Computational design of a specific heavy chain/κ light chain interface for expressing fully IgG bispecific antibodies.

The use of bispecific antibodies (BsAbs) to treat human diseases is on the rise. Increasingly complex and powerful therapeutic mechanisms made possible by BsAbs are spurring innovation of novel BsAb formats and methods for their production. The long-lived in vivo pharmacokinetics, optimal biophysical properties and potential effector functions of natural IgG monoclonal (and monospecific) antibodies has resulted in a push to generate fully IgG BsAb formats with the same quaternary structure as monoclonal IgGs. The production of fully IgG BsAbs is challenging because of the highly heterogeneous pairing of heavy chains (HCs) and light chains (LCs) when produced in mammalian cells with two IgG HCs and two LCs. A solution to the HC heterodimerization aspect of IgG BsAb production was first discovered two decades ago; however, addressing the LC mispairing issue has remained intractable until recently. Here, we use computational and rational engineering to develop novel designs to the HC/LC pairing issue, and particularly for κ LCs. Crystal structures of these designs highlight the interactions that provide HC/LC specificity. We produce and characterize multiple fully IgG BsAbs using these novel designs. We demonstrate the importance of specificity engineering in both the variable and constant domains to achieve robust HC/LC specificity within all the BsAbs. These solutions facilitate the production of fully IgG BsAbs for clinical use.

A comparative study of peptide models of the alpha-domain of alpha-lactalbumin, lysozyme, and alpha-lactalbumin/lysozyme chimeras allows the elucidation of critical factors that contribute to the ability to form stable partially folded states.

alpha-Lactalbumin (alpha LA) forms a well-populated equilibrium molten globule state, while the homologous protein hen lysozyme does not. alpha LA is a two-domain protein and the alpha-domain is more structured in the molten globule state than is the beta-domain. Peptide models derived from the alpha-subdomain that contain the A, B, D, and 3(10) helices of alpha LA are capable of forming a molten globule state in the absence of the remainder of the protein. Here we report comparative studies of a peptide model derived from the same region of hen lysozyme and a set of chimeric alpha-lactalbumin--lysozyme constructs. Circular dichroism, dynamic light scattering, sedimentation equilibrium, and fluorescence experiments indicate that the lysozyme construct does not fold. Chimeric constructs were prepared to probe the origins of the difference in the ability of the two isolated subdomains to fold. The first consists of the A and B helices of alpha LA cross-linked to the D and C-terminal 3(10) helices of lysozyme. This construct is highly helical, while a second construct that contains the A and B helices of lysozyme cross-linked to the D and 3(10) helices of alpha LA does not fold. Furthermore, the disulfide cross-linked homodimer of the alpha LA AB peptide is helical, while the homodimer of the lysozyme AB peptide is unstructured. Thus, the AB helix region of alpha LA appears to have an intrinsic ability to form structure as long as some relatively nonspecific interactions can be made with other regions of the protein. Our studies show that the A and B helices plays a key role in the ability of the respective alpha-subdomains to fold.

A protein dissection study demonstrates that two specific hydrophobic clusters play a key role in stabilizing the core structure of the molten globule state of human alpha-lactalbumin.

The molten globule state of alpha-lactalbumin (alpha LA) has served as a paradigm for understanding the role of these partially folded states in protein folding. We previously showed that a peptide construct consisting of the A and B helices (residues 1-38) cross-linked to the D- and C-terminal 3(10) helices (residues 101-120) of alpha LA is capable of folding to a stable molten globule-like state. Here, we report the study of three peptide constructs that are designed to investigate the contribution two short hydrophobic sequences located near the C-terminus of alpha LA make to the structure and stability of the alpha LA molten globule state. These regions of the protein have been shown to form stable non-native structures in isolation. The three peptide constructs contain residues 1-38 cross-linked to three separate C-terminal peptides via the native 28-111 disulfide bond. The C-terminal peptides consist of residues 101-114, 106-120, and 106-114. The results of CD, fluorescence, ANS binding, and urea denaturation experiments indicate that constructs that lack either of the hydrophobic sequences (residues 101-105 and 115-120) are significantly less structured. These results highlight the importance of long-range, mutually stabilizing interactions within the molten globule state of the protein. Proteins 2001;42:237-242.

Solution structure of a peptide model of a region important for the folding of alpha-lactalbumin provides evidence for the formation of nonnative structure in the denatured state.

Elucidating the properties of the denatured state of proteins under conditions relevant for their folding is a key factor in understanding the folding process. We show that a peptide corresponding to residues 111-120 of human alpha-lactalbumin has a pronounced propensity to adopt nonnative structure in aqueous solution. Two-dimensional NMR provides evidence for a structured, nonnative conformation in fast exchange with a random coil ensemble. A total of 78 Rotating Frame Overhauser Effects (ROEs) were used to calculate the conformation of the structured population. A nonnative cluster of hydrophobic residues involving the side chains of K114, W118, Ll119, and A120 is observed, which helps to stabilize a turn-like conformation in the vicinity of residues 115-118. The structure in 30% (vol/vol) TFE was also calculated. Interestingly, the addition of TFE did not simply amplify the population of the structured conformer observed in H2O, but instead induced a new conformation. The implications for the folding of the intact protein are discussed. We also discuss the implications of this study for the relevance of the use of mixed TFE/H2O solvent systems to study isolated peptides.

Local interactions and the role of the 6-120 disulfide bond in alpha-lactalbumin: implications for formation of the molten globule state.

Molten globule states are partially folded states of proteins which are compact and contain a high degree of secondary structure but which lack many of the fixed tertiary interactions associated with the native state. A set of peptides has been prepared in order to probe the role of local interactions in the vicinity of the Cys(6)-Cys(120) disulfide bond in stabilizing the molten globule state of human alpha-lactalbumin. Peptides derived from the N-terminal and C-terminal regions of human alpha-lactalbumin have been analyzed using nuclear magnetic resonance, circular dichroism, fluorescence spectroscopy and sedimentation equilibrium experiments. A peptide corresponding to the first helical region in the native protein, residues 1-13, is only slightly helical in isolation. Extending the peptide to include residues 14-18 results in a modest increase in helicity. A peptide derived from the C-terminal 12 residues, residues 112-123, is predominantly unstructured. Crosslinking the N- and C-terminal peptides by the native disulfide bond results in almost no increase in structure and there is no evidence for any significant cooperative structure formation over the range of pH 2.2-11.7. These results demonstrate that there is very little enhancement of local structure due to the formation of the Cys(6)-Cys(120) disulfide bond. This is in striking contrast to peptides derived from the region of the Cys(28)-Cys(111) disulfide.

Defining the core structure of the alpha-lactalbumin molten globule state.

Molten globules are partially folded states of proteins which are generally believed to mimic structures formed during the folding process. In order to determine the minimal requirements for the formation of a molten globule state, we have prepared a set of peptide models of the molten globule state of human alpha-lactalbumin (alphaLA). A peptide consisting of residues 1-38 crosslinked, via the native 28-111 disulfide bond, to a peptide corresponding to residues 95-120 forms a partially folded state at pH 2.8 which has all of the characteristics of the molten globule state of alphaLA as judged by near and far UV CD, fluorescence, ANS binding and urea denaturation experiments. The structure of the peptide construct is the same at pH 7.0. Deletion of residues 95-100 from the construct has little effect. Thus, less than half the sequence is required to form a molten globule. Further truncation corresponding to the selective deletion of the A (residues 1-19) or D (residues 101-110) helices or the C-terminal 310 helix (residues 112-120) leads to a significant loss of structure. The loss of structure which results from the deletion of any of these three regions is much greater than that which would be expected based upon the non-cooperative loss of local helical structure. Deletion of residues corresponding to the region of the D helix or C-terminal 310 helix region results in a peptide construct which is largely unfolded and contains no more helical structure than is expected from the sum of the helicity of the two reduced peptides. These experiments have defined the minimum core structure of the alphaLA molten globule state.

Local interactions drive the formation of nonnative structure in the denatured state of human alpha-lactalbumin: a high resolution structural characterization of a peptide model in aqueous solution.

There are a small number of peptides derived from proteins that have a propensity to adopt structure in aqueous solution which is similar to the structure they possess in the parent protein. There are far fewer examples of protein fragments which adopt stable nonnative structures in isolation. Understanding how nonnative interactions are involved in protein folding is crucial to our understanding of the topic. Here we show that a small, 11 amino acid peptide corresponding to residues 101-111 of the protein alpha-lactalbumin is remarkably structured in isolation in aqueous solution. The peptide has been characterized by 1H NMR, and 170 ROE-derived constraints were used to calculate a structure. The calculations yielded a single, high-resolution structure for residues 101-107 that is nonnative in both the backbone and side-chain conformations. In the pH 6.5 crystal structure, residues 101-105 are in an irregular turn-like conformation and residues 106-111 form an alpha-helix. In the pH 4.2 crystal structure, residues 101-105 form an alpha-helix, and residues 106-111 form a loopike structure. Both of these structures are significantly different from the conformation adopted by our peptide. The structure in the peptide model is primarily the result of local side-chain interactions that force the backbone to adopt a nonnative 310/turn-like structure in residues 103-106. The structure in aqueous solution was compared to the structure in 30% trifluoroethanol (TFE), and clear differences were observed. In particular, one of the side-chain interactions, a hydrophobic cluster involving residues 101-105, is different in the two solvents and residues 107-111 are considerably more ordered in 30% TFE. The implications of the nonnative structure for the folding of alpha-lactalbumin is discussed.

Peptide models of local and long-range interactions in the molten globule state of human alpha-lactalbumin.

alpha-Lactalbumin, a small calcium-binding protein, forms an equilibrium molten globule state under a variety of conditions. A set of four peptides designed to probe the role of local interactions and the role of potential long-range interactions in stabilizing the molten globule of alpha-lactalbumin has been prepared. The first peptide consists of residues 20 through 36 of human alpha-lactalbumin and includes the entire B-helix. This peptide is unstructured in solution as judged by CD. The second peptide is derived from residues 101 through 120 and contains both the D and 310 helices. When this peptide is crosslinked via the native 28 to 111 disulfide to the B-helix peptide, a dramatic increase in helicity is observed. The crosslinked peptide is monomeric, as judged by analytical ultracentrifugation. The peptide binds 1-anilinonaphthalene-8-sulphonate (ANS) and the fluorescence emission maximum of the construct is consistent with partial solvent exposure of the tryptophan residues. The peptide corresponding to residues 101 to 120 adopts significant non-random structure in aqueous solution at low pH. Two hydrophobic clusters, one involving residues 101 through 104 and the other residues 115 through 119 have been identified and characterized by NMR. The hydrophobic cluster formed by residues 101 through 104 is still present in a smaller peptide containing only residues 101 to 111 of alpha-lactalbumin. The cluster also persists in 6 M urea. A non-native, pH-dependent interaction between the Y103 and H107 side-chains that was previously identified in the acid-denatured molten globule state was examined. This interaction was found to be more prevalent at low pH and may therefore be an example of a local interaction that stabilizes preferentially the acid-induced molten globule state.