Widespread monoclonal IgE antibody convergence to an immunodominant, proanaphylactic Ara h 2 epitope in peanut allergy.

BACKGROUND: Despite their central role in peanut allergy, human monoclonal IgE antibodies have eluded characterization. OBJECTIVE: We sought to define the sequences, affinities, clonality, and functional properties of human monoclonal IgE antibodies in peanut allergy. METHODS: We applied our single-cell RNA sequencing-based SEQ SIFTER discovery platform to samples from allergic individuals who varied by age, sex, ethnicity, and geographic location in order to understand commonalities in the human IgE response to peanut allergens. Select antibodies were then recombinantly expressed and characterized for their allergen and epitope specificity, affinity, and functional properties. RESULTS: We found striking convergent evolution of IgE monoclonal antibodies (mAbs) from several clonal families comprising both memory B cells and plasmablasts. These antibodies bound with subnanomolar affinity to the immunodominant peanut allergen Ara h 2, specifically a linear, repetitive motif. Further characterization of these mAbs revealed their ability to single-handedly cause affinity-dependent degranulation of human mast cells and systemic anaphylaxis on peanut allergen challenge in humanized mice. Finally, we demonstrated that these mAbs, reengineered as IgGs, inhibit significant, but variable, amounts of Ara h 2- and peanut-mediated degranulation of mast cells sensitized with allergic plasma. CONCLUSIONS: Convergent evolution of IgE mAbs in peanut allergy is a common phenomenon that can reveal immunodominant epitopes on major allergenic proteins. Understanding the functional properties of these molecules is key to developing therapeutics, such as competitive IgG inhibitors, that are able to stoichiometrically outcompete endogenous IgE for allergen and thereby prevent allergic cascade in cases of accidental allergen exposure.

Cryptic-site-specific antibodies to the SARS-CoV-2 receptor binding domain can retain functional binding affinity to spike variants.

IMPORTANCE: Multiple SARS-CoV-2 variants of concern have emerged and caused a significant number of infections and deaths worldwide. These variants of concern contain mutations that might significantly affect antigen-targeting by antibodies. It is therefore important to further understand how antibody binding and neutralization are affected by the mutations in SARS-CoV-2 variants. We highlighted how antibody epitope specificity can influence antibody binding to SARS-CoV-2 spike protein variants and neutralization of SARS-CoV-2 variants. We showed that weakened spike binding and neutralization of Beta (B.1.351) and Omicron (BA.1) variants compared to wildtype are not universal among the panel of antibodies and identified antibodies of a specific binding footprint exhibiting consistent enhancement of spike binding and retained neutralization to Beta variant. These data and analysis can inform how antigen-targeting by antibodies might evolve during a pandemic and prepare for potential future sarbecovirus outbreaks.

Acute Treatment With XMetA Activates Hepatic Insulin Receptors and Lowers Blood Glucose in Normal Mice.

It has been proposed that monoclonal antibodies may become therapeutics for metabolic diseases such as diabetes mellitus. We have previously characterized an allosteric monoclonal antibody to the human insulin receptor (IR), XMetA, that activated metabolic signaling leading to enhanced glucose transport in cultured cells, and chronically reduced fasting blood glucose levels in mouse models of diabetes mellitus. Under acute dosing conditions, the large size of an IR-binding antibody like XMetA (∼ 150 kDa) could lead to a more rapid access into liver, an insulin sensitive tissue with well-fenestrated capillaries, when compared to other insulin sensitive tissues with non-fenestrated capillaries, such as muscle and adipose. Thus, in the present study we administered XMetA (10 mg/kg) and insulin (0.5 U/kg) via IV injection, and for 90 min compared their effects on blood glucose lowering and IR activation in three of the major insulin-sensitive tissues of the normal fasted mouse: liver, adipose, and muscle. Like insulin, XMetA lowered blood glucose levels, although the effect was less rapid. Insulin activated IR autophosphorylation and Akt phosphorylation in liver, fat, and muscle. In contrast, IR activation by XMetA was primarily observed in the liver. Both insulin and XMetA lowered ß-hydroxybutyrate levels in plasma; however, only insulin reduced both non-esterified fatty acids (NEFA) and glycerol concentrations. These data indicate that, in normal mice, acute glucose regulation by XMetA is largely mediated by its action on the liver.

Differential pathway coupling of the activated insulin receptor drives signaling selectivity by XMetA, an allosteric partial agonist antibody.

The monoclonal antibody XMetA is an allosteric partial agonist of the insulin receptor (IR), which activates the metabolic Akt kinase signaling pathway while having little or no effect on the mitogenic extracellular signal-regulated kinase (ERK) signaling pathway. To investigate the nature of this selective signaling, we have conducted a detailed investigation of XMetA to evaluate specific phosphorylation and activation of IR, Akt, and ERK in Chinese hamster ovary cell lines expressing either the short or long isoform of the human IR. Insulin activated both pathways, but the phosphorylation of Akt was more sensitive to the hormone than the phosphorylation of ERK. Maximally effective concentrations of XMetA elicited phosphorylation patterns similar to 40-100 pM insulin, which were sufficient for robust Akt phosphorylation, but had little effect on ERK phosphorylation. These data indicate that the preferential signaling of XMetA is due to an innate difference in pathway sensitivity of Akt versus ERK responses to IR activation and partial agonism by XMetA, rather than a separate pathway-biased mechanism. The metabolic selectivity of partial IR agonists like XMetA, if recapitulated in vivo, may be a desirable feature of therapeutic agents designed to regulate blood glucose levels while minimizing undesirable outcomes of excessive IR mitogenic activation.

Characterization of two affinity matured Anti-Yersinia pestis F1 human antibodies with medical countermeasure potential.

Yersinia pestis, the causative agent of plague and a biological threat agent, presents an urgent need for novel medical countermeasures due to documented cases of naturally acquired antibiotic resistance and potential person-to-person spread during a pneumonic infection. Immunotherapy has been proposed as a way to circumvent current and future antibiotic resistance. Here, we describe the development and characterization of two affinity matured human antibodies (αF1Ig AM2 and αF1Ig AM8) that promote survival of mice after exposure to aerosolized Y. pestis. We share details of the error prone PCR and yeast display technology-based affinity maturation process that we used. The resultant matured antibodies have nanomolar affinity for Y. pestis F1 antigen, are produced in high yield, and are resilient to 37°C stress for up to 6 months. Importantly, in vitro assays using a murine macrophage cell line demonstrated that αF1Ig AM2 and αF1Ig AM8 are opsonic. Even more importantly, in vivo studies using pneumonic plague mouse models showed that 100% of the mice receiving 500 µg of IgGs αF1Ig AM2 and αF1Ig AM8 survived lethal challenge with aerosolized Y. pestis CO92. Combined, these results provide evidence of the quality and robustness of αF1Ig AM2 and αF1Ig AM8 and support their development as potential medical countermeasures against plague.

Regulation of adipose branched-chain amino acid catabolism enzyme expression and cross-adipose amino acid flux in human obesity.

Elevated blood branched-chain amino acids (BCAA) are often associated with insulin resistance and type 2 diabetes, which might result from a reduced cellular utilization and/or incomplete BCAA oxidation. White adipose tissue (WAT) has become appreciated as a potential player in whole body BCAA metabolism. We tested if expression of the mitochondrial BCAA oxidation checkpoint, branched-chain α-ketoacid dehydrogenase (BCKD) complex, is reduced in obese WAT and regulated by metabolic signals. WAT BCKD protein (E1α subunit) was significantly reduced by 35-50% in various obesity models (fa/fa rats, db/db mice, diet-induced obese mice), and BCKD component transcripts significantly lower in subcutaneous (SC) adipocytes from obese vs. lean Pima Indians. Treatment of 3T3-L1 adipocytes or mice with peroxisome proliferator-activated receptor-γ agonists increased WAT BCAA catabolism enzyme mRNAs, whereas the nonmetabolizable glucose analog 2-deoxy-d-glucose had the opposite effect. The results support the hypothesis that suboptimal insulin action and/or perturbed metabolic signals in WAT, as would be seen with insulin resistance/type 2 diabetes, could impair WAT BCAA utilization. However, cross-tissue flux studies comparing lean vs. insulin-sensitive or insulin-resistant obese subjects revealed an unexpected negligible uptake of BCAA from human abdominal SC WAT. This suggests that SC WAT may not be an important contributor to blood BCAA phenotypes associated with insulin resistance in the overnight-fasted state. mRNA abundances for BCAA catabolic enzymes were markedly reduced in omental (but not SC) WAT of obese persons with metabolic syndrome compared with weight-matched healthy obese subjects, raising the possibility that visceral WAT contributes to the BCAA metabolic phenotype of metabolically compromised individuals.

Coronavirus Immunotherapeutic Consortium Database.

The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has seen multiple anti-SARS-CoV-2 antibodies being generated globally. It is difficult, however, to assemble a useful compendium of these biological properties if they are derived from experimental measurements performed at different sites under different experimental conditions. The Coronavirus Immunotherapeutic Consortium (COVIC) circumvents these issues by experimentally testing blinded antibodies side by side for several functional activities. To collect these data in a consistent fashion and make it publicly available, we established the COVIC database (COVIC-DB, https://covicdb.lji.org/). This database enables systematic analysis and interpretation of this large-scale dataset by providing a comprehensive view of various features such as affinity, neutralization, in vivo protection and effector functions for each antibody. Interactive graphs enable direct comparisons of antibodies based on select functional properties. We demonstrate how the COVIC-DB can be utilized to examine relationships among antibody features, thereby guiding the design of therapeutic antibody cocktails. Database URL  https://covicdb.lji.org/.

Healthy humans can be a source of antibodies countering COVID-19.

Here, we describe the isolation of 18 unique anti SARS-CoV-2 human single-chain antibodies from an antibody library derived from healthy donors. The selection used a combination of phage and yeast display technologies and included counter-selection strategies meant to direct the selection of the receptor-binding motif (RBM) of SARS-CoV-2 spike protein's receptor binding domain (RBD2). Selected antibodies were characterized in various formats including IgG, using flow cytometry, ELISA, high throughput SPR, and fluorescence microscopy. We report antibodies' RBD2 recognition specificity, binding affinity, and epitope diversity, as well as ability to block RBD2 binding to the human receptor angiotensin-converting enzyme 2 (ACE2) and to neutralize authentic SARS-CoV-2 virus infection in vitro. We present evidence supporting that: 1) most of our antibodies (16 out of 18) selectively recognize RBD2; 2) the best performing 8 antibodies target eight different epitopes of RBD2; 3) one of the pairs tested in sandwich assays detects RBD2 with sub-picomolar sensitivity; and 4) two antibody pairs inhibit SARS-CoV-2 infection at low nanomolar half neutralization titers. Based on these results, we conclude that our antibodies have high potential for therapeutic and diagnostic applications. Importantly, our results indicate that readily available non immune (naïve) antibody libraries obtained from healthy donors can be used to select high-quality monoclonal antibodies, bypassing the need for blood of infected patients, and offering a widely accessible and low-cost alternative to more sophisticated and expensive antibody selection approaches (e.g. single B cell analysis and natural evolution in humanized mice).

Kinetic approach to pathway attenuation using XOMA 052, a regulatory therapeutic antibody that modulates interleukin-1beta activity.

Many therapeutic antibodies act as antagonists to competitively block cellular signaling pathways. We describe here an approach for the therapeutic use of monoclonal antibodies based on context-dependent attenuation to reduce pathologically high activity while allowing homeostatic signaling in biologically important pathways. Such attenuation is achieved by modulating the kinetics of a ligand binding to its various receptors and regulatory proteins rather than by complete blockade of signaling pathways. The anti-interleukin-1beta (IL-1beta) antibody XOMA 052 is a potent inhibitor of IL-1beta activity that reduces the affinity of IL-1beta for its signaling receptor and co-receptor but not for its decoy and soluble inhibitory receptors. This mechanism shifts the effective dose response of the cytokine so that the potency of IL-1beta bound by XOMA 052 is 20-100-fold lower than that of IL-1beta in the absence of antibody in a variety of in vitro cell-based assays. We propose that by decreasing potency of IL-1beta while allowing binding to its clearance and inhibitory receptors, XOMA 052 treatment will attenuate IL-1beta activity in concert with endogenous regulatory mechanisms. Furthermore, the ability to bind the decoy receptor may reduce the potential for accumulation of antibody.target complexes. Regulatory antibodies like XOMA 052, which selectively modulate signaling pathways, may represent a new mechanistic class of therapeutic antibodies.

Defining variant-resistant epitopes targeted by SARS-CoV-2 antibodies: A global consortium study.

Antibody-based therapeutics and vaccines are essential to combat COVID-19 morbidity and mortality after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Multiple mutations in SARS-CoV-2 that could impair antibody defenses propagated in human-to-human transmission and spillover or spillback events between humans and animals. To develop prevention and therapeutic strategies, we formed an international consortium to map the epitope landscape on the SARS-CoV-2 spike protein, defining and structurally illustrating seven receptor binding domain (RBD)­directed antibody communities with distinct footprints and competition profiles. Pseudovirion-based neutralization assays reveal spike mutations, individually and clustered together in variants, that affect antibody function among the communities. Key classes of RBD-targeted antibodies maintain neutralization activity against these emerging SARS-CoV-2 variants. These results provide a framework for selecting antibody treatment cocktails and understanding how viral variants might affect antibody therapeutic efficacy.

Assessing the binding properties of the anti-PD-1 antibody landscape using label-free biosensors.

Here we describe an industry-wide collaboration aimed at assessing the binding properties of a comprehensive panel of monoclonal antibodies (mAbs) against programmed cell death protein 1 (PD-1), an important checkpoint protein in cancer immunotherapy and validated therapeutic target, with well over thirty unique mAbs either in clinical development or market-approved in the United States, the European Union or China. The binding kinetics of the PD-1/mAb interactions were measured by surface plasmon resonance (SPR) using a Carterra LSA instrument and the results were compared to data collected on a Biacore 8K. The effect of chip type on the SPR-derived binding rate constants and affinities were explored and the results compared with solution affinities from Meso Scale Discovery (MSD) and Kinetic Exclusion Assay (KinExA) experiments. When using flat chip types, the LSA and 8K platforms yielded near-identical kinetic rate and affinity constants that matched solution phase values more closely than those produced on 3D-hydrogels. Of the anti-PD-1 mAbs tested, which included a portion of those known to be in clinical development or approved, the affinities spanned from single digit picomolar to nearly 425 nM, challenging the dynamic range of our methods. The LSA instrument was also used to perform epitope binning and ligand competition studies which revealed over ten unique competitive binding profiles within this group of mAbs.

A natively paired antibody library yields drug leads with higher sensitivity and specificity than a randomly paired antibody library.

Deep sequencing and single-chain variable fragment (scFv) yeast display methods are becoming more popular for discovery of therapeutic antibody candidates in mouse B cell repertoires. In this study, we compare a deep sequencing and scFv display method that retains native heavy and light chain pairing with a related method that randomly pairs heavy and light chain. We performed the studies in a humanized mouse, using interleukin 21 receptor (IL-21R) as a test immunogen. We identified 44 high-affinity binder scFv with the native pairing method and 100 high-affinity binder scFv with the random pairing method. 30% of the natively paired scFv binders were also discovered with the randomly paired method, and 13% of the randomly paired binders were also discovered with the natively paired method. Additionally, 33% of the scFv binders discovered only in the randomly paired library were initially present in the natively paired pre-sort library. Thus, a significant proportion of "randomly paired" scFv were actually natively paired. We synthesized and produced 46 of the candidates as full-length antibodies and subjected them to a panel of binding assays to characterize their therapeutic potential. 87% of the antibodies were verified as binding IL-21R by at least one assay. We found that antibodies with native light chains were more likely to bind IL-21R than antibodies with non-native light chains, suggesting a higher false positive rate for antibodies from the randomly paired library. Additionally, the randomly paired method failed to identify nearly half of the true natively paired binders, suggesting a higher false negative rate. We conclude that natively paired libraries have critical advantages in sensitivity and specificity for antibody discovery programs.

Chickens with humanized immunoglobulin genes generate antibodies with high affinity and broad epitope coverage to conserved targets.

Transgenic animal platforms for the discovery of human monoclonal antibodies have been developed in mice, rats, rabbits and cows. The immune response to human proteins is limited in these animals by their tolerance to mammalian-conserved epitopes. To expand the range of epitopes that are accessible, we have chosen an animal host that is less phylogenetically related to humans. Specifically, we generated transgenic chickens expressing antibodies from immunoglobulin heavy and light chain loci containing human variable regions and chicken constant regions. From these birds, paired human light and heavy chain variable regions are recovered and cloned as fully human recombinant antibodies. The human antibody-expressing chickens exhibit normal B cell development and raise immune responses to conserved human proteins that are not immunogenic in mice. Fully human monoclonal antibodies can be recovered with sub-nanomolar affinities. Binning data of antibodies to a human protein show epitope coverage similar to wild type chickens, which we previously showed is broader than that produced from rodent immunizations.

Development and characterization of human monoclonal antibodies that neutralize multiple TGFß isoforms.

Transforming growth factor (TGF)ß levels are elevated in, and drive the progression of, numerous disease states such as advanced metastatic cancer and systemic and ocular fibrosis. There are 3 main isoforms, TGFß1, 2, and 3. As multiple TGFß isoforms are involved in disease processes, maximal therapeutic efficacy may require neutralization of 2 or more of the TGFß isoforms. Fully human antibody phage display libraries were used to discover a number of antibodies that bind and neutralize various combinations of TGFß1, 2 or 3. The primary panning did not yield any uniformly potent pan-isoform neutralizing antibodies; therefore, an antibody that displayed potent TGFß 1, 2 inhibition, but more modest affinity versus TGFß3, was affinity matured by shuffling with a light chain sub-library and further screening. This process yielded a high affinity pan-isoform neutralizing clone. Antibodies were analyzed and compared by binding affinity, as well as receptor and epitope competition by surface plasmon resonance methods. The antibodies were also shown to neutralize TGFß effects in vitro in 3 assays: 1) interleukin (IL)-4 induced HT-2 cell proliferation; 2) TGFß-mediated IL-11 release by A549 cells; and 3) decreasing SMAD2 phosphorylation in Detroit 562 cells. The antibodies' potency in these in vitro assays correlated well with their isoform-specific affinities. Furthermore, the ability of the affinity-matured clone to decrease tumor burden in a Detroit 562 xenograft study was superior to that of the parent clone. This affinity-matured antibody acts as a very potent inhibitor of all 3 main isoforms of TGFß and may have utility for therapeutic intervention in human disease.

Metabolic, anabolic, and mitogenic insulin responses: A tissue-specific perspective for insulin receptor activators.

Insulin acts as the major regulator of the fasting-to-fed metabolic transition by altering substrate metabolism, promoting energy storage, and helping activate protein synthesis. In addition to its glucoregulatory and other metabolic properties, insulin can also act as a growth factor. The metabolic and mitogenic responses to insulin are regulated by divergent post-receptor signaling mechanisms downstream from the activated insulin receptor (IR). However, the anabolic and growth-promoting properties of insulin require tissue-specific inter-relationships between the two pathways, and the nature and scope of insulin-regulated processes vary greatly across tissues. Understanding the nuances of this interplay between metabolic and growth-regulating properties of insulin would have important implications for development of novel insulin and IR modulator therapies that stimulate insulin receptor activation in both pathway- and tissue-specific manners. This review will provide a unique perspective focusing on the roles of "metabolic" and "mitogenic" actions of insulin signaling in various tissues, and how these networks should be considered when evaluating selective pharmacologic approaches to prevent or treat metabolic disease.

Selective allosteric antibodies to the insulin receptor for the treatment of hyperglycemic and hypoglycemic disorders.

Many therapeutic monoclonal antibodies act as antagonists to receptors by targeting and blocking the natural ligand binding site (orthosteric site). In contrast, the use of antibodies to target receptors at allosteric sites (distinct from the orthosteric site) has not been extensively studied. This approach is especially important in metabolic diseases in which endogenous ligand levels are dysregulated. Herein, we review our investigations of 3 categories of human monoclonal antibodies that bind allosterically to the insulin receptor (INSR) and affect its activity: XMetA, XMetS and XMetD. XMetA directly activates the INSR either alone or in combination with insulin. XMetS, in contrast, does not directly activate the INSR but markedly enhances the receptor's ability to bind insulin and potentiate insulin signaling. Both XMetA and XMetS are effective in controlling hyperglycemia in mouse models of diabetes. A third allosteric antibody, XMetD, is an inhibitor of INSR signaling. This antibody reverses insulin-induced hypoglycemia in a mouse model of hyperinsulinemia. These studies indicate, therefore, that allosteric antibodies to INSR can modulate its signaling and correct conditions of glucose dysregulation. These studies also raise the possibility that the use of allosteric antibodies can be expanded to other receptors for the treatment of metabolic disorders.

Improved glucose metabolism in vitro and in vivo by an allosteric monoclonal antibody that increases insulin receptor binding affinity.

Previously we reported studies of XMetA, an agonist antibody to the insulin receptor (INSR). We have now utilized phage display to identify XMetS, a novel monoclonal antibody to the INSR. Biophysical studies demonstrated that XMetS bound to the human and mouse INSR with picomolar affinity. Unlike monoclonal antibody XMetA, XMetS alone had little or no agonist effect on the INSR. However, XMetS was a strong positive allosteric modulator of the INSR that increased the binding affinity for insulin nearly 20-fold. XMetS potentiated insulin-stimulated INSR signaling ∼15-fold or greater including; autophosphorylation of the INSR, phosphorylation of Akt, a major enzyme in the metabolic pathway, and phosphorylation of Erk, a major enzyme in the growth pathway. The enhanced signaling effects of XMetS were more pronounced with Akt than with Erk. In cultured cells, XMetS also enhanced insulin-stimulated glucose transport. In contrast to its effects on the INSR, XMetS did not potentiate IGF-1 activation of the IGF-1 receptor. We studied the effect of XMetS treatment in two mouse models of insulin resistance and diabetes. The first was the diet induced obesity mouse, a hyperinsulinemic, insulin resistant animal, and the second was the multi-low dose streptozotocin/high-fat diet mouse, an insulinopenic, insulin resistant animal. In both models, XMetS normalized fasting blood glucose levels and glucose tolerance. In concert with its ability to potentiate insulin action at the INSR, XMetS reduced insulin and C-peptide levels in both mouse models. XMetS improved the response to exogenous insulin without causing hypoglycemia. These data indicate that an allosteric monoclonal antibody can be generated that markedly enhances the binding affinity of insulin to the INSR. These data also suggest that an INSR monoclonal antibody with these characteristics may have the potential to both improve glucose metabolism in insulinopenic type 2 diabetes mellitus and correct compensatory hyperinsulinism in insulin resistant conditions.

Inhibition of insulin receptor function by a human, allosteric monoclonal antibody: a potential new approach for the treatment of hyperinsulinemic hypoglycemia.

Novel therapies are needed for the treatment of hypoglycemia resulting from both endogenous and exogenous hyperinsulinema. To provide a potential new treatment option, we identified XMetD, an allosteric monoclonal antibody to the insulin receptor (INSR) that was isolated from a human antibody phage display library. To selectively obtain antibodies directed at allosteric sites, panning of the phage display library was conducted using the insulin-INSR complex. Studies indicated that XMetD bound to the INSR with nanomolar affinity. Addition of insulin reduced the affinity of XMetD to the INSR by 3-fold, and XMetD reduced the affinity of the INSR for insulin 3-fold. In addition to inhibiting INSR binding, XMetD also inhibited insulin-induced INSR signaling by 20- to 100-fold. These signaling functions included INSR autophosphorylation, Akt activation and glucose transport. These data indicated that XMetD was an allosteric antagonist of the INSR because, in addition to inhibiting the INSR via modulation of binding affinity, it also inhibited the INSR via modulation of signaling efficacy. Intraperitoneal injection of XMetD at 10 mg/kg twice weekly into normal mice induced insulin resistance. When sustained-release insulin implants were placed into normal mice, they developed fasting hypoglycemia in the range of 50 mg/dl. This hypoglycemia was reversed by XMetD treatment. These studies demonstrate that allosteric monoclonal antibodies, such as XMetD, can antagonize INSR signaling both in vitro and in vivo. They also suggest that this class of allosteric monoclonal antibodies has the potential to treat hyperinsulinemic hypoglycemia resulting from conditions such as insulinoma, congenital hyperinsulinism and insulin overdose.

Discovery of diverse and functional antibodies from large human repertoire antibody libraries.

Phage display antibody libraries have a proven track record for the discovery of therapeutic human antibodies, increasing the demand for large and diverse phage antibody libraries for the discovery of new therapeutics. We have constructed naïve antibody phage display libraries in both Fab and scFv formats, with each library having more than 250 billion clones that encompass the human antibody repertoire. These libraries show high fidelity in open reading frame and expression percentages, and their V-gene family distribution, VH-CDR3 length and amino acid usage mirror the natural diversity of human antibodies. Both the Fab and scFv libraries show robust sequence diversity in target-specific binders and differential V-gene usage for each target tested, supporting the use of libraries that utilize multiple display formats and V-gene utilization to maximize antibody-binding diversity. For each of the targets, clones with picomolar affinities were identified from at least one of the libraries and for the two targets assessed for activity, functional antibodies were identified from both libraries.

A fully human, allosteric monoclonal antibody that activates the insulin receptor and improves glycemic control.

Many patients with diabetes mellitus (both type 1 and type 2) require therapy to maintain normal fasting glucose levels. To develop a novel treatment for these individuals, we used phage display technology to target the insulin receptor (INSR) complexed with insulin and identified a high affinity, allosteric, human monoclonal antibody, XMetA, which mimicked the glucoregulatory, but not the mitogenic, actions of insulin. Biophysical studies with cultured cells expressing human INSR demonstrated that XMetA acted allosterically and did not compete with insulin for binding to its receptor. XMetA was found to function as a specific partial agonist of INSR, eliciting tyrosine phosphorylation of INSR but not the IGF-IR. Although this antibody activated metabolic signaling, leading to enhanced glucose uptake, it neither activated Erk nor induced proliferation of cancer cells. In an insulin resistant, insulinopenic model of diabetes, XMetA markedly reduced elevated fasting blood glucose and normalized glucose tolerance. After 6 weeks, significant improvements in HbA(1c), dyslipidemia, and other manifestations of diabetes were observed. It is noteworthy that hypoglycemia and weight gain were not observed during these studies. These studies indicate, therefore, that allosteric monoclonal antibodies have the potential to be novel, ultra-long acting, agents for the regulation of hyperglycemia in diabetes.