Antibodies against the erythroferrone N-terminal domain prevent hepcidin suppression and ameliorate murine thalassemia.

Erythroferrone (ERFE) is produced by erythroblasts in response to erythropoietin (EPO) and acts in the liver to prevent hepcidin stimulation by BMP6. Hepcidin suppression allows for the mobilization of iron to the bone marrow for the production of red blood cells. Aberrantly high circulating ERFE in conditions of stress erythropoiesis, such as in patients with ß-thalassemia, promotes the tissue iron accumulation that substantially contributes to morbidity in these patients. Here we developed antibodies against ERFE to prevent hepcidin suppression and to correct the iron loading phenotype in a mouse model of ß-thalassemia [Hbb(th3/+) mice] and used these antibodies as tools to further characterize ERFE's mechanism of action. We show that ERFE binds to BMP6 with nanomolar affinity and binds BMP2 and BMP4 with somewhat weaker affinities. We found that BMP6 binds the N-terminal domain of ERFE, and a polypeptide derived from the N terminus of ERFE was sufficient to cause hepcidin suppression in Huh7 hepatoma cells and in wild-type mice. Anti-ERFE antibodies targeting the N-terminal domain prevented hepcidin suppression in ERFE-treated Huh7 cells and in EPO-treated mice. Finally, we observed a decrease in splenomegaly and serum and liver iron in anti-ERFE-treated Hbb(th3/+) mice, accompanied by an increase in red blood cells and hemoglobin and a decrease in reticulocyte counts. In summary, we show that ERFE binds BMP6 directly and with high affinity, and that antibodies targeting the N-terminal domain of ERFE that prevent ERFE-BMP6 interactions constitute a potential therapeutic tool for iron loading anemias.

Erythroferrone inhibits the induction of hepcidin by BMP6.

Decreased hepcidin mobilizes iron, which facilitates erythropoiesis, but excess iron is pathogenic in ß-thalassemia. Erythropoietin (EPO) enhances erythroferrone (ERFE) synthesis by erythroblasts, and ERFE suppresses hepatic hepcidin production through an unknown mechanism. The BMP/SMAD pathway in the liver is critical for hepcidin control, and we show that EPO suppressed hepcidin and other BMP target genes in vivo in a partially ERFE-dependent manner. Furthermore, recombinant ERFE suppressed the hepatic BMP/SMAD pathway independently of changes in serum and liver iron. In vitro, ERFE decreased SMAD1, SMAD5, and SMAD8 phosphorylation and inhibited expression of BMP target genes. ERFE specifically abrogated the induction of hepcidin by BMP5, BMP6, and BMP7 but had little or no effect on hepcidin induction by BMP2, BMP4, BMP9, or activin B. A neutralizing anti-ERFE antibody prevented ERFE from inhibiting hepcidin induction by BMP5, BMP6, and BMP7. Cell-free homogeneous time-resolved fluorescence assays showed that BMP5, BMP6, and BMP7 competed with anti-ERFE for binding to ERFE. We conclude that ERFE suppresses hepcidin by inhibiting hepatic BMP/SMAD signaling via preferentially impairing an evolutionarily closely related BMP subgroup of BMP5, BMP6, and BMP7. ERFE can act as a natural ligand trap generated by stimulated erythropoiesis to regulate the availability of iron.

Augmented Binary Substitution: Single-pass CDR germ-lining and stabilization of therapeutic antibodies.

Although humanized antibodies have been highly successful in the clinic, all current humanization techniques have potential limitations, such as: reliance on rodent hosts, immunogenicity due to high non-germ-line amino acid content, v-domain destabilization, expression and formulation issues. This study presents a technology that generates stable, soluble, ultrahumanized antibodies via single-step complementarity-determining region (CDR) germ-lining. For three antibodies from three separate key immune host species, binary substitution CDR cassettes were inserted into preferred human frameworks to form libraries in which only the parental or human germ-line destination residue was encoded at each position. The CDR-H3 in each case was also augmented with 1 ± 1 random substitution per clone. Each library was then screened for clones with restored antigen binding capacity. Lead ultrahumanized clones demonstrated high stability, with affinity and specificity equivalent to, or better than, the parental IgG. Critically, this was mainly achieved on germ-line frameworks by simultaneously subtracting up to 19 redundant non-germ-line residues in the CDRs. This process significantly lowered non-germ-line sequence content, minimized immunogenicity risk in the final molecules and provided a heat map for the essential non-germ-line CDR residue content of each antibody. The ABS technology therefore fully optimizes the clinical potential of antibodies from rodents and alternative immune hosts, rendering them indistinguishable from fully human in a simple, single-pass process.

A Combination of Structural and Empirical Analyses Delineates the Key Contacts Mediating Stability and Affinity Increases in an Optimized Biotherapeutic Single-chain Fv (scFv).

Fully-human single-chain Fv (scFv) proteins are key potential building blocks of bispecific therapeutic antibodies, but they often suffer from manufacturability and clinical development limitations such as instability and aggregation. The causes of these scFv instability problems, in proteins that should be theoretically stable, remains poorly understood. To inform the future development of such molecules, we carried out a comprehensive structural analysis of the highly stabilized anti-CXCL13 scFv E10. E10 was derived from the parental 3B4 using complementarity-determining region (CDR)-restricted mutagenesis and tailored selection and screening strategies, and carries four mutations in VL-CDR3. High-resolution crystal structures of parental 3B4 and optimized E10 scFvs were solved in the presence and absence of human CXCL13. In parallel, a series of scFv mutants was generated to interrogate the individual contribution of each of the four mutations to stability and affinity improvements. In combination, these analyses demonstrated that the optimization of E10 was primarily mediated by removing clashes between both the VL and the VH, and between the VL and CXCL13. Importantly, a single, germline-encoded VL-CDR3 residue mediated the key difference between the stable and unstable forms of the scFv. This work demonstrates that, aside from being the critical mediators of specificity and affinity, CDRs may also be the primary drivers of biotherapeutic developability.

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.

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.

Drug use in pregnancy in Ireland's capital city: A decade of trends and outcomes.

OBJECTIVE: The aim of this study was to present contemporary trends in opiate use disorder (OUD) and substance use in pregnancy in Ireland, with associated obstetric outcomes, over the last ten years. STUDY DESIGN: This retrospective observational cohort study was conducted at an Irish tertiary maternity unit. All women with OUD or substance use in pregnancy delivered under this service between 2010 and 2019 were included. Drug-exposure was self-reported. Data was collected by combining electronic and hand-held patient records. Trends and outcomes were analysed by year of delivery. Approval for the study was granted by the institution's clinical governance committee. RESULTS: Of the 82,669 women delivered, 525 had OUD or substance use in pregnancy (1 in every 160 women booking). 11.6% were homeless, 20.0% were in full-time employment and 91.0% smoked tobacco in pregnancy. 66.3% had a history of psychiatric disorders. Over the ten years, there was a significant reduction in women delivered with OUD or substance use in pregnancy (0.8 % to 0.4 %, RR 0.55, 95 % CI 0.36-0.85), significant reduction in the proportion of women on Opioid-Substitute-Treatment (OST, RR 0.66 95 % CI 0.51-0.87) and an increase in mean maternal age (30.7to32.0 years). Rates of cocaine and cannabis consumption increased (20.6 %, RR 3.8, 95 % CI 1.57-9.44: 24.0 %, RR 3.7, 95 % CI 1.58-8.86 respectively). The maternal mortality rate was 380.9:100,000 births. The perinatal mortality rate was 15.6:1000 births. The preterm birth rate was 17.9 %, with a mean birth weight of 2832 g. The rate of NICU admission was 52.0 % and the mean length of stay was 22.4 days. Amongst the smaller OUD population, the rate of NICU admission for Neonatal Abstinence Syndrome (NAS) and treatment for NAS increased over the study timeframe (36.0 %, RR 2.97, 95 % CI 1.86-4.75: 28.5 %, RR 2.92, 95 % CI 1.70-5.0 respectively). CONCLUSIONS: The obstetric population attending an Irish antenatal service with opiate use disorder or substance exposure is reducing in size with older patients, less opioid substitute therapy and increasing cocaine and cannabis use. These women have high rates of maternal and perinatal morbidity and mortality. Specialist antenatal addiction services, coordinated by the drug-liaison midwife, are critical in adapting care to respond to this dynamic and vulnerable patient cohort.

uPAR-induced cell adhesion and migration: vitronectin provides the key.

Expression of the membrane receptor uPAR induces profound changes in cell morphology and migration, and its expression correlates with the malignant phenotype of cancers. To identify the molecular interactions essential for uPAR function in these processes, we carried out a complete functional alanine scan of uPAR in HEK293 cells. Of the 255 mutant receptors characterized, 34 failed to induce changes in cell morphology. Remarkably, the molecular defect of all of these mutants was a specific reduction in integrin-independent cell binding to vitronectin. A membrane-tethered plasminogen activator inhibitor-1, which has the same binding site in vitronectin as uPAR, replicated uPAR-induced changes. A direct uPAR-vitronectin interaction is thus both required and sufficient to initiate downstream changes in cell morphology, migration, and signal transduction. Collectively these data demonstrate a novel mechanism by which a cell adhesion molecule lacking inherent signaling capability evokes complex cellular responses by modulating the contact between the cell and the matrix without the requirement for direct lateral protein-protein interactions.

Crystal structure of ultra-humanized anti-pTau Fab reveals how germline substitutions humanize CDRs without loss of binding'.

Administration of therapeutic antibodies can elicit adverse immune responses in patients through the generation of anti-drug antibodies that, in turn, reduce the efficacy of the therapeutic. Removal of foreign amino acid content by humanization can lower the immunogenic risk of the therapeutic mAb. We previously developed the ultra-humanization technology "Augmented Binary Substitution" (ABS) which enables single-step CDR germlining of antibodies. The application of ABS to a chicken anti-pTau antibody generated an ultra-humanized variant, anti-pTau C21-ABS, with increased human amino acid content in the CDRs and reduced in-silico predicted immunogenicity risk. Here, we report the high-resolution crystal structure of anti-pTau C21-ABS Fab in complex with the pTau peptide (7KQK). This study examines how ultra-humanization, via CDR germlining, is facilitated while maintaining near-identical antigen affinity (within 1.6-fold). The co-complex structure reveals that the ABS molecule targets the same antigenic epitope, accommodated by structurally-similar changes in the paratope. These findings confirm that ABS enables the germlining of amino acids within CDRs by exploiting CDR plasticity, to reduce non-human amino acid CDR content, with few alterations to the overall mechanism of binding.

Discovery and multi-parametric optimization of a high-affinity antibody against interleukin-25 with neutralizing activity in a mouse model of skin inflammation.

Background: Interleukin (IL)25 has been implicated in tissue homeostasis at barrier surfaces and the initiation of type two inflammatory signaling in response to infection and cell injury across multiple organs. We sought to discover and engineer a high affinity neutralizing antibody and evaluate the antibody functional activity in vitro and in vivo. Methods: In this study, we generated a novel anti-IL25 antibody (22C7) and investigated the antibody's therapeutic potential for targeting IL25 in inflammation. Results: A novel anti-IL25 antibody (22C7) was generated with equivalent in vitro affinity and potency against the human and mouse orthologs of the cytokine. This translated into in vivo potency in an IL25-induced air pouch model where 22C7 inhibited the recruitment of monocytes, macrophages, neutrophils and eosinophils. Furthermore, 22C7 significantly reduced ear swelling, acanthosis and disease severity in the Aldara mouse model of psoriasiform skin inflammation. Given the therapeutic potential of IL25 targeting in inflammatory conditions, 22C7 was further engineered to generate a highly developable, fully human antibody while maintaining the affinity and potency of the parental molecule. Conclusions: The generation of 22C7, an anti-IL25 antibody with efficacy in a preclinical model of skin inflammation, raises the therapeutic potential for 22C7 use in the spectrum of IL25-mediated diseases.

Polyreactivity and polyspecificity in therapeutic antibody development: risk factors for failure in preclinical and clinical development campaigns.

Antibody-based drugs, which now represent the dominant biologic therapeutic modality, are used to modulate disparate signaling pathways across diverse disease indications. One fundamental premise that has driven this therapeutic antibody revolution is the belief that each monoclonal antibody exhibits exquisitely specific binding to a single-drug target. Herein, we review emerging evidence in antibody off-target binding and relate current key findings to the risk of failure in therapeutic development. We further summarize the current state of understanding of structural mechanisms underpining the different phenomena that may drive polyreactivity and polyspecificity, and highlight current thinking on how de-risking studies may be best implemented in the screening triage. We conclude with a summary of what we believe to be key observations in the field to date, and a call for the wider antibody research community to work together to build the tools needed to maximize our understanding in this nascent area.

Combining random mutagenesis, structure-guided design and next-generation sequencing to mitigate polyreactivity of an anti-IL-21R antibody.

Despite substantial technological advances in antibody library and display platform development, the number of approved biotherapeutics from displayed libraries remains limited. In vivo, 20-50% of peripheral B cells undergo a process of receptor editing, which modifies the variable and junctional regions of light chains to delete auto-reactive clones. However, in vitro antibody evolution relies primarily on interaction with antigen, with no in-built checkpoints to ensure that the selected antibodies have not acquired additional specificities or biophysical liabilities during the optimization process. We had previously observed an enrichment of positive charge in the complementarity-determining regions of an anti-IL-21 R antibody during affinity optimization, which correlated with more potent IL-21 neutralization, but poor in vivo pharmacokinetics (PK). There is an emerging body of data that has correlated antibody nonspecificity with poor PK in vivo, and established a series of screening assays that are predictive of this behavior. In this study we revisit the challenge of developing an anti-IL-21 R antibody that can effectively compete with IL-21 for its highly negatively charged paratope while maintaining favorable biophysical properties. In vitro deselection methods that included an excess of negatively charged membrane preparations, or deoxyribonucleic acid, during phage selection of optimization libraries were unsuccessful in avoiding enrichment of highly charged, nonspecific antibody variants. However, a combination of structure-guided rational library design, next-generation sequencing of library outputs and application of linear regression models resulted in the identification of an antibody that maintained high affinity for IL-21 R and exhibited a desirable stability and biophysical profile.

Discovery and optimization of a novel anti-GUCY2c x CD3 bispecific antibody for the treatment of solid tumors.

We report here the discovery and optimization of a novel T cell retargeting anti-GUCY2C x anti-CD3ε bispecific antibody for the treatment of solid tumors. Using a combination of hybridoma, phage display and rational design protein engineering, we have developed a fully humanized and manufacturable CD3 bispecific antibody that demonstrates favorable pharmacokinetic properties and potent in vivo efficacy. Anti-GUCY2C and anti-CD3ε antibodies derived from mouse hybridomas were first humanized into well-behaved human variable region frameworks with full retention of binding and T-cell mediated cytotoxic activity. To address potential manufacturability concerns, multiple approaches were taken in parallel to optimize and de-risk the two antibody variable regions. These approaches included structure-guided rational mutagenesis and phage display-based optimization, focusing on improving stability, reducing polyreactivity and self-association potential, removing chemical liabilities and proteolytic cleavage sites, and de-risking immunogenicity. Employing rapid library construction methods as well as automated phage display and high-throughput protein production workflows enabled efficient generation of an optimized bispecific antibody with desirable manufacturability properties, high stability, and low nonspecific binding. Proteolytic cleavage and deamidation in complementarity-determining regions were also successfully addressed. Collectively, these improvements translated to a molecule with potent single-agent in vivo efficacy in a tumor cell line adoptive transfer model and a cynomolgus monkey pharmacokinetic profile (half-life>4.5 days) suitable for clinical development. Clinical evaluation of PF-07062119 is ongoing.

Investigating the clearance of VWF A-domains using site-directed PEGylation and novel N-linked glycosylation.

BACKGROUND: Previous studies have demonstrated that the A1A2A3 domains of von Willebrand factor (VWF) play a key role in regulating macrophage-mediated clearance in vivo. In particular, the A1-domain has been shown to modulate interaction with macrophage low-density lipoprotein receptor-related protein-1 (LRP1) clearance receptor. Furthermore, N-linked glycans within the A2-domain have been shown to protect VWF against premature LRP1-mediated clearance. Importantly, however, the specific regions within A1A2A3 that enable macrophage binding have not been defined. OBJECTIVE AND METHODS: To address this, we utilized site-directed PEGylation and introduced novel targeted N-linked glycosylation within A1A2A3-VWF and subsequently examined VWF clearance. RESULTS: Conjugation with a 40-kDa polyethylene glycol (PEG) moiety significantly extended the half-life of A1A2A3-VWF in VWF-/- mice in a site-specific manner. For example, PEGylation at specific sites within the A1-domain (S1286) and A3-domain (V1803, S1807) attenuated VWF clearance in vivo, compared to wild-type A1A2A3-VWF. Furthermore, PEGylation at these specific sites ablated binding to differentiated THP-1 macrophages and LRP1 cluster II and cluster IV in-vitro. Conversely, PEGylation at other positions (Q1353-A1-domain and M1545-A2-domain) had limited effects on VWF clearance or binding to LRP1.Novel N-linked glycan chains were introduced at N1803 and N1807 in the A3-domain. In contrast to PEGylation at these sites, no significant extension in half-life was observed with these N-glycan variants. CONCLUSIONS: These novel data demonstrate that site specific PEGylation but not site specific N-glycosylation modifies LRP1-dependent uptake of the A1A2A3-VWF by macrophages. This suggests that PEGylation, within the A1- and A3-domains in particular, may be used to attenuate LRP1-mediated clearance of VWF.

In vitro affinity optimization of an anti-BDNF monoclonal antibody translates to improved potency in targeting chronic pain states in vivo.

The role of brain-derived neurotrophic factor (BDNF) signaling in chronic pain has been well documented. Given the important central role of BDNF in long term plasticity and memory, we sought to engineer a high affinity, peripherally-restricted monoclonal antibody against BDNF to modulate pain. BDNF shares 100% sequence homology across human and rodents; thus, we selected chickens as an alternative immune host for initial antibody generation. Here, we describe the affinity optimization of complementarity-determining region-grafted, chicken-derived R3bH01, an anti-BDNF antibody specifically blocking the TrkB receptor interaction. Antibody optimization led to the identification of B30, which has a > 300-fold improvement in affinity based on BIAcore, an 800-fold improvement in potency in a cell-based pERK assay and demonstrates exquisite selectivity over related neurotrophins. Affinity improvements measured in vitro translated to in vivo pharmacological activity, with B30 demonstrating a 30-fold improvement in potency over parental R3bH01 in a peripheral nerve injury model. We further demonstrate that peripheral BDNF plays a role in maintaining the plasticity of sensory neurons following nerve damage, with B30 reversing neuron hyperexcitability associated with heat and mechanical stimuli in a dose-dependent fashion. In summary, our data demonstrate that effective sequestration of BDNF via a high affinity neutralizing antibody has potential utility in modulating the pathophysiological mechanisms that drive chronic pain states.

Microglia and the urokinase plasminogen activator receptor/uPA system in innate brain inflammation.

The urokinase plasminogen activator (uPA) receptor (uPAR) is a GPI-linked cell surface protein that facilitates focused plasmin proteolytic activity at the cell surface. uPAR has been detected in macrophages infiltrating the central nervous system (CNS) and soluble uPAR has been detected in the cerebrospinal fluid during a number of CNS pathologies. However, its expression by resident microglial cells in vivo remains uncertain. In this work, we aimed to elucidate the murine CNS expression of uPAR and uPA as well as that of tissue plasminogen activator and plasminogen activator inhibitor 1 (PAI-1) during insults generating distinct and well-characterized inflammatory responses; acute intracerebral lipopolysaccharide (LPS), acute kainate-induced neurodegeneration, and chronic neurodegeneration induced by prion disease inoculation. All three insults induced marked expression of uPAR at both mRNA and protein level compared to controls (naïve, saline, or control inoculum-injected). uPAR expression was microglial in all cases. Conversely, uPA transcription and activity was only markedly increased during chronic neurodegeneration. Dissociation of uPA and uPAR levels in acute challenges is suggestive of additional proteolysis-independent roles for uPAR. PAI-1 was most highly expressed upon LPS challenge, whereas tissue plasminogen activator mRNA was constitutively present and less responsive to all insults studied. These data are novel and suggest much wider involvement of the uPAR/uPA system in CNS function and pathology than previously supposed.

A simple high-throughput purification method for hit identification in protein screening.

Phage and ribosome display technologies have emerged as important tools in the high-throughput screening of protein pharmaceuticals. However, a challenge created by the implementation of such tools is the need to purify large numbers of proteins for screening. While some assays may be compatible with crude bacterial lysates or periplasmic extracts, many functional assays, particularly cell-based assays, require protein of high purity and concentration. Here we evaluate several methods for small-scale, high-throughput protein purification. From our initial assessment we identified the HIS-Select 96-well filter plate system as the method of choice for further evaluation. This method was optimized and used to produce scFvs that were tested in cell-based functional assays. The behavior of HIS-Select purified scFvs in these assays was found to be similar to scFvs purified using a traditional large-scale 2-step purification method. The HIS-Select method allows high-throughput purification of hundreds of scFvs with yields in the 50-100 microg range, and of sufficient purity to allow evaluation in a cell-based proliferation assay. In addition, the use of a similar 96-well-based method facilitates the purification and subsequent screening of large numbers of IgGs and Fc fusion proteins generated through reformatting of scFv fragments.

Parallel Evolution of Antibody Affinity and Thermal Stability for Optimal Biotherapeutic Development.

Naïve antibody libraries provide a rich resource for the identification of binding domains against targets of therapeutic interest. Being naïve in nature means a lack in antigen bias, resulting in a breadth of diversity with respect to epitopes that can be successfully targeted. In combination with display-based technology platforms, selection strategies allow for the generation of ortholog cross-reactive binding domains which enable critical preclinical proof-of-concept studies. However, naïve binding domains often suffer from low target affinity. In addition, construction of large naïve libraries results in non-native pairing of heavy and light v-domains which can present a challenge to molecular stability. Here we describe effective methods for the parallel evolution of antibody affinity and thermal stability which couple mutant antibody library phage display with carefully designed selection strategies.

Establishing in vitro in vivo correlations to screen monoclonal antibodies for physicochemical properties related to favorable human pharmacokinetics.

Implementation of in vitro assays that correlate with in vivo human pharmacokinetics (PK) would provide desirable preclinical tools for the early selection of therapeutic monoclonal antibody (mAb) candidates with minimal non-target-related PK risk. Use of these tools minimizes the likelihood that mAbs with unfavorable PK would be advanced into costly preclinical and clinical development. In total, 42 mAbs varying in isotype and soluble versus membrane targets were tested in in vitro and in vivo studies. MAb physicochemical properties were assessed by measuring non-specific interactions (DNA- and insulin-binding ELISA), self-association (affinity-capture self-interaction nanoparticle spectroscopy) and binding to matrix-immobilized human FcRn (surface plasmon resonance and column chromatography). The range of scores obtained from each in vitro assay trended well with in vivo clearance (CL) using both human FcRn transgenic (Tg32) mouse allometrically projected human CL and observed human CL, where mAbs with high in vitro scores resulted in rapid CL in vivo. Establishing a threshold value for mAb CL in human of 0.32 mL/hr/kg enabled refinement of thresholds for each in vitro assay parameter, and using a combinatorial triage approach enabled the successful differentiation of mAbs at high risk for rapid CL (unfavorable PK) from those with low risk (favorable PK), which allowed mAbs requiring further characterization to be identified. Correlating in vitro parameters with in vivo human CL resulted in a set of in vitro tools for use in early testing that would enable selection of mAbs with the greatest likelihood of success in the clinic, allowing costly late-stage failures related to an inadequate exposure profile, toxicity or lack of efficacy to be avoided.

Optimized Generation of High-Affinity, High-Specificity Single-Chain Fv Antibodies from Multi-Antigen Immunized Chickens.

High-affinity, highly specific binding proteins are a key class of molecules used in the development of new affinity chromatography methods. Traditionally, antibody-based methods have relied on the use of immunoglobulins purified from immune animal sera, from egg yolks, or from murine monoclonal hybridoma supernatants. To accelerate and refine the reagent antibody generation process, we have developed optimized methods that allow the rapid assembly of scFv libraries from chickens immunized with pools of immunogens. These methods allow the simplified generation of a single, moderately sized library of single chain Fv (scFv) and the subsequent isolation of diverse, high affinity, and high specificity monoclonals for each individual immunogen, via phage display. Using these methods, antibodies can be derived that exhibit the desired selectivity, including exquisite specificity or cross-reactivity to multiple orthologues of the same protein.