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R Blake Pepinsky,
Laura Silvian,
Steven A Berkowitz,
Graham Farrington,
Alexey Lugovskoy,
Lee Walus, John Eldredge,
Allan Capili,
Sha Mi,
Christilyn Graff,
Ellen Garber
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ABSTRACT: Monoclonal antibodies (Mabs) are a favorite drug platform of the biopharmaceutical industry. Currently, over 20 Mabs have been approved and several hundred others are in clinical trials. The anti-LINGO-1 Mab Li33 was selected from a large panel of antibodies by Fab phage display technology based on its extraordinary biological activity in promoting oligodendrocyte differentiation and myelination in vitro and in animal models of remyelination. However, the Li33 Fab had poor solubility when converted into a full antibody in an immunoglobulin G1 framework. A detailed analysis of the biochemical and structural features of the antibody revealed several possible reasons for its propensity to aggregate. Here, we successfully applied three molecular approaches (isotype switching, targeted mutagenesis of complementarity determining region residues, and glycosylation site insertion mutagenesis) to address the solubility problem. Through these efforts we were able to improve the solubility of the Li33 Mab from 0.3 mg/mL to >50 mg/mL and reduce aggregation to an acceptable level. These strategies can be readily applied to other proteins with solubility issues.
Protein Science 03/2010; 19(5):954-66. · 2.80 Impact Factor
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ABSTRACT: A design approach was taken to investigate the feasibility of replacing single complementarity determining region (CDR) antibody loops. This approach may complement simpler mutation-based strategies for rational antibody design by expanding conformation space. Enormous crystal structure diversity is available, making CDR loops logical targets for structure-based design. A detailed analysis for the L1 loop shows that each loop length takes a distinct conformation, thereby allowing control on a length scale beyond that accessible to simple mutations. The L1 loop in the anti-VLA1 antibody was replaced with the L2 loop residues longer in an attempt to add an additional hydrogen bond and fill space on the antibody-antigen interface. The designs expressed well, but failed to improve affinity. In an effort to learn more, one design was crystallized and data were collected at 1.9 A resolution. The designed L1 loop takes the qualitatively desired conformation; confirming that loop replacement by design is feasible. The crystal structure also shows that the outermost loop (residues Leu51-Ser68) is domain swapped with another monomer. Tryptophan fluorescence measurements were used to monitor unfolding as a function of temperature and indicate that the loop involved in domain swapping does not unfold below 60 degrees C. The domain-swapping is not directly responsible for the affinity loss, but is likely a side-effect of the structural instability which may contribute to affinity loss. A second round of design was successful in eliminating the dimerization through mutation of a residue (Leu51Ser) at the joint of the domain-swapped loop.
Protein Engineering Design and Selection 01/2009; 22(2):93-101. · 2.94 Impact Factor
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ABSTRACT: LTbetaR is a member of the TNF receptor family of proteins. It binds to two different cell surface ligands, LIGHT, a homotypic trimer, and LTalpha1beta2, a heterotypic trimer. We have measured the affinities of the dimeric IgG fusion protein, LTbetaRIgG, and monomeric LTbetaR protein binding to both LIGHT and LTalpha1beta2 using surface plasmon resonance and found values of <0.1 and 38 nM for LIGHT and <0.1 and 48 nM for LTalpha1beta2, respectively. We also determined the stoichiometries of binding for both forms of the receptor LTbetaRIgG and LTbetaR binding to LIGHT. The data obtained from several biophysical methods are consistent with receptor polypeptide to trimeric ligand ratios of 2:1. The determined masses of the complexes using SEC-LS corresponded to a single LTbetaRIgG bound to a LIGHT trimer, or two LTbetaR bound per LIGHT. Sedimentation velocity of varied ratios of LTbetaR to a fixed concentration of LIGHT were analyzed by SEDANAL and were successfully fit with a model with two tight binding sites on LIGHT and one poor affinity site. Isothermal calorimetric titration of LIGHT with either LTbetaR or LTbetaRIgG also demonstrated stoichiometries of 1:2 and 1:1, respectively. The binding of LTbetaR to LIGHT was endothermic and, hence, entropy-driven. TNFR p55 (extracellular domain) complexed with the trimeric ligand, TNFbeta, exhibits a 3:1 receptor/ligand stoichiometry. This complex has been used as the prototypical model setting the receptor-ligand complexation paradigm for the entire TNF family. The LTbetaR/LIGHT binding stoichiometry of 2:1 demonstrated here does not fit the paradigm. This has numerous implications for cell biology including signaling requiring only dimerization of LTbetaR rather than trimerization as expected from the structural paradigm.
Biochemistry 09/2006; 45(33):10117-28. · 3.42 Impact Factor
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ABSTRACT: LTβR is a member of the TNF receptor family of proteins. It binds to two different cell surface ligands, LIGHT, a homotypic trimer, and LTα1β2, a heterotypic trimer. We have measured the affinities of the dimeric IgG fusion protein, LTβRIgG, and monomeric LTβR protein binding to both LIGHT and LTα1β2 using surface plasmon resonance and found values of <0.1 and 38 nM for LIGHT and <0.1 and 48 nM for LTα1β2, respectively. We also determined the stoichiometries of binding for both forms of the receptor LTβRIgG and LTβR binding to LIGHT. The data obtained from several biophysical methods are consistent with receptor polypeptide to trimeric ligand ratios of 2:1. The determined masses of the complexes using SEC−LS corresponded to a single LTβRIgG bound to a LIGHT trimer, or two LTβR bound per LIGHT. Sedimentation velocity of varied ratios of LTβR to a fixed concentration of LIGHT were analyzed by SEDANAL and were successfully fit with a model with two tight binding sites on LIGHT and one poor affinity site. Isothermal calorimetric titration of LIGHT with either LTβR or LTβRIgG also demonstrated stoichiometries of 1:2 and 1:1, respectively. The binding of LTβR to LIGHT was endothermic and, hence, entropy-driven. TNFR p55 (extracellular domain) complexed with the trimeric ligand, TNFβ, exhibits a 3:1 receptor/ligand stoichiometry. This complex has been used as the prototypical model setting the receptor−ligand complexation paradigm for the entire TNF family. The LTβR/LIGHT binding stoichiometry of 2:1 demonstrated here does not fit the paradigm. This has numerous implications for cell biology including signaling requiring only dimerization of LTβR rather than trimerization as expected from the structural paradigm.
07/2006;
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Louis A Clark,
P Ann Boriack-Sjodin, John Eldredge,
Christopher Fitch,
Bethany Friedman,
Karl J M Hanf,
Matthew Jarpe,
Stefano F Liparoto,
You Li,
Alexey Lugovskoy,
Stephan Miller,
Mia Rushe,
Woody Sherman,
Kenneth Simon,
Herman Van Vlijmen
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ABSTRACT: Improving the affinity of a high-affinity protein-protein interaction is a challenging problem that has practical applications in the development of therapeutic biomolecules. We used a combination of structure-based computational methods to optimize the binding affinity of an antibody fragment to the I-domain of the integrin VLA1. Despite the already high affinity of the antibody (Kd approximately 7 nM) and the moderate resolution (2.8 A) of the starting crystal structure, the affinity was increased by an order of magnitude primarily through a decrease in the dissociation rate. We determined the crystal structure of a high-affinity quadruple mutant complex at 2.2 A. The structure shows that the design makes the predicted contacts. Structural evidence and mutagenesis experiments that probe a hydrogen bond network illustrate the importance of satisfying hydrogen bonding requirements while seeking higher-affinity mutations. The large and diverse set of interface mutations allowed refinement of the mutant binding affinity prediction protocol and improvement of the single-mutant success rate. Our results indicate that structure-based computational design can be successfully applied to further improve the binding of high-affinity antibodies.
Protein Science 06/2006; 15(5):949-60. · 2.80 Impact Factor
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Teresa G Cachero,
Ian M Schwartz,
Fang Qian,
Eric S Day,
Claudia Bossen,
Karine Ingold,
Aubry Tardivel,
Dennis Krushinskie, John Eldredge,
Laura Silvian,
Alexey Lugovskoy,
Graham K Farrington,
Kathy Strauch,
Pascal Schneider,
Adrian Whitty
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ABSTRACT: The oligomeric state of BAFF (B cell activing factor), a tumor necrosis factor (TNF) family cytokine that plays a critical role in B cell development and survival, has been the subject of recent debate. Myc-tagged BAFF starting at residue Gln136 was previously reported to crystallize as trimers at pH 4.5, whereas a histidine-tagged construct of BAFF, starting at residue Ala134, formed a virus-like cluster containing 60 monomers when crystallized at pH 9.0. The formation of the BAFF 60-mer was pH dependent, requiring pH >or= 7.0. More recently, 60-mer formation was suggested to be artificially induced by the histidine tag, and it was proposed that BAFF, like all other TNF family members, is trimeric. We report here that a construct of BAFF with no amino-terminal tag (Ala134-BAFF) can form a 60-mer in solution. Using size exclusion chromatography and static light scattering to monitor trimer to 60-mer ratios in BAFF preparations, we find that 60-mer formation is pH-dependent and requires histidine 218 within the DE loop of BAFF. Biacore measurements established that the affinity of Ala134-BAFF for the BAFF receptor BAFFR/BR3 is similar to that of myc-Gln136-BAFF, which is exclusively trimeric in solution. However, Ala134-BAFF is more efficacious than myc-Gln136-BAFF in inducing B cell proliferation in vitro. We additionally show that BAFF that is processed and secreted by 293T cells transfected with full-length BAFF, or by a histiocytic lymphoma cell line (U937) that expresses BAFF endogenously, forms a pH-dependent 60-mer in solution. Our results indicate that the formation of the 60-mer in solution by the BAFF extracellular domain is an intrinsic property of the protein, and therefore that this more active form of BAFF may be physiologically relevant.
Biochemistry 02/2006; 45(7):2006-13. · 3.42 Impact Factor
