Kimberly E. Cook

Modulation of the effector functions of a human IgG1 through engineering of its hinge region.

We report here the engineering of a humanized anti-human EphA2 mAb (mAb 12G3H11) in an effort to explore the relationship between the hinge of a human IgG1 and its effector functions. mAb 12G3H11, used here as a model, is directed against the human receptor tyrosine kinase EphA2, which is an actively investigated target for cancer therapy due to its up-regulation in many cancer cells. Various rational modifications were introduced into the hinge region of mAb 12G3H11. These mutations were predicted to modulate the hinge’s length, flexibility, and/or biochemical properties. We show that the upper and middle hinge both play important, although functionally distinct roles. In particular, middle hinge modifications predicted to decrease its rigidity or length as well as eliminating either one of its two cysteine residues had a strong negative impact on C1q binding and complement-dependent cytotoxicity. Disruption of covalent bonds between both H chains may account in part for these effects. We also describe middle hinge mutants with a significantly decreased ability to bind FcγRIIIA and trigger Ab-dependent cell-mediated cytotoxicity. Conversely, we also generated upper hinge mutants exhibiting an increase in C1q binding and complement-dependent cytotoxicity activity. Therefore, this approach represents a novel strategy to fine-tune the biological activity of a given human IgG1. We also define, for the first time in such a systematic fashion, the relationship between various characteristics of the middle and upper hinge and the corresponding effector functions.

Structural Insights Into Neonatal Fc Receptor-Based Recycling Mechanisms

Background: The neonatal Fc receptor (FcRn) regulates the serum half-life of human IgGs and serum albumin (SA). Results: Structures of human FcRn bound to its two ligands shed new lights on the corresponding interactions. Conclusion: Molecular mechanisms for IgG/SA recycling and increased IgG/FcRn binding affinity are presented. Significance: Understanding the molecular basis of the IgG/SA/FcRn interaction is crucial to develop biologics exhibiting beneficial pharmacokinetics properties. We report the three-dimensional structure of human neonatal Fc receptor (FcRn) bound concurrently to its two known ligands. More particularly, we solved the crystal structure of the complex between human FcRn, wild-type human serum albumin (HSA), and a human Fc engineered for improved pharmacokinetics properties (Fc-YTE). The crystal structure of human FcRn bound to wild-type HSA alone is also presented. HSA domain III exhibits an extensive interface of contact with FcRn, whereas domain I plays a lesser role. A molecular explanation for the HSA recycling mechanism is provided with the identification of FcRn His161 as the only potential direct contributor to the corresponding pH-dependent process. At last, this study also allows an accurate structural definition of residues considered for decades as important to the human IgG/FcRn interaction and reveals Fc His310 as a significant contributor to pH-dependent binding. Finally, we explain various structural mechanisms by which several Fc mutations (including YTE) result in increased human IgG binding to FcRn. Our study provides an unprecedented relevant understanding of the molecular basis of human Fc interaction with human FcRn.

A Novel Anti-PcrV Antibody Providing Enhanced Protection against Pseudomonas aeruginosa in Multiple Animal Infection Models

ABSTRACT Pseudomonas aeruginosa is a major cause of hospital-acquired infections, particularly in mechanically ventilated patients, and it is the leading cause of death in cystic fibrosis patients. A key virulence factor associated with disease severity is the P. aeruginosa type III secretion system (T3SS), which injects bacterial toxins directly into the cytoplasm of host cells. The PcrV protein, located at the tip of the T3SS injectisome complex, is required for T3SS function and is a well-validated target in animal models of immunoprophylactic strategies targeting P. aeruginosa. In an effort to identify a highly potent and protective monoclonal antibody (MAb) that inhibits the T3SS, we generated and characterized a panel of novel anti-PcrV MAbs. Interestingly, some MAbs exhibiting potent inhibition of T3SS in vitro failed to provide protection in a mouse model of P. aeruginosa infection, suggesting that effective in vivo inhibition of T3SS with anti-PcrV MAbs is epitope dependent. V2L2MD, while not the most potent MAb as assessed by in vitro cytotoxicity inhibition assays, provided strong prophylactic protection in several murine infection models and a postinfection therapeutic model. V2L2MD mediated significantly (P < 0.0001) better in vivo protection than that provided by a comparator antibody, MAb166, a well-characterized anti-PcrV MAb and the progenitor of a clinical candidate, KB001-A. The results described here support further development of a V2L2MD-containing immunotherapeutic and may suggest even greater potential than was previously recognized for the prevention and treatment of P. aeruginosa infections in high-risk populations.

A highly potent extended half-life antibody as a potential RSV vaccine surrogate for all infants.

Development of a highly potent anti-RSV monoclonal antibody with extended half-life intended to be used as RSV prophylaxis for all infants. Pan-RSV prophylaxis The common respiratory syncytial virus (RSV) can progress to a very dangerous lower respiratory infection in some infants. A protective monoclonal antibody is available but is not recommended for general use. Zhu et al. describe the selection and optimization of a human monoclonal antibody able to neutralize a wide array of RSV A and B viruses and protect cotton rats at lower doses than the currently approved antibody. The antibody was optimized to persist in circulation, and data indicate that infants could be given a single dose and be protected for the entirety of the RSV season. If administered widely, this antibody could potentially prevent the hospitalization of thousands of children each year. Prevention of respiratory syncytial virus (RSV) illness in all infants is a major public health priority. However, no vaccine is currently available to protect this vulnerable population. Palivizumab, the only approved agent for RSV prophylaxis, is limited to high-risk infants, and the cost associated with the requirement for dosing throughout the RSV season makes its use impractical for all infants. We describe the development of a monoclonal antibody as potential RSV prophylaxis for all infants with a single intramuscular dose. MEDI8897*, a highly potent human antibody, was optimized from antibody D25, which targets the prefusion conformation of the RSV fusion (F) protein. Crystallographic analysis of Fab in complex with RSV F from subtypes A and B reveals that MEDI8897* binds a highly conserved epitope. MEDI8897* neutralizes a diverse panel of RSV A and B strains with >50-fold higher activity than palivizumab. At similar serum concentrations, prophylactic administration of MEDI8897* was ninefold more potent than palivizumab at reducing pulmonary viral loads by >3 logs in cotton rats infected with either RSV A or B subtypes. MEDI8897 was generated by the introduction of triple amino acid substitutions (YTE) into the Fc domain of MEDI8897*, which led to more than threefold increased half-life in cynomolgus monkeys compared to non-YTE antibody. Considering the pharmacokinetics of palivizumab in infants, which necessitates five monthly doses for protection during an RSV season, the high potency and extended half-life of MEDI8897 support its development as a cost-effective option to protect all infants from RSV disease with once-per-RSV-season dosing in the clinic.

Structural insights into the interaction of human IgG1 with FcγRI: no direct role of glycans in binding

In an effort to identify the critical structural features responsible for the high-affinity interaction of IgG1 Fc with FcγRI, the structure of the corresponding complex was solved at a resolution of 2.4 Å.

Targeting the junction of CɛmX and ɛ-migis for the specific depletion of mIgE-expressing B cells

Monoclonal antibodies targeting the extracellular region of the human IgE heavy chain membrane-tethering domain have been proposed for treating allergies caused by hyperproliferative monoclonal expansion of IgE-producing B cells. Antibodies against this target are expected to deplete membrane IgE (mIgE) displaying B cells and leave B cells of other immunoglobulin isotypes intact. Because of alternative splicing, the mIgE heavy chain has two isoforms that differ in their membrane-proximal segment. In the long isoform, the CH4 domain is followed by a 67-amino acid-long extracellular portion. Out of these 67 amino acids, the first 52 amino acids following the CH4 domain constitute the CɛmX segment while the rest of the 15 amino acids immediately adjacent to the membrane constitute the ɛ-migis. In the short isoform the CɛmX segment is absent and the CH4 domain is followed only by the 15-amino acid-long ɛ-migis segment. Using antibodies derived from a phage display library, we investigated: (1) ɛ-migis and (2) the junction of CɛmX and ɛ-migis (CɛmX.migis), as potential therapeutic antibody targets. Our results indicate that antibodies obtained from our phage library that target ɛ-migis bind to a variety of human cells irrespective of mIgE expression, possibly due to homology between ɛ-migis and a region of phosphoinositide-binding protein (ARAP3). In contrast, antibodies specific for the CɛmX.migis junctional region, bound specifically to transfected and primary B cells expressing human mIgE and elicited antibody-dependent cellular cytotoxicity and reduction in IgE production. These antibodies did not bind secreted IgE or the mIgE isoform in which CɛmX is absent. These results suggest that CɛmX.migis junctional region is a promising antibody target and the human antibodies we describe warrant further evaluation.

Crystallization and preliminary X-ray diffraction analysis of the complex of a human anti-ephrin type-A receptor 2 antibody fragment and its cognate antigen

The recombinant N-terminal domain of human ephrin type-A receptor 2 (rEphA2) has been crystallized in complex with the recombinantly produced Fab fragment of a fully human antibody (1C1; IgG1/kappa). These are the first reported crystals of an ephrin receptor bound to an antibody. The orthorhombic crystals belonged to space group C222(1) (the 00l reflections obey the l = 2n rule), with unit-cell parameters a = 78.93, b = 120.79, c = 286.20 A. The diffraction of the crystals extended to 2.0 A resolution. However, only data to 2.55 A resolution were considered to be useful owing to spot overlap caused by the long unit-cell parameter. The asymmetric unit is most likely to contain two 1C1 Fab-rEphA2 complexes. This corresponds to a crystal volume per protein weight (V(M)) of 2.4 A(3) Da(-1) and a solvent content of 49.5%. The three-dimensional structure of this complex will shed light on the molecular basis of 1C1 specificity. This will also contribute to a better understanding of the mechanism of action of this antibody, the current evaluation of which as an antibody-drug conjugate in cancer therapy makes it a particularly interesting case study.

Molecular basis for the mechanism of action of an anti-TACE antibody

ABSTRACT Inhibitors of tumor necrosis factor-α converting enzyme (TACE) have potential as therapeutics for various diseases. Many small molecule inhibitors, however, exhibit poor specificity profiles because they target the highly conserved catalytic cleft of TACE. We report for the first time the molecular interaction of a highly specific anti-TACE antagonistic antibody (MEDI3622). We characterized the binding of MEDI3622 using mutagenesis, as well as structural modeling and docking approaches. We show that MEDI3622 recognizes a unique surface loop of sIVa-sIVb β-hairpin on TACE M-domain, but does not interact with the conserved catalytic cleft or its nearby regions. The exquisite specificity of MEDI3622 is mediated by this distinct structural feature on the TACE M-domain. These findings may aid the design of antibody therapies against TACE.

Molecular basis for the antagonistic activity of an anti-CXCR4 antibody

Antagonistic antibodies targeting the G-protein C-X-C chemokine receptor 4 (CXCR4) hold promising therapeutic potential in various diseases. We report for the first time the detailed mechanism of action at a molecular level of a potent anti-CXCR4 antagonistic antibody (MEDI3185). We characterized the MEDI3185 paratope using alanine scanning on all 6 complementary-determining regions (CDRs). We also mapped its epitope using CXCR4 mutagenesis to assess the relative importance of the CXCR4 N-terminal peptide, extracellular loops (ECL) and ligand-binding pocket. We show that the interaction between MEDI3185 and CXCR4 is mediated mostly by CDR3H in MEDI3185 and ECL2 in CXCR4. The MEDI3185 epitope comprises the entire ECL2 sequence, lacks any so-called ‘hot-spot’ and is remarkably resistant to mutations. The structure of MEDI3185 variable domains was modeled, and suggested a β-strand/β-strand interaction between MEDI3185 CDR3H and CXCR4 ECL2, resulting in direct steric hindrance with CXCR4 ligand SDF-1. These findings may have important implications for designing antibody therapies against CXCR4.

Crystallization and preliminary X-ray diffraction analysis of the complex between a human anti-interferon antibody fragment and human interferon α-2A

Crystals of the complex between the Fab fragment of a human anti-interferon α therapeutic antibody and human interferon α-2A have been obtained and diffracted to 3.0 Å resolution.