Hemagglutinin from multiple divergent influenza A and B viruses bind to a distinct branched, sialylated poly-LacNAc glycan by surface plasmon resonance.

Influenza viruses initiate infection via specific interactions of hemagglutinin (HA) with host cell surface sialic acid-containing glycans. Antigenic drift has resulted in HA amino acid sequence changes that affect binding properties for sialic acids. Further, viral propagation in eggs and cell culture for vaccine production can yield variants with mutations that affect the conformation and affinity of HA for sialic acids. Therefore, influenza vaccine researchers and manufacturers need robust analytical methods to assess directly the ability of vaccine candidates to bind to their specific sialic acid ligand. We developed a surface plasmon resonance method that uses an extended, biantennary glycan terminating with α-2,6 linked sialic acids to bind influenza HA and assess this interaction. Recombinant HA (rHA) from both influenza A and B viruses isolated from 1999 to 2017 strongly and specifically bind this sialic acid ligand, suggesting the binding ability of divergent HA for this ligand is resistant to antigenic drift. Importantly, the method can differentiate between wild type and mutant rHA for which binding to this sialylated glycan and red blood cells in hemagglutination assays is compromised. We believe this method can be a powerful tool to screen influenza A and B vaccine candidates and final vaccine preparations for their functional ability to bind sialic acids, which allows manufacturers to identify preparations in which mutations that affect sialic acid binding have arisen during propagation. Evaluation of vaccine rHA antigen integrity by confirmation of the receptor binding site functionality is a prudent cautionary step to assure the antigenic quality of seasonal influenza vaccines.

Combining phage display with de novo protein sequencing for reverse engineering of monoclonal antibodies.

The enormous diversity created by gene recombination and somatic hypermutation makes de novo protein sequencing of monoclonal antibodies a uniquely challenging problem. Modern mass spectrometry-based sequencing will rarely, if ever, provide a single unambiguous sequence for the variable domains. A more likely outcome is computation of an ensemble of highly similar sequences that can satisfy the experimental data. This outcome can result in the need for empirical testing of many candidate sequences, sometimes iteratively, to identity one which can replicate the activity of the parental antibody. Here we describe an improved approach to antibody protein sequencing by using phage display technology to generate a combinatorial library of sequences that satisfy the mass spectrometry data, and selecting for functional candidates that bind antigen. This approach was used to reverse engineer 2 commercially-obtained monoclonal antibodies against murine CD137. Proteomic data enabled us to assign the majority of the variable domain sequences, with the exception of 3-5% of the sequence located within or adjacent to complementarity-determining regions. To efficiently resolve the sequence in these regions, small phage-displayed libraries were generated and subjected to antigen binding selection. Following enrichment of antigen-binding clones, 2 clones were selected for each antibody and recombinantly expressed as antigen-binding fragments (Fabs). In both cases, the reverse-engineered Fabs exhibited identical antigen binding affinity, within error, as Fabs produced from the commercial IgGs. This combination of proteomic and protein engineering techniques provides a useful approach to simplifying the technically challenging process of reverse engineering monoclonal antibodies from protein material.

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

The three-dimensional structure of a human IgG1 Fc fragment bound to wild-type human FcγRI is reported. The structure of the corresponding complex was solved at a resolution of 2.4 Šusing molecular replacement; this is the highest resolution achieved for an unmutated FcγRI molecule. This study highlights the critical structural and functional role played by the second extracellular subdomain of FcγRI. It also explains the long-known major energetic contribution of the Fc `LLGG' motif at positions 234-237, and particularly of Leu235, via a `lock-and-key' mechanism. Finally, a previously held belief is corrected and a differing view is offered on the recently proposed direct role of Fc carbohydrates in the corresponding interaction. Structural evidence is provided that such glycan-related effects are strictly indirect.

Structural Insights into the Neutralization Properties of the Fully Human, Anti-interferon Monoclonal Antibody Sifalimumab.

We report the three-dimensional structure of human interferon α-2A (IFN-α2A) bound to the Fab fragment of a therapeutic monoclonal antibody (sifalimumab; IgG1/κ). The structure of the corresponding complex was solved at a resolution of 3.0 Å using molecular replacement and constitutes the first reported structure of a human type I IFN bound to a therapeutic antibody. This study revealed the major contribution made by the first complementarity-determining region in each of sifalimumab light and heavy chains. These data also provided the molecular basis for sifalimumab mechanism of action. We propose that its interferon-neutralizing properties are the result of direct competition for IFN-α2A binding to the IFN receptor subunit 1 (IFNAR1) and do not involve inhibiting IFN-α2A binding to the IFN receptor subunit 2 (IFNAR2).

The design and characterization of oligospecific antibodies for simultaneous targeting of multiple disease mediators.

Monoclonal antibodies are traditionally used to block the function of a specific target in a given disease. However, some diseases are the consequence of multiple components or pathways and not the result of a single mediator; thus, blocking at a single point may not optimally control disease. Antibodies that simultaneously block the functions of two or more disease-associated targets are now being developed. Herein, we describe the design, expression, and characterization of several oligospecific antibody formats that are capable of binding simultaneously to two or three different antigens. These constructs were generated by genetically linking single-chain Fv fragments to the N-terminus of the antibody heavy and light chains and to the C-terminus of the antibody C(H)3 domain. The oligospecific antibodies were expressed in mammalian cells, purified to homogeneity, and characterized for binding to antigens, Fcgamma receptors, FcRn, and C1q. In addition, the oligospecific antibodies were assayed for effector function, protease susceptibility, thermal stability, and size distribution. We demonstrate that these oligospecific antibody formats maintain high expression level, thermostability, and protease resistance. The in vivo half-life, antibody-dependent cellular cytotoxicity function, and binding ability to Fcgamma receptors and C1q of the test oligospecific antibodies remain similar to the corresponding properties of their parental IgG antibodies. The excellent expression, biophysical stability, and potential manufacturing feasibility of these multispecific antibody formats suggest that they will provide a scaffold template for the construction of similar molecules to target multiple antigens in complex diseases.

Structural characterization of a human Fc fragment engineered for extended serum half-life.

The first three-dimensional structure of a human Fc fragment genetically engineered for improved pharmacokinetics properties is reported. When introduced into the C(H)2 domain of human immunoglobulin G (IgG) molecules, the triple mutation M252Y/S254T/T256E ('YTE') causes an about 10-fold increase in their binding to the human neonatal Fc receptor (FcRn). This translates into an almost 4-fold increase in the serum half-life of YTE-containing human IgGs in cynomolgus monkeys. A recombinantly produced human Fc/YTE fragment was crystallized and its structure solved at a resolution of 2.5A using molecular replacement. This revealed that Fc/YTE three-dimensional structure is very similar to that of other human Fc fragments in the experimentally visible region spanning residues 236-444. We propose that the enhanced interaction between Fc/YTE and human FcRn is likely mediated by local effects at the substitutions sites. Molecular modeling suggested that potential favorable hydrogen bonds along with an increase in the surface of contact between the two partners may account in part for the corresponding increase in affinity.

Development of motavizumab, an ultra-potent antibody for the prevention of respiratory syncytial virus infection in the upper and lower respiratory tract.

Respiratory syncytial virus (RSV) is the leading cause of viral bronchiolitis and pneumonia in infants and children. Currently, palivizumab is the only approved monoclonal antibody (mAb) for prophylaxis of RSV. However, a small percentage of patients are not protected by palivizumab; in addition, palivizumab does not inhibit RSV replication effectively in the upper respiratory tract. We report here the development and characterization of motavizumab, an ultra-potent, affinity-matured, humanized mAb derived from palivizumab. Several palivizumab variants that enhanced the neutralization of RSV in vitro by up to 44-fold were generated; however, in vivo prophylaxis of cotton rats with these antibodies conferred only about a twofold improvement in potency over palivizumab. This unexpected small increase of in vivo potency was caused by poor serum pharmacokinetics and lung bio-availability that resulted from unexpectedly broad tissue binding. Subsequent analyses revealed that changes at three amino acids arising from the affinity maturation markedly increased the non-specific binding to various tissues. Our results suggested that k(on)-driven mutations are more likely to initiate non-specific binding events than k(off)-driven mutations. Reversion of these three residues to the original sequences greatly diminished the tissue binding. The resulting mAb, motavizumab, binds to RSV F protein 70-fold better than palivizumab, and exhibits about a 20-fold improvement in neutralization of RSV in vitro. In cotton rats, at equivalent concentrations, motavizumab reduced pulmonary RSV titers to up to 100-fold lower levels than did palivizumab and, unlike palivizumab, motavizumab very potently inhibited viral replication in the upper respiratory tract. This affinity-enhanced mAb is being investigated in pivotal clinical trials. Importantly, our engineering process offers precious insights into the improvement of other therapeutic mAbs.

Isolation and characterization of monoclonal antibodies which neutralize human metapneumovirus in vitro and in vivo.

Human metapneumovirus (hMPV) is a recently described member of the Paramyxoviridae family/Pneumovirinae subfamily and shares many common features with respiratory syncytial virus (RSV), another member of the same subfamily. hMPV causes respiratory tract illnesses that, similar to human RSV, occur predominantly during the winter months and have symptoms that range from mild to severe cough, bronchiolitis, and pneumonia. Like RSV, the hMPV virus can be subdivided into two genetic subgroups, A and B. With RSV, a single monoclonal antibody directed at the fusion (F) protein can prevent severe lower respiratory tract RSV infection. Because of the high level of sequence conservation of the F protein across all the hMPV subgroups, this protein is likely to be the preferred antigenic target for the generation of cross-subgroup neutralizing antibodies. Here we describe the generation of a panel of neutralizing monoclonal antibodies that bind to the hMPV F protein. A subset of these antibodies has the ability to neutralize prototypic strains of both the A and B hMPV subgroups in vitro. Two of these antibodies exhibited high-affinity binding to the F protein and were shown to protect hamsters against infection with hMPV. The data suggest that a monoclonal antibody could be used prophylactically to prevent lower respiratory tract disease caused by hMPV.

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 FcgammaRIIIA 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.

Antibody humanization by framework shuffling.

We report here the humanization of a mouse monoclonal antibody (mAb B233) using a new technique which we call framework shuffling. mAb B233 was raised against the human receptor tyrosine kinase EphA2 which is selectively up-regulated in many cancer cell lines and as such constitutes an attractive target for cancer therapy. The six CDRs of B233 were fused in-frame to pools of corresponding individual human frameworks. These human frameworks encompassed all known heavy and light (kappa) chain human germline genes. The resulting Fab combinatorial libraries were then screened for binding to the antigen. A two-step selection process, in which the light and heavy chains of the parental mAb were successively humanized, resulted in the identification of several humanized variants that retained binding to EphA2. More precisely, after conversion to human IgG1, the dissociation constants of three select fully humanized variants ranged from 3 to 48 nM. This brings the best framework-shuffled, humanized binder within 5-fold of the avidity of parental mAb B233. Importantly, these humanized IgGs also possessed biochemical activities similar to those of parental mAb B233 as judged by induction of EphA2 phosphorylation. Thus, without requiring any rational design or structural information, this new humanization approach allows to rapidly identify various human framework combinations able to support the structural feature(s) of the CDRs which are essential for binding and functional activity.

Analysis of human and primate CD2 molecules by protein sequence and epitope mapping with anti-human CD2 antibodies.

A panel of anti-human CD2 monoclonal antibodies (mAb) and soluble human CD58 (LFA-3) were tested for binding to human peripheral blood mononuclear cells (PBMCs), recombinant human CD2 and mononuclear cells from Cynomolgus, Rhesus and African green monkey, Stump-tail, Pig-tail and Assamese macaque, Chimpanzee and Baboon. This analysis revealed that whilst some antibodies recognized all species, there were differential binding profiles with others. Three antibodies, MEDI-507, 6F10.3 and 4B2, recognized CD2 from human and Chimpanzee but not that from the other primates. We have cloned eight of the previously unknown primate CD2 molecules and report here their sequences for the first time. This analysis revealed that 12 amino acids formed a common set of residues in the extra cellular domain of human and Chimpanzee CD2. Using a "knock-in" mutagenesis approach starting with Baboon CD2, which does not bind MEDI-507, 6F10.3 and 4B2, we have identified three residues in the adhesion domain of human CD2 which are critical for its binding to these mAbs. These residues, N18, K55 and T59 define a region located outside of the previously described binding regions on CD2. Affinity measurements of the mutants revealed a variety of degrees of binding restoration for MEDI-507, 6F10.3 and 4B2, indicating that there are fine differences within a given epitope. Furthermore, the analysis of the competition of several of the anti-human CD2 antibodies with each other and CD58 demonstrated the existence of a continuum of overlapping epitopes on human CD2, which is in contrast to the commonly held belief that epitopes on human CD2 are clearly segregated.

Increasing the affinity of a human IgG1 for the neonatal Fc receptor: biological consequences.

Many biological functions, including control of the homeostasis and maternofetal transfer of serum gamma-globulins, are mediated by the MHC class I-related neonatal FcR (FcRn). A correlation exists in mice between the binding affinity of IgG1/Fc fragments to FcRn at pH 6.0 and their serum t(1/2). To expand this observation, phage display of mutagenized Fc fragments derived from a human IgG1 was used to increase their affinity to both murine and human FcRn. Ten variants were identified that have a higher affinity toward murine and human FcRn at pH 6.0, with DeltaDeltaG (DeltaG(wild type) - DeltaG(mutant)) from 1.0 to 2.0 kcal/mol and from 0.6 to 2.4 kcal/mol, respectively. Those variants exhibit a parallel increase in binding at pH 7.4 to murine, but not human, FcRn. Although not degraded in blood in vitro, accumulated in tissues, nor excreted in urine, their serum concentration in mice is decreased. We propose that higher affinity to FcRn at pH 7.4 adversely affects release into the serum and offsets the benefit of the enhanced binding at pH 6.0.