assessment in infants only. Post-vaccination fecal shedding of vaccine-virus rotavirus strains was also determined in adults and infants.Results: In this study, HSRV, when compared with placebo, did not result in increase in solicited adverse events (solicited AEs) in adults. In infants, HSRV had a safety profile similar to comparator vis-à-vis solicited AEs. In infants, fecal shedding of vaccine-virus strains was not detected in HSRV recipients but was observed in two comparator recipients. Percentage of infants exhibiting threefold rise in serum anti-rotavirus IgA titers from baseline to 1-month post-dose 3 in HSRV group was 88% (22/25) and 84% (21/25) in comparator group.Conclusion: HSRV was found to be generally well-tolerated in both adults and infants and immunogenic in infants.

Rotavirus vaccine efficacy: current status and areas for improvement.

The difference noted in Rotavirus vaccine efficiency between high and low income countries correlates with the lack of universal access to clean water and higher standards of hygiene. Overcoming these obstacles will require great investment and also time, therefore more effective vaccines should be developed to meet the needs of those who would benefit the most from them. Increasing our current knowledge of mucosal immunity, response to Rotavirus infection and its modulation by circadian rhythms could point at actionable pathways to improve vaccination efficacy, especially in the case of individuals affected by environmental enteropathy. Also, a better understanding and validation of Rotavirus entry factors as well as the systematic monitoring of dominant strains could assist in tailoring vaccines to individual's needs. Another aspect that could improve vaccine efficiency is targeting to M cells, for which new ligands could potentially be sought. Finally, alternative mucosal adjuvants and vaccine expression, storage and delivery systems could have a positive impact in the outcome of Rotavirus vaccination.

Rational design of heat stable lyophilized rotavirus vaccine formulations.

To develop a safe and efficacious heat-stable rotavirus vaccine, new lyophilized formulations were developed using rotavirus serotypes constituting RotaTeq®. A series of formulation compositions, differing in buffering agents, bulking agents, cryoprotectants, amino acids and divalent cations, were screened for their ability to provide stability to rotavirus serotypes during lyophilization and when stored under elevated temperatures for extended periods. Lead formulations and lyophilization cycles were further optimized. Stability profiles of thus optimized formulations showed their ability to retain the potency of rotavirus for > 36 months at 5°C, 20 months at 37°C, and 7 months at 45°C. The heat-stable lyophilized rotavirus formulations developed met the all critical quality attributes for appearance, heat-stability during storage, moisture content as well as pH, viability and stability after reconstitution and has great potential to be used as vaccine candidates for improving access in low-income countries.

Bidirectional relationships between cigarette use and spinal pain in adolescents accounting for psychosocial functioning.

OBJECTIVES: The objective of this study was to determine the presence of possible bidirectional causal pathways between cigarette use and spinal pain in adolescents controlling for psychosocial functioning, using a prospective longitudinal research design. DESIGN: The data for this study was collected from a cohort of Australian adolescents at 14 (n = 1596) and 17 (n = 1291) years of age. METHODS: Multivariable logistic regression was used to assess i) if cigarette use at 14 predicted low back pain (LBP), mid back pain (MBP) or neck shoulder pain (NSP) at 17 in those adolescents without each condition at 14, and ii) if back pain only (BP), neck shoulder pain only (NSP) or comorbid BP and NSP at 14 predicted cigarette use at 17 in those adolescents who did not smoke at 14. RESULTS: After controlling for psychosocial factors, cigarette use at 14 predicted MBP at 17 (OR = 3.05, p = 0.049, 95% CI [1.01-9.24]). BP only at 14 was a significant risk factor for smoking at 17 (OR = 1.84, p = 0.006, 95% CI [1.19-2.84]) after controlling for psychosocial factors. CONCLUSION: The findings indicate that there are bi-directional relationships between cigarette use and spinal pain and that these relationships vary with pain location.

Atypical antigen recognition mode of a shark immunoglobulin new antigen receptor (IgNAR) variable domain characterized by humanization and structural analysis.

The immunoglobulin new antigen receptors (IgNARs) are a class of Ig-like molecules of the shark immune system that exist as heavy chain-only homodimers and bind antigens by their single domain variable regions (V-NARs). Following shark immunization and/or in vitro selection, V-NARs can be generated as soluble, stable, and specific high affinity monomeric binding proteins of ∼12 kDa. We have previously isolated a V-NAR from an immunized spiny dogfish shark, named E06, that binds specifically and with high affinity to human, mouse, and rat serum albumins. Humanization of E06 was carried out by converting over 60% of non-complementarity-determining region residues to those of a human germ line Vκ1 sequence, DPK9. The resulting huE06 molecules have largely retained the specificity and affinity of antigen binding of the parental V-NAR. Crystal structures of the shark E06 and its humanized variant (huE06 v1.1) in complex with human serum albumin (HSA) were determined at 3- and 2.3-Å resolution, respectively. The huE06 v1.1 molecule retained all but one amino acid residues involved in the binding site for HSA. Structural analysis of these V-NARs has revealed an unusual variable domain-antigen interaction. E06 interacts with HSA in an atypical mode that utilizes extensive framework contacts in addition to complementarity-determining regions that has not been seen previously in V-NARs. On the basis of the structure, the roles of various elements of the molecule are described with respect to antigen binding and V-NAR stability. This information broadens the general understanding of antigen recognition and provides a framework for further design and humanization of shark IgNARs.

Comprehensive interrogation of a minimalist synthetic CDR-H3 library and its ability to generate antibodies with therapeutic potential.

We have generated large libraries of single-chain Fv antibody fragments (>10(10) transformants) containing unbiased amino acid diversity that is restricted to the central combining site of the stable, well-expressed DP47 and DPK22 germline V-genes. Library WySH2A was constructed to examine the potential for synthetic complementarity-determining region (CDR)-H3 diversity to act as the lone source of binding specificity. Library WySH2B was constructed to assess the necessity for diversification in both the H3 and L3. Both libraries provided diverse, specific antibodies, yielding a total of 243 unique hits against 7 different targets, but WySH2B produced fewer hits than WySH2A when selected in parallel. WySH2A also consistently produced hits of similar quality to WySH2B, demonstrating that the diversification of the CDR-L3 reduces library fitness. Despite the absence of deliberate bias in the library design, CDR length was strongly associated with the number of hits produced, leading to a functional loop length distribution profile that mimics the biases observed in the natural repertoire. A similar trend was also observed for the CDR-L3. After target selections, several key amino acids were enriched in the CDR-H3 (e.g., small and aromatic residues) while others were reduced (e.g., strongly charged residues) in a manner that was specific to position, preferentially occurred in CDR-H3 stem positions, and tended towards residues associated with loop stabilization. As proof of principle for the WySH2 libraries to produce viable lead candidate antibodies, 114 unique hits were produced against Delta-like ligand 4 (DLL4). Leads exhibited nanomolar binding affinities, highly specific staining of DLL4+ cells, and biochemical neutralization of DLL4-NOTCH1 interaction.

An ultra-specific avian antibody to phosphorylated tau protein reveals a unique mechanism for phosphoepitope recognition.

Highly specific antibodies to phosphoepitopes are valuable tools to study phosphorylation in disease states, but their discovery is largely empirical, and the molecular mechanisms mediating phosphospecific binding are poorly understood. Here, we report the generation and characterization of extremely specific recombinant chicken antibodies to three phosphoepitopes on the Alzheimer disease-associated protein tau. Each antibody shows full specificity for a single phosphopeptide. The chimeric IgG pT231/pS235_1 exhibits a K(D) of 0.35 nm in 1:1 binding to its cognate phosphopeptide. This IgG is murine ortholog-cross-reactive, specifically recognizing the pathological form of tau in brain samples from Alzheimer patients and a mouse model of tauopathy. To better understand the underlying binding mechanisms allowing such remarkable specificity, we determined the structure of pT231/pS235_1 Fab in complex with its cognate phosphopeptide at 1.9 Å resolution. The Fab fragment exhibits novel complementarity determining region (CDR) structures with a "bowl-like" conformation in CDR-H2 that tightly and specifically interacts with the phospho-Thr-231 phosphate group, as well as a long, disulfide-constrained CDR-H3 that mediates peptide recognition. This binding mechanism differs distinctly from either peptide- or hapten-specific antibodies described to date. Surface plasmon resonance analyses showed that pT231/pS235_1 binds a truly compound epitope, as neither phosphorylated Ser-235 nor free peptide shows any measurable binding affinity.

Characterization of the immunoglobulin repertoire of the spiny dogfish (Squalus acanthias).

The cartilaginous fish (chimeras, sharks, skates and rays) are the oldest group relative to mammals in which an adaptive immune system founded upon immunoglobulins has been found. In this manuscript we characterize the immunoglobulins of the spiny dogfish (Squalus acanthias) at both the molecular and expressed protein levels. Despite the presence of hundreds of IgM clusters in this species the serum levels of this isotype are comparatively low. However, analysis of cDNA sequences and serum protein suggests microheterogeneity in the IgM heavy chains and supports the proposal that different clusters are preferentially used in the two forms (monomer or pentamer) of this isotype. We also found that the IgNAR isotype in this species exists in a previously unknown multimeric format in serum. Finally, we identified a new form of the IgW isotype (the shark IgD orthologue), in which the leader is spliced directly to the first constant domain, resulting in a molecule lacking an antigen-binding domain.

Improving the pharmacokinetic properties of biologics by fusion to an anti-HSA shark VNAR domain.

Advances in recombinant antibody technology and protein engineering have provided the opportunity to reduce antibodies to their smallest binding domain components and have concomitantly driven the requirement for devising strategies to increase serum half-life to optimise drug exposure, thereby increasing therapeutic efficacy. In this study, we adopted an immunization route to raise picomolar affinity shark immunoglobulin new antigen receptors (IgNARs) to target human serum albumin (HSA). From our model shark species, Squalus acanthias, a phage display library encompassing the variable binding domain of IgNAR (VNAR) was constructed, screened against target, and positive clones were characterized for affinity and specificity. N-terminal and C-terminal molecular fusions of our lead hit in complex with a naïve VNAR domain were expressed, purified and exhibited the retention of high affinity binding to HSA, but also cross-selectivity to mouse, rat and monkey serum albumin both in vitro and in vivo. Furthermore, the naïve VNAR had enhanced pharmacokinetic (PK) characteristics in both N- and C-terminal orientations and when tested as a three domain construct with naïve VNAR flanking the HSA binding domain at both the N and C termini. Molecules derived from this platform technology also demonstrated the potential for clinical utility by being available via the subcutaneous route of delivery. This study thus demonstrates the first in vivo functional efficacy of a VNAR binding domain with the ability to enhance PK properties and support delivery of multifunctional therapies.

Fundamental characteristics of the immunoglobulin VH repertoire of chickens in comparison with those of humans, mice, and camelids.

Examination of 1269 unique naive chicken V(H) sequences showed that the majority of positions in the framework (FW) regions were maintained as germline, with high mutation rates observed in the CDRs. Many FW mutations could be clearly related to the modulation of CDR structure or the V(H)-V(L) interface. CDRs 1 and 2 of the V(H) exhibited frequent mutation in solvent-exposed positions, but conservation of common structural residues also found in human CDRs at the same positions. In comparison with humans and mice, the chicken CDR3 repertoire was skewed toward longer sequences, was dominated by small amino acids (G/S/A/C/T), and had higher cysteine (chicken, 9.4%; human, 1.6%; and mouse, 0.25%) but lower tyrosine content (chicken, 9.2%; human, 16.8%; and mouse 26.4%). A strong correlation (R(2) = 0.97) was observed between increasing CDR3 length and higher cysteine content. This suggests that noncanonical disulfides are strongly favored in chickens, potentially increasing CDR stability and complexity in the topology of the combining site. The probable formation of disulfide bonds between CDR3 and CDR1, FW2, or CDR2 was also observed, as described in camelids. All features of the naive repertoire were fully replicated in the target-selected, phage-displayed repertoire. The isolation of a chicken Fab with four noncanonical cysteines in the V(H) that exhibits 64 nM (K(D)) binding affinity for its target proved these constituents to be part of the humoral response, not artifacts. This study supports the hypothesis that disulfide bond-constrained CDR3s are a structural diversification strategy in the restricted germline v-gene repertoire of chickens.

Necrotizing enterocolitis in a 16-day-old, term neonate.

Necrotizing enterocolitis in a term neonate is a rare presentation to the ED. Prompt diagnosis and management is critical because of its rarity and the reported mortality of 5-20%. We report a case of necrotizing enterocolitis in a term neonate who presented at 16 days of age with bloody stool.

High-throughput measurement, correlation analysis, and machine-learning predictions for pH and thermal stabilities of Pfizer-generated antibodies.

Generating stable antibodies is an important goal in the development of antibody-based drugs. Often, thermal stability is assumed predictive of overall stability. To test this, we used different internally created antibodies and first studied changes in antibody structure as a function of pH, using the dye ANS. Comparison of the pH(50) values, the midpoint of the transition from the high-pH to the low-pH conformation, allowed us for the first time to rank antibodies based on their pH stability. Next, thermal stability was probed by heating the protein in the presence of the dye Sypro Orange. A new data analysis method allowed extraction of all three antibody unfolding transitions and showed close correspondence to values obtained by differential scanning calorimetry. T(1%) , the temperature at which 1% of the protein is unfolded, was also determined. Importantly, no correlations could be found between thermal stability and pH(50) , suggesting that to accurately quantify antibody stability, different measures of protein stability are necessary. The experimental data were further analyzed using a machine-learning approach with a trained model that allowed the prediction of biophysical stability using primary sequence alone. The pH stability predictions proved most successful and were accurate to within pH ±0.2.

Coupling of aggregation and immunogenicity in biotherapeutics: T- and B-cell immune epitopes may contain aggregation-prone regions.

Biotherapeutics, including recombinant or plasma-derived human proteins and antibody-based molecules, have emerged as an important class of pharmaceuticals. Aggregation and immunogenicity are among the major bottlenecks during discovery and development of biotherapeutics. Computational tools that can predict aggregation prone regions as well as T- and B-cell immune epitopes from protein sequence and structure have become available recently. Here, we describe a potential coupling between aggregation and immunogenicity: T-cell and B-cell immune epitopes in therapeutic proteins may contain aggregation-prone regions. The details of biological mechanisms behind this observation remain to be understood. However, our observation opens up an exciting potential for rational design of de-immunized novel, as well as follow on biotherapeutics with reduced aggregation propensity.

Complex pharmacokinetics of a humanized antibody against human amyloid beta peptide, anti-abeta Ab2, in nonclinical species.

PURPOSE: Anti-Aß Ab2 (Ab2) is a humanized monoclonal antibody against amino acids 3-6 of primate (but not rodent) amyloid ß (Aß) and is being evaluated for the treatment of Alzheimer's disease (AD). This study was conducted to predict the human pharmacokinetics of Ab2. METHODS: In vivo PK profile of Ab2 in preclinical species and in vitro mechanistic studies in preclinical and human systems were used for pharmacokinetic predictions. RESULTS: In Tg2576 and PSAPP mice that have ~100-fold higher circulating levels of human Aß compared to humans, elimination of Ab2 was target-mediated, such that exposure was 5-10 fold lower compared to wild-type rodents or to PDAPP mice that have human Aß concentrations in plasma similar to humans. In cynomolgus monkeys, the t(1/2) of Ab2 was faster (<2.5 days) compared to that of the control antibody (~13 days). The fast elimination of Ab2 in cynomolgus monkeys was linked to off-target binding to cynomolgus monkey fibrinogen that was also causing incomplete recovery of Ab2 in cynomolgus monkey serum in blood partitioning experiments. Ab2 had significantly weaker to undetectable binding to human (and mouse) fibrinogen and had good recovery in human serum in blood partitioning experiments. CONCLUSIONS: These data predict that elimination of Ab2 in healthy or AD humans is expected to be slow, with t(1/2) similar to that observed for other humanized antibodies.

Distinct in vitro binding properties of the anti-CD20 small modular immunopharmaceutical 2LM20-4 result in profound and sustained in vivo potency in cynomolgus monkeys.

OBJECTIVES: To characterize the in vitro binding and effector function properties of CD20-directed small modular immunopharmaceutical (SMIP) 2LM20-4, and to compare its in vivo B-cell depletion activity with the mutated 2LM20-4 P331S [no in vitro complement-dependent cytotoxicity (CDC)] and rituximab in cynomolgus monkeys. METHODS: Direct binding is examined in flow cytometry, confocal microscopy, scatchard and lipid raft assays. Effector function assays include CDC and Fc-mediated cellular toxicity. In the 6-month-long in vivo B-cell depletion study, single i.v. dosages of 1 or 10 mg/kg of anti-CD20 proteins were administered to monkeys and B-cell counts were monitored in peripheral blood, bone marrow and lymph nodes. RESULTS: 2LM20-4 has lower saturation binding to human primary B cells and recruits fewer CD20 molecules into lipid rafts compared with rituximab; however, it induces higher in vitro CDC. In competitive binding, 2LM20-4 only partially displaces rituximab, suggesting that it binds to a fraction of CD20 molecules within certain locations of the plasma membrane as compared with rituximab. In monkeys, 2LM20-4 had more sustained B-cell depletion activity than rituximab in peripheral blood and had significantly more profound and sustained activity than 2LM20-4 P331S and rituximab in the lymph nodes. CONCLUSIONS: SMIP 2LM20-4, which binds to a fraction of CD20 molecules as compared with rituximab, has more potent in vitro CDC, and more potent and sustained B-cell depletion activity in cynomolgus monkeys. Our work has considerable clinical relevance since it provides novel insights related to the emerging B-cell depletion therapies in autoimmune diseases.

In vitro potency, pharmacokinetic profiles, and pharmacological activity of optimized anti-IL-21R antibodies in a mouse model of lupus.

Using phage display, we generated a panel of optimized neutralizing antibodies against the human and mouse receptors for interleukin 21 (IL-21), a cytokine that is implicated in the pathogenesis of many types of autoimmune disease. Two antibodies, Ab-01 and Ab-02, which differed by only four amino acids in V(L) CDR3, showed potent inhibition of human and mouse IL-21R in cell-based assays and were evaluated for their pharmacological and pharmacodynamic properties. Ab-01, but not Ab-02, significantly reduced a biomarker of disease (anti-dsDNA antibodies) and IgG deposits in the kidney in the MRL-Fas(lpr) mouse model of lupus, suggesting that anti-IL-21R antibodies may prove useful in the treatment of lupus. Ab-01 also had a consistently higher exposure (AUC(0-infinity)) than Ab-02 following a single dose in rodents or cynomolgus monkeys (2-3-fold or 4-7-fold, respectively). Our data demonstrate that small differences in CDR3 sequences of optimized antibodies can lead to profound differences in in vitro and in vivo properties, including differences in pharmacological activity and pharmacokinetic profiles. The lack of persistent activity of Ab-02 in the MRL-Fas(lpr) mouse lupus model may have been a consequence of faster elimination, reduced potency in blocking the effects of mouse IL-21R, and more potent/earlier onset of the anti-product response relative to Ab-01.