High-affinity Human Antibodies Research Articles

The novel high-affinity humanized antibody IMM40H targets CD70, eliminates tumors via Fc-mediated effector functions, and interrupts CD70/CD27 signaling.

A significant level of CD70 can be detected in various types of tumor tissues and CD27 is expressed on Treg cells, but CD70 expression is low in normal tissues. The interaction between CD70 and CD27 can stimulate the proliferation and survival of cancer cells and increase the level of soluble CD27, which is associated with poor prognosis in patients with lymphoma and certain solid tumors. Thus, it is a promising therapeutic target for the treatment of many major CD70+ cancer indications, including CD70+ lymphoma, RCC, NSCLC, HNSCC and OC. IMM40H was obtained through hybridoma screening and antibody humanization techniques. IMM40H was evaluated for its binding, blocking, Fc-dependent effector functions and antitumor activity characteristics in various in vitro and in vivo systems. The safety and tolerability profile of IMM40H were evaluated through single and repeated administration in cynomolgus monkeys. In vitro cell-based assays demonstrated that IMM40H had considerably stronger CD70-binding affinity than competitor anti-CD70 antibodies, including cusatuzumab, which enabled it to block the interaction of between CD70 and CD27 more effectively. IMM40H also exhibited potent Fc-dependent effector functions (ADCC/CDC/ADCP), and could make a strong immune attack on tumor cells and enhance therapeutic efficacy. Preclinical findings showed that IMM40H had potent antitumor activity in multiple myeloma U266B1 xenograft model, and could eradicate subcutaneously established tumors at a low dose of 0.3 mg/kg. IMM40H (0.3 mg/kg) showed therapeutic effects faster than cusatuzumab (1 mg/kg). A strong synergistic effect between IMM01 (SIRPα-Fc fusion protein) and IMM40H was recorded in Burkitt's lymphoma Raji and renal carcinoma cell A498 tumor models. In cynomolgus monkeys, the highest non-severely toxic dose (HNSTD) for repeat-dose toxicity was up to 30 mg/kg, while the maximum tolerated dose (MTD) for single-dose toxicity was up to 100 mg/kg, confirming that IMM40H had a good safety and tolerability profile. IMM40H is a high-affinity humanized IgG1 specifically targeting the CD70 monoclonal antibody with enhanced Fc-dependent activities. IMM40H has a dual mechanism of action: inducing cytotoxicity against CD70+ tumor cells via various effector functions (ADCC, ADCP and CDC) and obstructs the proliferation and activation of Tregs by inhibiting CD70/CD27 signaling.

A TREM2-activating antibody with a blood–brain barrier transport vehicle enhances microglial metabolism in Alzheimer’s disease models

Loss-of-function variants of TREM2 are associated with increased risk of Alzheimer’s disease (AD), suggesting that activation of this innate immune receptor may be a useful therapeutic strategy. Here we describe a high-affinity human TREM2-activating antibody engineered with a monovalent transferrin receptor (TfR) binding site, termed antibody transport vehicle (ATV), to facilitate blood–brain barrier transcytosis. Upon peripheral delivery in mice, ATV:TREM2 showed improved brain biodistribution and enhanced signaling compared to a standard anti-TREM2 antibody. In human induced pluripotent stem cell (iPSC)-derived microglia, ATV:TREM2 induced proliferation and improved mitochondrial metabolism. Single-cell RNA sequencing and morphometry revealed that ATV:TREM2 shifted microglia to metabolically responsive states, which were distinct from those induced by amyloid pathology. In an AD mouse model, ATV:TREM2 boosted brain microglial activity and glucose metabolism. Thus, ATV:TREM2 represents a promising approach to improve microglial function and treat brain hypometabolism found in patients with AD.

A rabies virus-vectored vaccine expressing two copies of the Marburg virus glycoprotein gene induced neutralizing antibodies against Marburg virus in humanized mice

ABSTRACT Marburg virus disease (MVD) is a lethal viral haemorrhagic fever caused by Marburg virus (MARV) with a case fatality rate as high as 88%. There is currently no vaccine or antiviral therapy approved for MVD. Due to high variation among MARV isolates, vaccines developed against one strain fail to protect against other strains. Here we report that three recombinant rabies virus (RABV) vector vaccines encoding two copies of GPs covering both MARV lineages induced pseudovirus neutralizing antibodies in BALB/c mice. Furthermore, high-affinity human neutralizing antibodies were isolated from a humanized mouse model. The three vaccines produced a Th1-biased serological response similar to that of human patients. Adequate sequential immunization enhanced the production of neutralizing antibodies. Virtual docking suggested that neutralizing antibodies induced by the Angola strain seemed to be able to hydrogen bond to the receptor-binding site (RBS) in the GP of the Ravn strain through hypervariable regions 2 (CDR2) and CDR3 of the VH region. These findings demonstrate that three inactivated vaccines are promising candidates against different strains of MARV, and a novel fully humanized neutralizing antibody against MARV was isolated.

Development of a potent high-affinity human therapeutic antibody via novel application of recombination signal sequence–based affinity maturation

Therapeutic antibody development requires discovery of an antibody molecule with desired specificities and drug-like properties. For toxicological studies, a therapeutic antibody must bind the ortholog antigen with a similar affinity to the human target to enable relevant dosing regimens, and antibodies falling short of this affinity design goal may not progress as therapeutic leads. Herein, we report the novel use of mammalian recombination signal sequence (RSS)–directed recombination for complementarity-determining region–targeted protein engineering combined with mammalian display to close the species affinity gap of human interleukin (IL)-13 antibody 731. This fully human antibody has not progressed as a therapeutic in part because of a 400-fold species affinity gap. Using this nonhypothesis-driven affinity maturation method, we generated multiple antibody variants with improved IL-13 affinity, including the highest affinity antibody reported to date (34 fM). Resolution of a cocrystal structure of the optimized antibody with the cynomolgus monkey (or nonhuman primate) IL-13 protein revealed that the RSS-derived mutations introduced multiple successive amino-acid substitutions resulting in a de novo formation of a π–π stacking–based protein–protein interaction between the affinity-matured antibody heavy chain and helix C on IL-13, as well as an introduction of an interface-distant residue, which enhanced the light chain–binding affinity to target. These mutations synergized binding of heavy and light chains to the target protein, resulting in a remarkably tight interaction, and providing a proof of concept for a new method of protein engineering, based on synergizing a mammalian display platform with novel RSS-mediated library generation.

A high-affinity human TCR-like antibody detects celiac disease gluten peptide-MHC complexes and inhibits T cell activation.

Antibodies specific for peptides bound to human leukocyte antigen (HLA) molecules are valuable tools for studies of antigen presentation and may have therapeutic potential. Here, we generated human T cell receptor (TCR)-like antibodies toward the immunodominant signature gluten epitope DQ2.5-glia-α2 in celiac disease (CeD). Phage display selection combined with secondary targeted engineering was used to obtain highly specific antibodies with picomolar affinity. The crystal structure of a Fab fragment of the lead antibody 3.C11 in complex with HLA-DQ2.5:DQ2.5-glia-α2 revealed a binding geometry and interaction mode highly similar to prototypic TCRs specific for the same complex. Assessment of CeD biopsy material confirmed disease specificity and reinforced the notion that abundant plasma cells present antigen in the inflamed CeD gut. Furthermore, 3.C11 specifically inhibited activation and proliferation of gluten-specific CD4+ T cells in vitro and in HLA-DQ2.5 humanized mice, suggesting a potential for targeted intervention without compromising systemic immunity.

Antibody Therapy Targeting RAN Proteins Rescues C9 ALS/FTD Phenotypes in C9orf72 Mouse Model

The intronic C9orf72 G4C2 expansion, the most common genetic cause of ALS and FTD, produces sense- and antisense-expansion RNAs and six dipeptide repeat-associated, non-ATG (RAN) proteins, but their roles in disease are unclear. We generated high-affinity human antibodies targeting GA or GP RAN proteins. These antibodies cross the blood-brain barrier and co-localize with intracellular RANaggregatesin C9-ALS/FTD BAC mice. In cells, α-GA1 interacts with TRIM21, and α-GA1 treatment reduced GAlevels, increased GA turnover, and decreased RAN toxicity and co-aggregation of proteasome and autophagy proteins to GA aggregates. InC9-BAC mice, α-GA1 reduced GA as well as GP and GR proteins, improved behavioral deficits, decreased neuroinflammation and neurodegeneration, and increased survival. Glycosylation of the Fc region of α-GA1 is important for cell entry and efficacy. These data demonstrate that RAN proteins drive C9-ALS/FTD in C9-BAC transgenic mice and establish a novel therapeutic approach for C9orf72 ALS/FTD and other RAN-protein diseases.

Abstract 1533: IMGC936, a first-in-class ADAM9-targeting antibody-drug conjugate, demonstrates promising anti-tumor activity

Abstract Dysregulation of ADAM9, a member of the ADAM (a disintegrin and metalloproteinase) family of proteases, has been implicated in tumor progression and metastasis, as well as pathological neovascularization. ADAM9 overexpression correlates with poor prognosis in multiple cancers. We have shown that ADAM9 is overexpressed in multiple solid tumor indications and that anti-ADAM9 antibodies are efficiently internalized and degraded by tumor cell lines making ADAM9 an attractive target for antibody-drug conjugate (ADC) development. Here, we describe IMGC936, the first ADAM9-targeting ADC to enter preclinical development. IMGC936 is comprised of a high-affinity humanized antibody site-specifically conjugated to DM21, a next-generation linker-payload that combines a maytansinoid microtubule-disrupting payload with a stable peptide linker at a drug-antibody ratio of two. To maximize the potential for IMGC936 activity, the M252Y/S254T/T256E (YTE) mutation was introduced into the CH2 domain of the antibody to increase in vivo plasma half-life and exposure. In vitro studies demonstrated targeted cytotoxicity of IMGC936 across a panel of ADAM9-positve tumor cell lines with activity at least 2 logs greater than a non-targeting conjugate. Consistent with the in vitroactivity, an anti-ADAM9-DM21 conjugate displayed compelling anti-tumor activity in multiple xenograft models representing non-small cell lung, gastric and colorectal cancers. For example, in the EBC-1 non-small cell lung cancer subcutaneous xenograft model with only moderate ADAM9 expression (H-score of 130), anti-ADAM9-DM21 not only induced tumor growth delay but produced complete and durable remissions in 6/6 mice following a single intravenous dose of 8.6 mg Ab/kg (100 ug DM21/kg). IMGC936 demonstrated a favorable pharmacokinetic profile with good conjugate stability in non-human primates. Importantly, IMGC936 was well-tolerated following repeat dosing in cynomolgus monkeys with no ADAM9 target-related toxicities identified at doses exceeding the levels required for anti-tumor activity in murine xenograft models. Based on the totality of the preclinical data, IMGC936 represents a promising therapeutic candidate to target a wide range of ADAM9-expressing tumors. Citation Format: Stuart Hicks, Deryk Loo, Kerstin Sinkevicius, Juniper Scribner, Bhaswati Barat, Nicholas Yoder, Christopher Espelin, Marian Themeles, Francine Chen, Jacquelynn Lucas, Jennifer Brown, Bahar Matin, Megan Fuller, Jenny Lee, Paulin Salomon, Juliet Costoplus, Sadiqa Yancey, Gundo Diedrich, Sergey Gorlatov, Thomas Son, Michael Chiechi, Pam Li, Michael Spliedt, Valentina Ciccarone, Jeff Hooley, Nadia Gantt, James Tamura, Kerry Donahue, Paul Moore, Syd Johnson, Thomas Chittenden, Richard Gregory, Ezio Bonvini. IMGC936, a first-in-class ADAM9-targeting antibody-drug conjugate, demonstrates promising anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1533.

Selection of Antibodies with Tailored Properties by Application of High-Throughput Multiparameter Fluorescence-Activated Cell Sorting of Yeast-Displayed Immune Libraries

In this study, we present a multiparameter screening procedure for the identification of target-specific antibodies with prescribed properties. Based on B cell receptor gene repertoires from transgenic rats, yeast surface display libraries were generated, and high-affinity human antibodies were readily isolated. We demonstrate that specific desirable features, i.e., species’ cross-reactivity and a broad epitope coverage can be integrated into the screening procedure using high-throughput fluorescence-activated cell sorting. We show that the applied screening stringencies translate directly into binding properties of isolated human antibody variants.

Development of a new high-affinity human antibody with antitumor activity against solid and blood malignancies.

mAbs have emerged as a promising strategy for the treatment of cancer. However, in several malignancies, no effective antitumor mAbs are yet available. Identifying therapeutic mAbs that recognize common tumor antigens could render the treatment widely applicable. Here, a human single-chain variable fragment (scFv) antibody library was sequentially affinity selected against a panel of human cancer cell lines and an antibody fragment (named MS5) that bound to solid and blood cancer cells was identified. The MS5 scFv was fused to the human IgG1 Fc domain to generate an antibody (MS5-Fc fusion) that induced antibody-dependent cellular cytotoxicity and phagocytosis of cancer cells by macrophages. In addition, the MS5-Fc antibody bound to primary leukemia cells and induced antibody-dependent cellular cytotoxicity. In the majority of analyzed cancer cells, the MS5-Fc antibody induced cell surface redistribution of the receptor complexes, but not internalization, thus maximizing the accessibility of the IgG1 Fc domain to immune effector cells. In vitro stability studies showed that the MS5-Fc antibody was stable after 6 d of incubation in human serum, retaining ∼60% of its initial intact form. After intravenous injections, the antibody localized into tumor tissues and inhibited the growth of 3 different human tumor xenografts (breast, lymphoma, and leukemia). These antitumor effects were associated with tumor infiltration by macrophages and NK cells. In the Ramos B-cell lymphoma xenograft model, the MS5-Fc antibody exhibited a comparable antitumor effect as rituximab, a chimeric anti-CD20 IgG1 mAb. These results indicate that human antibodies with pan-cancer abilities can be generated from phage display libraries, and that the engineered MS5-Fc antibody could be an attractive agent for further clinical investigation.-Sioud, M., Westby, P., Vasovic, V., Fløisand, Y., Peng, Q. Development of a new high-affinity human antibody with antitumor activity against solid and blood malignancies.

Engineering a high-affinity humanized anti-CD24 antibody to target hepatocellular carcinoma by a novel CDR grafting design.

Cluster of differentiation 24 (CD24) is a specific surface marker involved in the tumorigenesis and progression of hepatocellular carcinoma (HCC). However, all reported anti-CD24 antibodies are murine ones with inevitable immunogenicity. To address this, a method using both molecular structure and docking-based complementarity determining region (CDR) grafting was employed for humanization. After xenogeneic CDR grafting into a human antibody, three types of canonical residues (in the VL/VH interface core, in the loop foundation, and interaction with loop residues) that support loop conformation and residues involved in the antigen-binding interface were back-mutated. Four engineered antibodies were produced, among which hG7-BM3 has virtually identical 3-D structure and affinity parameters with the parental chimeric antibody cG7. In vitro, hG7-BM3 demonstrated superior immunogenicity and serum stability to cG7. Antibody-dependent cellular cytotoxicity (ADCC), tumor cell internalization and in vivo targeting assays indicate that hG7-BM3 has the potential for development as an antibody-drug conjugate (ADC). We therefore generated the hG7-BM3-VcMMAE conjugate, which was shown to induce tumor cell apoptosis and effectively suppress nude mice bearing HCC xenografts. In conclusion, our study provides new inspiration for antibody humanization and an ADC candidate for laboratory study and clinical applications.

A Therapeutic Antibody for Cancer, Derived from Single Human B Cells.

Some patients with cancer never develop metastasis, and their host response might provide cues for innovative treatment strategies. We previously reported an association between autoantibodies against complement factor H (CFH) and early-stage lung cancer. CFH prevents complement-mediated cytotoxicity (CDC) by inhibiting formation of cell-lytic membrane attack complexes on self-surfaces. In an effort to translate these findings into a biologic therapy for cancer, we isolated and expressed DNA sequences encoding high-affinity human CFH antibodies directly from single, sorted B cells obtained from patients with the antibody. The co-crystal structure of a CFH antibody-target complex shows a conformational change in the target relative to the native structure. This recombinant CFH antibody causes complement activation and release of anaphylatoxins, promotes CDC of tumor cell lines, and inhibits tumor growth invivo. The isolation of anti-tumor antibodies derived from single human B cells represents an alternative paradigm in antibody drug discovery.

Selection and characterization of high affinity VEGFR1 antibodies from a novel human binary code scFv phage library

VEGFR1 is a receptor tyrosine kinase that has been implicated in cancer pathogenesis. It is upregulated in angiogenic endothelial cells and expressed on human tumor cells as well. VEGFR1 positive hematopoietic progenitor cells home to sites of distant metastases prior to the arrival of the tumor cells thus establishing a pre-metastatic niche. To discover high affinity human antibodies selective for VEGFR1 molecular imaging or for molecularly targeted therapy, a novel phage display scFv library was assembled and characterized. The library was constructed from the humanized 4D5 framework that was mostly comprised tyrosine and serine residues in four complimentarity determining regions (CDRs). The library produced diverse and functional antibodies against a panel of proteins, some of which are of biomedical interest including, CD44, VEGFA, and VEGFR1. After panning, these antibodies had affinity strong enough for molecular imaging or targeted drug delivery without the need for affinity maturation. One of the anti-VEGFR1 scFvs recognized its cognate receptor and was selective for the VEGFR1.

Abstract 634: Novel antibody-drug conjugates targeting Axl show anti-tumor activity in solid cancer xenograft models

Abstract Axl is a receptor tyrosine kinase of the TAM family, which is structurally characterized by the presence of two extracellular immunoglobulin-like domains and two fibronectin type III domains, a single-pass transmembrane region and an intracellular tyrosine kinase domain. Axl is aberrantly expressed and activated in both solid and hematological malignancies and has been associated with poor clinical prognosis in a number of cancers, including non-small cell lung cancer, breast cancer, pancreatic cancer and acute myeloid lymphoma. Functionally, Axl has been implicated in multiple pathological processes, such as tumor cell motility, adherence and migration, epithelial-to-mesenchymal transition, metastasis, angiogenesis and resistance to tyrosine kinase inhibitors. With the aim to target Axl expressing cancers, a panel of Axl-specific antibody-drug conjugates (ADC) was generated. First, high affinity human Axl antibodies were generated in HuMAb mice. Binding studies using Axl mutants, in which specific human Axl domains had been replaced with their murine analogs, demonstrated that the Axl antibody panel consisted of antibodies against different Axl extracellular domains. Second, the Axl antibodies were conjugated with the microtubule disrupting agent monomethyl auristatin E (MMAE) using the protease cleavable valine citrulline linker. Axl ADCs showed cytotoxic activity in vitro, which was dependent on target expression and on conjugation with cytotoxic payload. The anti-tumor activity of Axl ADCs was confirmed in vivo. Axl ADCs induced tumor regression in cell line derived xenograft models for lung cancer and epidermoid carcinoma. Anti-tumor activity was especially efficient in the lung cancer xenograft model, in which inhibition of tumor growth was observed upon treatment with a single dose of 0.5 mg/kg Axl ADC. In the same model, 1 mg/kg Axl ADC was sufficient to induce complete tumor regression. Importantly, Axl ADCs also showed efficient anti-tumor activity in patient-derived xenograft (PDX) models that showed heterogeneous target expression. In summary, Axl is a novel target for antibody-mediated delivery of cytotoxic agents into tumor cells. The promising results obtained in preclinical tumor models support further development of Axl-specific ADCs. Citation Format: Esther C.W. Breij, Sandra Verploegen, Andreas Lingnau, Edward N. van den Brink, Maarten Janmaat, Mischa Houtkamp, Wim K. Bleeker, David Satijn, Paul W.H.I. Parren. Novel antibody-drug conjugates targeting Axl show anti-tumor activity in solid cancer xenograft models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 634. doi:10.1158/1538-7445.AM2015-634

Selective targeting of the IL23 pathway: Generation and characterization of a novel high-affinity humanized anti-IL23A antibody

Herein, we describe the generation and characterization of BI 655066, a novel, highly potent neutralizing anti-interleukin-23 (IL23) monoclonal antibody in clinical development for autoimmune conditions, including psoriasis and Crohn's disease. IL23 is a key driver of the differentiation, maintenance, and activity of a number of immune cell subsets, including T helper 17 (Th17) cells, which are believed to mediate the pathogenesis of several immune-mediated disorders. Thus, IL23 neutralization is an attractive therapeutic approach. Designing an antibody for clinical activity and convenience for the patient requires certain properties, such as high affinity, specificity, and solubility. These properties were achieved by directed design of the immunization, lead identification, and humanization procedures. Favorable substance and pharmacokinetic properties were established by biophysical assessments and studies in cynomolgus monkeys.

Generation and characterization of tabalumab, a human monoclonal antibody that neutralizes both soluble and membrane-bound B-cell activating factor.

B-cell activating factor (BAFF) is a B-cell survival factor with a key role in B-cell homeostasis and tolerance. Dysregulated BAFF expression may contribute to autoimmune diseases or B-cell malignancies via effects on abnormal B-lymphocyte activation, proliferation, survival, and immunoglobulin secretion. Monoclonal antibodies were generated against human BAFF, characterized for species specificity and affinity, and screened for the ability to neutralize both membrane-bound and soluble BAFF. In addition, studies were undertaken to determine the relative potency of membrane-bound and soluble BAFF. Tabalumab has a high affinity for human, cynomolgus monkey, and rabbit BAFF. No binding to mouse BAFF was detected. Tabalumab was able to neutralize soluble human, cynomolgus monkey, or rabbit BAFF with equal potency. Our data demonstrate that membrane-bound BAFF can be a more potent stimulus for B-cells than soluble BAFF, and tabalumab also neutralized membrane-bound BAFF. Tabalumab prevented BAFF from binding to BAFF receptors and demonstrated pharmacodynamic effects in human BAFF transgenic mice. Tabalumab is a high-affinity human antibody with neutralizing activity against membrane-bound and soluble BAFF. Given our findings that membrane-bound BAFF can have greater in vitro potency than soluble BAFF, neutralization of both forms of BAFF is likely to be important for optimal therapeutic effect.

Peripheral administration of a humanized anti-PrP antibody blocks Alzheimer's disease Aβ synaptotoxicity.

Alzheimer's disease (AD) is associated with pathological assembly states of amyloid-β protein (Aβ). Aβ-related synaptotoxicity can be blocked by anti-prion protein (PrP) antibodies, potentially allowing therapeutic targeting of this aspect of AD neuropathogenesis. Here, we show that intravascular administration of a high-affinity humanized anti-PrP antibody to rats can prevent the plasticity-disrupting effects induced by exposure to soluble AD brain extract. These results provide an in vivo proof of principle for such a therapeutic strategy.

Mammalian Cell Display and Somatic Hypermutation In Vitro for Human Antibody Discovery

Human therapeutic antibody discovery has utilized a variety of systems, from in vivo immunization of human immunoglobulin-expressing mice, to in vitro display of antibody libraries. Of the in vitro antibody display technologies, mammalian cell display provides a number of advantages with the ability to co-select immunoglobulin molecules for high expression level in mammalian cells, native folding, and biophysical properties appropriate for drug development. Mammalian cell display has been achieved using either transient or stable expression systems, using a number of alternate transmembrane domains to present antibody on the cell surface. The unique capability of mammalian cells to present IgG in its fully post-translationally modified format also allows selection of antibodies for functional properties. One limitation of mammalian cell based systems, however, has been the smaller library size that can be presented compared to phage display approaches. Until recently, this has necessitated the use of libraries biased toward a particular antigen, such as libraries derived from immunized donors, to achieve success. An alternative approach has now been developed which recapitulates key aspects of the in vivo immune system through reproducing somatic hypermutation (SHM) in vitro. Libraries representing a naïve human B lymphocyte antibody repertoire are created by PCR amplification of the rearranged (D)J segments of heavy and light chain variable regions from human donors and incorporating the resulting sequence diversity into panels of human germline VH and VL genes. The resulting antibodies are presented as full length IgG on the surface of HEK293 cells. After isolation of antibodies binding to individual target antigens, subsequent affinity maturation using in vitro SHM is induced by expression of activation-induced cytidine deaminase (AID). Selection of antibodies from naïve fully human libraries using mammalian cell display coupled with in vitro SHM is an efficient methodology for the generation of high affinity human antibodies with excellent properties for drug development.

Inhibitory Mechanism of an Allosteric Antibody Targeting the Glucagon Receptor

Elevated glucagon levels and increased hepatic glucagon receptor (GCGR) signaling contribute to hyperglycemia in type 2 diabetes. We have identified a monoclonal antibody that inhibits GCGR, a class B G-protein coupled receptor (GPCR), through a unique allosteric mechanism. Receptor inhibition is mediated by the binding of this antibody to two distinct sites that lie outside of the glucagon binding cleft. One site consists of a patch of residues that are surface-exposed on the face of the extracellular domain (ECD) opposite the ligand-binding cleft, whereas the second binding site consists of residues in the αA helix of the ECD. A docking model suggests that the antibody does not occlude the ligand-binding cleft. We solved the crystal structure of GCGR ECD containing a naturally occurring G40S mutation and found a shift in the register of the αA helix that prevents antibody binding. We also found that alterations in the αA helix impact the normal function of GCGR. We present a model for the allosteric inhibition of GCGR by a monoclonal antibody that may form the basis for the development of allosteric modulators for the treatment of diabetes and other class B GPCR-related diseases.

Human 4F5 single-chain Fv antibody recognizing a conserved HA1 epitope has broad neutralizing potency against H5N1 influenza A viruses of different clades

Influenza A viruses present a significant threat to public health worldwide. High-affinity human scFv antibodies against a conserved epitope can potentially provide immunity to diverse viruses and protect against future pandemic viruses. A library of phage-displayed human scFv containing 6.0×108 members was generated from lymphocytes of H5N1 virus vaccinated individuals. Using the recombinant H5N1 virus hemagglutinin ectodomain (HA1), 4F5 scFv was identified with neutralizing activity against both clade 2 and 9 H5N1 viruses. In embryonated chicken eggs, the antiviral activity of 4F5 scFv conferred a 100% survival rate and at least a 62.5% survival rate against different clades of H5N1 viruses by pre-treatment and post-treatment, respectively. 4F5 scFv belongs to the VH-3-43 family according to the IMGT database, and a peptide (76)WLLGNP(81) containing half of an α-helix in HA1 was identified as the binding pocket. The conserved binding epitope of this novel broadly neutralizing scFv may become key in the design and implementation of vaccines or RNA interference against H5N1 viruses.

Humanization of Antibodies Using Heavy Chain Complementarity-determining Region 3 Grafting Coupled with in Vitro Somatic Hypermutation

A method for simultaneous humanization and affinity maturation of monoclonal antibodies has been developed using heavy chain complementarity-determining region (CDR) 3 grafting combined with somatic hypermutation in vitro. To minimize the amount of murine antibody-derived antibody sequence used during humanization, only the CDR3 region from a murine antibody that recognizes the cytokine hβNGF was grafted into a nonhomologous human germ line V region. The resulting CDR3-grafted HC was paired with a CDR-grafted light chain, displayed on the surface of HEK293 cells, and matured using in vitro somatic hypermutation. A high affinity humanized antibody was derived that was considerably more potent than the parental antibody, possessed a low pm dissociation constant, and demonstrated potent inhibition of hβNGF activity in vitro. The resulting antibody contained half the heavy chain murine donor sequence compared with the same antibody humanized using traditional methods.