[show abstract][hide abstract] ABSTRACT: The CTLA4-Ig fusion proteins abatacept and belatacept are clinically proven immunosuppressants used for rheumatoid arthritis and renal transplant, respectively. Given that both biologics are typically administered chronically by infusion, a need exists for a next-generation CTLA4-Ig with more convenient dosing. We used structure-based protein engineering to optimize the affinity of existing CTLA4-Ig therapeutics for the ligands CD80 and CD86, and for the neonatal Fc receptor, FcRn. From a rationally designed library, we identified four substitutions that enhanced binding to human CD80 and CD86. Coupled with two IgG1 Fc substitutions that enhanced binding to human FcRn, these changes comprise the novel CTLA4-Ig fusion protein, XPro9523. Compared with abatacept, XPro9523 demonstrated 5.9-fold, 23-fold, and 12-fold increased binding to CD80, CD86, and FcRn, respectively; compared with belatacept, CD80, CD86, and FcRn binding increased 1.5-fold, 7.7-fold, and 11-fold, respectively. XPro9523 and belatacept suppressed human T cell proliferation and IL-2 production more potently than abatacept. XPro9523 also suppressed inflammation in the mouse collagen-induced arthritis model. In cynomolgus monkeys, XPro9523 saturated CD80 and CD86 more effectively than abatacept and belatacept, potently inhibited IgM and IgG immunization responses, and demonstrated longer half-life. Pharmacokinetic modeling of its increased potency and persistence suggests that, in humans, XPro9523 may demonstrate superior efficacy and dosing convenience compared with abatacept and belatacept.
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: Bispecific antibodies based on full-length antibody structures are more optimal than fragment-based formats because they benefit from the favorable properties of the Fc region. However, the homodimeric nature of Fc effectively imposes bivalent binding on all current full-length bispecific antibodies, an attribute that can result in nonspecific activation of cross-linked receptors. We engineered a novel bispecific format, referred to as mAb-Fv, that utilizes a heterodimeric Fc region to enable monovalent co-engagement of a second target antigen in a full-length context. mAb-Fv constructs co-targeting CD16 and CD3 were expressed and purified as heterodimeric species, bound selectively to their co-target antigens, and mediated potent cytotoxic activity by NK cells and T cells, respectively. The capacity to co-engage distinct target antigens simultaneously with different valencies is an improved feature for bispecific antibodies with promising therapeutic implications.
[show abstract][hide abstract] ABSTRACT: 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.
Experimental Cell Research 03/2011; 317(9):1278-85. · 3.56 Impact Factor
[show abstract][hide abstract] ABSTRACT: Fully human monoclonal antibodies (mAbs) derived from transgenic mice or human antibody libraries are the current state of the art for reducing the immunogenicity risk of antibody drugs. Here, we describe a novel method for generating fully human mAbs from nonhuman variable regions using information from the human germline repertoire. Central to our strategy is the rational engineering of residues within and proximal to CDRs and the V(H)/V(L) interface by iteratively exploring substitutions to the closest human germline sequences using semi-automated computational methods. Starting from the parent murine variable regions of three currently marketed mAbs targeting CD25, vascular endothelial growth factor, and tumor necrosis factor alpha, we have generated fully human antibodies with 59, 46, and 45 substitutions, respectively, compared to the parent murine sequences. A large number of these substitutions were in the CDRs, which are typically avoided in humanization methods. Antigen affinities of the fully human variants were comparable to the chimeric mAbs in each case. Furthermore, in vitro functional characterization indicated that all retain potency of the chimeric mAbs and have comparable activity to their respective marketed drugs daclizumab, bevacizumab, and infliximab. Based on local and global sequence identity, the sequences of our engineered mAbs are indistinguishable from those of fully human mAbs isolated from transgenic mice or human antibody libraries. This work establishes a simple rational engineering methodology for generating fully human antibody therapeutics from murine mAbs produced from standard hybridoma technology.
Journal of Molecular Biology 03/2010; 396(5):1474-90. · 3.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: Engineering the antibody Fc region to enhance the cytotoxic activity of therapeutic antibodies is currently an active area of investigation. The contribution of complement to the mechanism of action of some antibodies that target cancers and pathogens makes a compelling case for its optimization. Here we describe the generation of a series of Fc variants with enhanced ability to recruit complement. Variants enhanced the cytotoxic potency of an anti-CD20 antibody up to 23-fold against tumor cells in CDC assays, and demonstrated a correlated increase in C1q binding affinity. Complement-enhancing substitutions combined additively, and in one case synergistically, with substitutions previously engineered for improved binding to Fc gamma receptors. The engineered combinations provided a range of effector function activities, including simultaneously enhanced CDC, ADCC, and phagocytosis. Variants were also effective at boosting the effector function of antibodies targeting the antigens CD40 and CD19, in the former case enhancing CDC over 600-fold, and in the latter case imparting complement-mediated activity onto an IgG1 antibody that was otherwise incapable of it. This work expands the toolkit of modifications for generating monoclonal antibodies with improved therapeutic potential and enables the exploration of optimized synergy between Fc gamma receptors and complement pathways for the destruction of tumors and infectious pathogens.
[show abstract][hide abstract] ABSTRACT: Improved affinity for the neonatal Fc receptor (FcRn) is known to extend antibody half-life in vivo. However, this has never been linked with enhanced therapeutic efficacy. We tested whether antibodies with half-lives extended up to fivefold in human (h)FcRn transgenic mice and threefold in cynomolgus monkeys retain efficacy at longer dosing intervals. We observed that prolonged exposure due to FcRn-mediated enhancement of half-life improved antitumor activity of Fc-engineered antibodies in an hFcRn/Rag1(-/-) mouse model. This bridges the demand for dosing convenience with the clinical necessity of maintaining efficacy.
[show abstract][hide abstract] ABSTRACT: The humoral immune response requires antigen-specific B cell activation and subsequent terminal differentiation into plasma cells. Engagement of B cell antigen receptor (BCR) on mature B cells activates an intracellular signaling cascade, including calcium mobilization, which leads to cell proliferation and differentiation. Coengagement by immune complex of BCR with the inhibitory Fc receptor FcgammaRIIb, the only IgG receptor expressed on B cells, inhibits B cell activation signals through a negative feedback loop. We now describe antibodies that mimic the inhibitory effects of immune complex by high-affinity coengagement of FcgammaRIIb and the BCR coreceptor complex on human B cells. We engineered the Fc domain of an anti-CD19 antibody to generate variants with up to approximately 430-fold greater affinity to FcgammaRIIb. Relative to native IgG1, the FcgammaRIIb binding-enhanced (IIbE) variants strongly inhibited BCR-induced calcium mobilization and viability in primary human B cells. Inhibitory effects involved phosphorylation of SH2-containing inositol polyphosphate 5-phosphatase (SHIP), which is known to be involved in FcgammaRIIb-induced negative feedback of B cell activation by immune complex. Coengagement of BCR and FcgammaRIIb by IIbE variants also overcame the anti-apoptotic effects of BCR activation. The use of a single antibody to suppress B cell functions by coengagement of BCR and FcgammaRIIb may represent a novel approach in the treatment of B cell-mediated autoimmune diseases.
[show abstract][hide abstract] ABSTRACT: The contribution of Fc-mediated effector functions to the therapeutic efficacy of some monoclonal antibodies has motivated efforts to enhance interactions with Fcgamma receptors (FcgammaR). Although an early goal has been enhanced FcgammaRIIIa binding and natural killer (NK) cell antibody-dependent cell-mediated cytotoxicity (ADCC), other relevant cell types such as macrophages are dependent on additional activating receptors such as FcgammaRIIa. Here, we describe a set of engineered Fc variants with diverse FcgammaR affinities, including a novel substitution G236A that provides selectively enhanced binding to FcgammaRIIa relative to FcgammaRIIb. Variants containing this substitution have up to 70-fold greater FcgammaRIIa affinity and 15-fold improvement in FcgammaRIIa/FcgammaRIIb ratio and mediate enhanced phagocytosis of antibody-coated target cells by macrophages. Specific double and triple combination variants with this substitution are simultaneously capable of exhibiting high NK-mediated ADCC and high macrophage phagocytosis. In addition, we have used this unique set of variants to quantitatively probe the relative contributions of individual FcgammaR to effector functions mediated by NK cells and macrophages. These experiments show that FcgammaRIIa plays the most influential role for macrophages and, surprisingly, that the inhibitory receptor FcgammaRIIb has little effect on effector function. The enhancements in phagocytosis described here provide the potential to improve the performance of therapeutic antibodies targeting cancers.
Molecular Cancer Therapeutics 09/2008; 7(8):2517-27. · 5.60 Impact Factor
[show abstract][hide abstract] ABSTRACT: A unique property of monoclonal antibodies, and a principal reason for their success as cancer therapeutics, is their ability to engage the immune system. A growing set of data supporting the relevance of Fc-mediated effector functions to anti-tumor efficacy has motivated efforts to enhance the interactions between antibodies and Fc receptors expressed on immune cells. Although current approaches have considerable promise for improved clinical performance, the immunobiology of tumors, antibodies, and Fc receptors continues to evolve. In this review we discuss what is known and what is not known about the interactions between therapeutic antibodies and the immune system, with the goal being progress toward clear target profiles for effector engineering efforts.
Drug Discovery Today 12/2007; 12(21-22):898-910. · 6.55 Impact Factor
[show abstract][hide abstract] ABSTRACT: We introduce a new method of humanization based on a novel and immunologically relevant metric of antibody humanness, termed human string content (HSC), that quantifies a sequence at the level of potential MHC/T-cell epitopes. Use of this quantity rather than global identity as an optimization goal enables the sampling of human diversity from distinct human germline sequences across the framework and CDR regions, and allows for the generation of multiple diverse candidate sequences. As a result engineering is carried out at finer sequence resolution relative to standard CDR grafting methods, providing for the optimization of antibody properties beyond immunogenicity such as antigen affinity and solution behavior. We have applied this method to the humanization of four antibodies with different antigen specificities. The resulting variable domains differ fundamentally from CDR-grafted antibodies in that they are immunologically more human and their humanness is derived from several discrete germline sequences. Furthermore, these antibodies bind their respective antigens better than or comparable to those of the parent antibodies without the need for affinity maturation.
[show abstract][hide abstract] ABSTRACT: Antibody-dependent cell-mediated cytotoxicity, a key effector function for the clinical efficacy of monoclonal antibodies, is mediated primarily through a set of closely related Fcgamma receptors with both activating and inhibitory activities. By using computational design algorithms and high-throughput screening, we have engineered a series of Fc variants with optimized Fcgamma receptor affinity and specificity. The designed variants display >2 orders of magnitude enhancement of in vitro effector function, enable efficacy against cells expressing low levels of target antigen, and result in increased cytotoxicity in an in vivo preclinical model. Our engineered Fc regions offer a means for improving the next generation of therapeutic antibodies and have the potential to broaden the diversity of antigens that can be targeted for antibody-based tumor therapy.
Proceedings of the National Academy of Sciences 04/2006; 103(11):4005-10. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Journal of Biological Chemistry 03/2006; 281(8):5042-9. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Two recent papers describe noncanonical strategies for improvement of the pharmacokinetic properties of important therapeutic proteins. Although the detailed biologies of the two systems are very different, the unifying feature of both studies is the use of negative design principles. Rather than enhancing specific activity, the central strategy was the removal of states that compromise efficacy. These studies highlight the use of rational engineering and the benefits of firmly understanding the in vivo clearance biology of a biotherapeutic.
Trends in Biotechnology 11/2003; 21(10):425-7. · 9.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: Protein design is becoming an increasingly useful tool for optimizing protein drugs and creating novel biotherapeutics. Recent progress includes the engineering of monoclonal antibodies, cytokines, enzymes and viral fusion inhibitors.
Current Opinion in Structural Biology 09/2003; 13(4):513-8. · 8.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: An increasing number of engineered protein therapeutics are currently being developed, tested in clinical trials and marketed for use. Many of these proteins arose out of hit-and-miss efforts to discover specific mutations, fusion partners or chemical modifications that confer desired properties. Through these efforts, several useful strategies have emerged for rational optimization of therapeutic candidates. The controlled manipulation of the physical, chemical and biological properties of proteins enabled by structure-based simulation is now being used to refine established rational engineering approaches and to advance new strategies. These methods provide clear, hypothesis-driven routes to solve problems that plague many proteins and to create novel mechanisms of action. We anticipate that rational protein engineering will shape the field of protein therapeutics dramatically by improving existing products and enabling the development of novel therapeutic agents.
Drug Discovery Today 04/2003; 8(5):212-21. · 6.55 Impact Factor
[show abstract][hide abstract] ABSTRACT: The recent merger of computation and protein design has resulted in a burst of success in the generation of novel proteins with native-like properties. A critical component of this coupling between theory and experiment is a detailed analysis of the structures and stabilities of designed proteins to assess and improve the accuracy of design algorithms.
Here we report the solution structure of a hydrophobic core variant of ubiquitin, referred to as 1D7, which was designed with the core-repacking algorithm ROC. As a measure of conformational specificity, we also present amide exchange protection factors and backbone and sidechain dynamics. The results indicate that 1D7 is similar to wild-type (WT) ubiquitin in backbone structure and degree of conformational specificity. We also observe a good correlation between experimentally determined sidechain structures and those predicted by ROC. However, evaluation of the core sidechain conformations indicates that, in general, 1D7 has more sidechains in less statistically favorable conformations than WT.
Our results provide an explanation for the lower stability of 1D7 compared to WT, and suggest modifications to design algorithms that may improve the accuracy with which structure and stability are predicted. The results also demonstrate that core packing can affect conformational flexibility in subtle ways that are likely to be important for the design of function and protein-ligand interactions.
[show abstract][hide abstract] ABSTRACT: 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.
Current Opinion in Chemical Biology 01/1999; 2(6):675-9. · 9.47 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Protein Science 12/1998; 8(12):2598 - 2610. · 2.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Protein Science 07/1997; 6(6):1167-78. · 2.74 Impact Factor