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Antibodies are fundamental effectors of humoral immunity, and have become a highly successful class of therapeutics. There is increasing evidence that antibodies utilize transient homotypic interactions to enhance function, and elucidation of such interactions can provide insights into their biology and new opportunities for their optimization as drugs. Yet the transitory nature of weak interactions makes them difficult to investigate. Capitalizing on their rich structural data and high conservation, we have characterized all the ways that antibody fragment antigen-binding (Fab) regions interact crystallographically. This approach led to the discovery of previously unrealized interfaces between antibodies. While diverse interactions exist, β-sheet dimers and variable-constant elbow dimers are recurrent motifs. Disulfide engineering enabled interactions to be trapped and investigated structurally and functionally, providing experimental validation of the interfaces and illustrating their potential for optimization. This work provides first insight into previously undiscovered oligomeric interactions between antibodies, and enables new opportunities for their biotherapeutic optimization.

Developability assessment of therapeutic antibody candidates assists drug discovery by enabling early identification of undesirable instabilities. Rapid chemical stability screening of antibody variants can accelerate the identification of potential solutions. We describe here the development of a high-throughput assay to characterize asparagine deamidation. We applied the assay to identify a mutation that unexpectedly stabilizes a critical asparagine. Ninety antibody variants were incubated under thermal stress in order to induce deamidation and screened for both affinity and total binding capacity. Surprisingly, a mutation five residues downstream from the unstable asparagine greatly reduced deamidation. Detailed assessment by LC-MS analysis confirmed the predicted improvement. This work describes both a high-throughput method for antibody stability screening during the early stages of antibody discovery and highlights the value of broad searches of antibody sequence space.

HM1.24, an immunologic target for multiple myeloma (MM) cells, has not been effectively targeted with therapeutic monoclonal antibodies (mAbs). In this study, we investigated in vitro and in vivo anti-MM activities of XmAb5592, a humanized anti-HM1.24 mAb with Fc-domain engineered to significantly enhance FcγR binding and associated immune effector functions. XmAb5592 increased antibody-dependent cellular cytotoxicity (ADCC) several fold relative to the anti-HM1.24 IgG1 analog against both MM cell lines and primary patient myeloma cells. XmAb5592 also augmented antibody dependent cellular phagocytosis (ADCP) by macrophages. Natural killer (NK) cells became more activated by XmAb5592 than the IgG1 analog, evidenced by increased cell surface expression of granzyme B–dependent CD107a and MM cell lysis, even in the presence of bone marrow stromal cells. XmAb5592 potently inhibited tumor growth in mice bearing human MM xenografts via FcγR-dependent mechanisms, and was significantly more effective than the IgG1 analog. Lenalidomide synergistically enhanced in vitro ADCC against MM cells and in vivo tumor inhibition induced by XmAb5592. A single dose of 20 mg/kg XmAb5592 effectively depleted both blood and bone marrow plasma cells in cynomolgus monkeys. These results support clinical development of XmAb5592, both as a monotherapy and in combination with lenalidomide, to improve patient outcome of MM.

Over the past few years, we have witnessed exciting advances in protein design. Several groups have reported success in the design of hydrophobic cores, and the principles developed in these studies have been recently applied to the full sequence design of a small protein motif and the design of a catalytically active metal center. These successes suggest that designing large, functional proteins in computero is more feasible than ever before.

We have previously reported the development and evaluation of a computational program to assist in the design of hydrophobic cores of proteins. In an effort to investigate the role of core packing in protein structure, we have used this program, referred to as Repacking of Cores (ROC), to design several variants of the protein ubiquitin. Nine ubiquitin variants containing from three to eight hydrophobic core mutations were constructed, purified, and characterized in terms of their stability and their ability to adopt a uniquely folded native-like conformation. In general, designed ubiquitin variants are more stable than control variants in which the hydrophobic core was chosen randomly. However, in contrast to previous results with 434 cro, all designs are destabilized relative to the wild-type (WT) protein. This raises the possibility that beta-sheet structures have more stringent packing requirements than alpha-helical proteins. A more striking observation is that all variants, including random controls, adopt fairly well-defined conformations, regardless of their stability. This result supports conclusions from the cro studies that non-core residues contribute significantly to the conformational uniqueness of these proteins while core packing largely affects protein stability and has less impact on the nature or uniqueness of the fold. Concurrent with the above work, we used stability data on the nine ubiquitin variants to evaluate and improve the predictive ability of our core packing algorithm. Additional versions of the program were generated that differ in potential function parameters and sampling of side chain conformers. Reasonable correlations between experimental and predicted stabilities suggest the program will be useful in future studies to design variants with stabilities closer to that of the native protein. Taken together, the present study provides further clarification of the role of specific packing interactions in protein structure and stability, and demonstrates the benefit of using systematic computational methods to predict core packing arrangements for the design of proteins.

Several novel technologies have evolved over the last decade for the modification of antibodies to enhance their inherent effector functions. All focus on the constant Fc domain and utilize either amino acid substitutions or glycoform perturbations to modulate their interaction with Fc receptors and the effector cells that bear them. We review these technologies with an emphasis on their validation with animal models and human clinical data.

The interaction between integrin lymphocyte function-associated antigen-1 (LFA-1) and its ligand intercellular adhesion molecule-1 (ICAM-1) is critical in immunological and inflammatory reactions but, like other adhesive interactions, is of low affinity. Here, multiple rational design methods were used to engineer ICAM-1 mutants with enhanced affinity for LFA-1. Five amino acid substitutions 1) enhance the hydrophobicity and packing of residues surrounding Glu-34 of ICAM-1, which coordinates to a Mg2+ in the LFA-1 I domain, and 2) alter associations at the edges of the binding interface. The affinity of the most improved ICAM-1 mutant for intermediate- and high-affinity LFA-1 I domains was increased by 19-fold and 22-fold, respectively, relative to wild type. Moreover, potency was similarly enhanced for inhibition of LFA-1-dependent ligand binding and cell adhesion. Thus, rational design can be used to engineer novel adhesion molecules with high monomeric affinity; furthermore, the ICAM-1 mutant holds promise for targeting LFA-1-ICAM-1 interaction for biological studies and therapeutic purposes.

The heat shock protein Hsp90 plays a key, but poorly understood role in the folding, assembly and activation of a large number of signal transduction molecules, in particular kinases and steroid hormone receptors. In carrying out these functions Hsp90 hydrolyses ATP as it cycles between ADP- and ATP-bound forms, and this ATPase activity is regulated by the transient association with a variety of co-chaperones. Cdc37 is one such co-chaperone protein that also has a role in client protein recognition, in that it is required for Hsp90-dependent folding and activation of a particular group of protein kinases. These include the cyclin-dependent kinases (Cdk) 4/6 and Cdk9, Raf-1, Akt and many others. Here, the biochemical details of the interaction of human Hsp90β and Cdc37 have been characterised. Small angle X-ray scattering (SAXS) was then used to study the solution structure of Hsp90 and its complexes with Cdc37. The results suggest a model for the interaction of Cdc37 with Hsp90, whereby a Cdc37 dimer binds the two N-terminal domain/linker regions in an Hsp90 dimer, fixing them in a single conformation that is presumably suitable for client protein recognition.

We present direct evidence for a change in protein structural specificity due to hydrophobic core packing. High resolution structural analysis of a designed core variant of ubiquitin reveals that the protein is in slow exchange between two conformations. Examination of side-chain rotamers indicates that this dynamic response and the lower stability of the protein are coupled to greater strain and mobility in the core. The results suggest that manipulating the level of side-chain strain may be one way of fine tuning the stability and specificity of proteins.

CD30 (also known as Ki-1), a member of the TNF-receptor superfamily, is normally expressed at low levels on activated lymphocytes and has been implicated in cell death and T-cell proliferation. CD30 is highly expressed in Hodgkin’s disease, (HD) and in Anaplastic Large Cell Lymphoma (ALCL). Unmodified CD30 antibodies as well as anti-CD30 based bi-specific antibodies, antibody-toxin conjugates, and radioimmuno-therapeutics have examined CD30 as a therapeutic target in preclinical and clinical studies. Unmodified antibodies have met with limited success and a lack of engagement of immune effector cells may be one of the major short-comings. Although bispecific antibodies proved among the most clinically effective through the recruiting of host effector functions to tumor cells, they pose significant manufacturing challenges. Similarly, toxin- and radio-conjugates require complicated manufacture and handling. Recent advances in antibody engineering have led to the development of “naked” antibodies with greatly enhanced effector function through mutagenesis at the Fc-receptor binding interface. This approach has been applied to a humanized antibody specific for CD30 to produce an antibody with enhanced potency and efficacy coupled with the ease of manufacture and handling of a traditional IgG antibody. The murine:human chimeric antibody cAC10 was humanized using the novel method of human string content optimization. The humanized antibody (hAC10) has an affinity for antigen 4-fold higher than that of the corresponding chimeric antibody. The humanized variable domain was then combined with a modified Fc region and exhibited an approximately 20 fold increase in affinity for the FcγRIIIA receptor resulting in the therapeutic lead XmAb2513. Expression levels from a stable cell-line were close to 1 gm/L in preliminary development. The cytotoxic activity of XmAb2513 was measured by Antibody Dependent Cell-mediated Cytotoxicity (ADCC) assay. ADCC assays used PBMC’s isolated from peripheral blood as effector cells and the human Hodgkin’s cell line L540 which expresses high levels of CD30 as the target at an effector:target ratio of 25:1; cytotoxicity was measured by release of LDH or preloaded TDA. The activity of XmAb2513 was compared to that of cAC10 with no Fc-receptor binding enhancement (IgG1) as well as to the antibody 5F11 (also human IgG1). Significant improvements were observed in both potency (concentration of antibody required to effect 50% of maximal lysis) and efficacy (maximal percent lysis at saturating antibody concentration). The potency of XmAb2513 is ~3 fold higher than that of cAC10-IgG1 and 10-fold higher than 5F11 with an increase in efficacy of 4-fold relative to cAC10-IgG1. XmAb2513 has advantageous properties for a therapeutic compound against CD30-positive lymphomas including high levels of cytotoxicity and ease of manufacture and handling. The promising results reported herein clearly warrant further investigation.

Antibody-dependent cell-mediated cytotoxicity (ADCC) is a key effector function for the clinical efficacy of monoclonal antibodies (Cartron et al., 2002; Weng and Levy, 2003), and is mediated primarily through a set of closely related Fc receptors (FcRs) with both activating and inhibitory activities. Using computational design algorithms and high-throughput screening, we have engineered a series of IgG1 Fc variants with optimized FcR affinity and specificity. These variant Fc domains are readily combined with the variable domains of any antibody and consist of from 1–5 changes in the amino acid sequence. With rituximab as a model system, we have generated variants with greater affinity for the activating receptor FcgammaRIIIa and weaker affinity for the inhibitory receptor FcgammaRIIb. These variants show a marked increase (over 2 logs) in ADCC using purified natural killer cells or PMBCs from healthy human donors against WIL2-S cells, a CD20-bearing tumor cell line. Moreover, PBMC genotyping indicates that this effect occurs irrespective of whether the donor is a high responder (FcgammaRIIIa dimorphism 158V homozygous) or low responder (FcgammaRIIIa dimorphism 158F homozygous or heterozygous) to rituximab therapy. In vivo studies show that our variants deplete circulating B-cells more effectively than rituximab in cynomolgus monkeys with an improvement in half-maximal potency of over 10-fold as measured by flow cytometry. The macaques showed no unusual clinical signs or depletion of other cells types. These agents hold promise as more potent and more efficacious treatments for NHL. Because these Fc mutations display a comparable effector function enhancement in other antibodies such as anti-CD52, anti-EGFR1 and anti-Her2, our technology provides a broadly applicable means for improving the efficacy of the next generation of therapeutic antibodies.