Truncation of the constant domain drives amyloid formation by immunoglobulin light chains.

AL amyloidosis is a life-threatening disease caused by deposition of immunoglobulin light chains. While the mechanisms underlying light chains amyloidogenesis in vivo remain unclear, several studies have highlighted the role that tissue environment and structural amyloidogenicity of individual light chains have in the disease pathogenesis. AL natural deposits contain both full-length light chains and fragments encompassing the variable domain (VL) as well as different length segments of the constant region (CL), thus highlighting the relevance that proteolysis may have in the fibrillogenesis pathway. Here, we investigate the role of major truncated species of the disease-associated AL55 light chain that were previously identified in natural deposits. Specifically, we study structure, molecular dynamics, thermal stability, and capacity to form fibrils of a fragment containing both the VL and part of the CL (133-AL55), in comparison with the full-length protein and its variable domain alone, under shear stress and physiological conditions. Whereas the full-length light chain forms exclusively amorphous aggregates, both fragments generate fibrils, although, with different kinetics, aggregate structure, and interplay with the unfragmented protein. More specifically, the VL-CL 133-AL55 fragment entirely converts into amyloid fibrils microscopically and spectroscopically similar to their ex vivo counterpart and increases the amorphous aggregation of full-length AL55. Overall, our data support the idea that light chain structure and proteolysis are both relevant for amyloidogenesis in vivo and provide a novel biocompatible model of light chain fibrillogenesis suitable for future mechanistic studies.

An antibody with Fab-constant domains exchanged for a pair of CH3 domains.

We have designed a complete antibody-like construct where the CH1 and Cκ domains are exchanged for a pair of the CH3 domains and efficient pairing of the heavy and light variable domain is achieved using "Knobs-into-Holes" strategy. This construct, composed of only naturally occurring immunoglobulin sequences without artificial linkers, expressed at a high level in mammalian cells, however exhibited low solubility. Rational mutagenesis aimed at the amino acid residues located at the interface of the variable domains and the exchanged CH3 domains was applied to improve the biophysical properties of the molecule. The domain-exchanged construct, including variable domains of the HER2/neu specific antibody trastuzumab, was able to bind to the surface of the strongly HER2/neu positive cell line SK-BR3 4-fold weaker than trastuzumab, but could nevertheless incite a more potent response in an antibody-dependent cell cytotoxicity (ADCC) reporter assay with FcγRIIIa-overexpressing T-cells. This could be explained with a stronger binding to the FcγRIIIa. Importantly, the novel construct could mediate a specific ADCC effect with natural killer cells similar to the parental antibody.

Engineering Bifunctional Antibodies with Constant Region Fusion Architectures.

We report a method to generate bifunctional antibodies by grafting full-length proteins into constant region loops of a full-length antibody or an antigen-binding fragment (Fab). The fusion proteins retain the antigen binding activity of the parent antibody but have an additional activity associated with the protein insert. The engineered antibodies have excellent in vitro activity, physiochemical properties, and stability. Among these, a Her2 × CD3 bispecific antibody (BsAb) was constructed by inserting an anti-Her2 single-chain variable fragment (ScFv) into an anti-CD3 Fab. This bispecific antibody efficiently induces targeted cell lysis in the presence of effector cells at as low as sub-picomolar concentrations in vitro. Moreover, the Her2 × CD3 BsAb shows potent in vivo antitumor activity in mouse Her22+ and Her21+ xenograft models. These results demonstrate that insertion of a full-length protein into non-CDR loops of antibodies provides a feasible approach to generate multifunctional antibodies for therapeutic applications.

Engineered antibody CH2 domains binding to nucleolin: Isolation, characterization and improvement of aggregation.

Smaller recombinant antibody fragments are now emerging as alternatives of conventional antibodies. Especially, immunoglobulin (Ig) constant CH2 domain and engineered CH2 with improved stability are promising as scaffolds for selection of specific binders to various antigens. We constructed a yeast display library based on an engineered human IgG1 CH2 scaffold with diversified loop regions. A group of CH2 binders were isolated from this yeast display library by panning against nucleolin, which is a tumor-associated antigen involved in cell proliferation, tumor cell growth and angiogenesis. Out of 20 mutants, we selected 3 clones exhibiting relatively high affinities to nucleolin on yeasts. However, recombinant CH2 mutants aggregated when they were expressed. To find the mechanism of the aggregation, we employed computational prediction approaches through structural homology models of CH2 binders. The analysis of potential aggregation prone regions (APRs) and solvent accessible surface areas (ASAs) indicated two hydrophobic residues, Val264 and Leu309, in the ß-sheet, in which replacement of both charged residues led to significant decrease of the protein aggregation. The newly identified CH2 binders could be improved to use as candidate therapeutics or research reagents in the future.

Improving the CH1-CK heterodimerization and pharmacokinetics of 4Dm2m, a novel potent CD4-antibody fusion protein against HIV-1.

We previously described 4Dm2m, an exceptionally potent broadly neutralizing CD4-antibody fusion protein against HIV-1. It was generated by fusing the engineered single human CD4 domain mD1.22 to both the N and C termini of the human IgG1 heavy chain constant region and the engineered single human antibody domain m36.4, which targets the CD4-induced coreceptor binding site of the viral envelope glycoprotein, to the N terminus of the human antibody kappa light chain constant region via the (G4S)3 polypeptide linkers. However, therapeutic use of 4Dm2m was limited by its short in vivo half-life. Here, we show that a combination of three approaches have successfully increased the persistence of 4Dm2m in mice. First, to stabilize the scaffold, we enhanced heterodimerization between the heavy chain constant domain 1 (CH1) and kappa light chain constant domain (CK) by using structure-guided design and phage-display library technologies. Second, to address the possibility that long polypeptide linkers might render fusion proteins more susceptible to proteolysis, we shortened the (G4S)3 linkers or replaced them with the human IgG1 hinge sequence, which is naturally designed for both flexibility and stability. Third, we introduced two amino acid mutations into the crystallizable fragment (Fc) of the scaffold previously shown to increase antibody binding to the neonatal Fc receptor (FcRn) and prolong half-lives in vivo. Collectively, these approaches markedly increased the serum concentrations of 4Dm2m in mice while not affecting other properties of the fusion protein. The new 4Dm2m variants are promising candidates for clinical development to prevent or treat HIV-1 infection. To our knowledge, this is the first report on stabilized CH1-CK, which is potentially useful as a new heterodimerization scaffold for generation of bispecific and multispecific antibodies or proteins with a more favorable pharmacokinetic profile.

Human myeloma immunoglobulins of the fourth subclass (IgG4 MAM) contain a fraction with different properties of CH2 domains.

A long-lived metastable minor fraction has been detected and characterized in myeloma protein IgG4 MAM by hydro- and thermodynamic methods. The sedimentation constants of the minor and the major protein fractions are different. The stability of the two CH2 domains in the minor fraction varies. The unique characteristics of these IgG4 MAM conformers arise from the fact that on exchange of the heavy chains between IgG4 molecules, in some of them only one noncanonical bond Cys226-Cys229 is formed in the central part of the "hinge region" instead of two canonical interchain disulfide bonds Cys226-Cys226 and Cys229-Cys229. This leads to asymmetric structure of the IgG4 MAM molecules.

A new tool for monoclonal antibody analysis: application of IdeS proteolysis in IgG domain-specific characterization.

Monoclonal antibody (mAb) products are extraordinarily heterogeneous due to the presence of a variety of enzymatic and chemical modifications, such as deamidation, isomerization, oxidation, glycosylation, glycation, and terminal cyclization. The modifications in different domains of the antibody molecule can result in different biological consequences. Therefore, characterization and routine monitoring of domain-specific modifications are essential to ensure the quality of the therapeutic antibody products. For this purpose, a rapid and informative methodology was developed to examine the heterogeneity of individual domains in mAb products. A recently discovered endopeptidase, IdeS, cleaves heavy chains below the hinge region, producing F(ab') 2 and Fc fragments. Following reduction of disulfide bonds, three antibody domains (LC, Fd, and Fc/2) can be released for further characterization. Subsequent analyses by liquid chromatography/mass spectrometry, capillary isoelectric focusing, and glycan mapping enable domain-specific profiling of oxidation, charge heterogeneity, and glycoform distribution. When coupled with reversed phase chromatography, the unique chromatographic profile of each molecule offers a simple strategy for an identity test, which is an important formal test for biopharmaceutical quality control purposes. This methodology is demonstrated for a number of IgGs of different subclasses (IgG1, IgG2, IgG4), as well as an Fc fusion protein. The presented technique provides a convenient platform approach for scientific and formal therapeutic mAb product characterization. It can also be applied in regulated drug substance batch release and stability testing of antibody and Fc fusion protein products, in particular for identity and routine monitoring of domain-specific modifications.

Quantification of peptides from immunoglobulin constant and variable regions by LC-MRM MS for assessment of multiple myeloma patients.

PURPOSE: Quantitative MS assays for Igs are compared with existing clinical methods in samples from patients with plasma cell dyscrasias, for example, multiple myeloma (MM). EXPERIMENTAL DESIGN: Using LC-MS/MS data, Ig constant region peptides, and transitions were selected for LC-MRM MS. Quantitative assays were used to assess Igs in serum from 83 patients. RNA sequencing and peptide-based LC-MRM are used to define peptides for quantification of the disease-specific Ig. RESULTS: LC-MRM assays quantify serum levels of Igs and their isoforms (IgG1-4, IgA1-2, IgM, IgD, and IgE, as well as kappa (κ) and lambda (λ) light chains). LC-MRM quantification has been applied to single samples from a patient cohort and a longitudinal study of an IgE patient undergoing treatment, to enable comparison with existing clinical methods. Proof-of-concept data for defining and monitoring variable region peptides are provided using the H929 MM cell line and two MM patients. CONCLUSIONS AND CLINICAL RELEVANCE: LC-MRM assays targeting constant region peptides determine the type and isoform of the involved Ig and quantify its expression; the LC-MRM approach has improved sensitivity compared with the current clinical method, but slightly higher inter-assay variability. Detection of variable region peptides is a promising way to improve Ig quantification, which could produce a dramatic increase in sensitivity over existing methods, and could further complement current clinical techniques.

[Correlation between macro- and micro-stability ch2 domains of human IGG2 and their biological activity. 1. Ansalysis the calorimetric and optical melting curves].

Human myeloma immunoglobulin second subclass LOM and SIN, their Fc fragment and firstly obtained hFc fragment in which there is not only low portion of the hinge region, but also its core portion (Cys-Cys-Val-Glu-Cys-Pro-Pro-Cys), have been studied by number of physical methods (scanning calorimetry, fluorescence spectroscopy, analytical centrifugation). Joint analysis of calorimetric and optical melting curves revealed that only first (low-temperature) heat absorption peak at all the melting curves corresponds to the melting of the two CH2 domains. It was shown that CH2 domains of intact IgG2 are destabilized relative to those domains in hFc and Fc fragments.

Destabilization of CH2 domains in intact IgG2 is accompanied by reduced ability to inhibit complement system factor C1.

Fc fragments (hFc) of human myeloma IgG2 proteins LOM and SIN having core hinge (Cys-Cys-Val-Glu-Cys-Pro-Pro-Cys) were first obtained by a modified proteolytic procedure. The thermostability of CH2 domains inside of standard Fc, hFc fragments, and intact IgG2 LOM and SIN was studied by fluorescence spectroscopy. It was found that CH2 domains of intact IgG2 are destabilized. The destabilization is accompanied by reduced ability of IgG2 to inhibit the activation of complement system by classical pathway. This could be due to the decrease in the affinity of CH2 domains to factor C1q.

Extracellular IgC2 constant domains of CEACAMs mediate PI3K sensitivity during uptake of pathogens.

BACKGROUND: Several pathogenic bacteria utilize receptors of the CEACAM family to attach to human cells. Binding to different members of this receptor family can result in uptake of the bacteria. Uptake of Neisseria gonorrhoeae, a gram-negative human pathogen, via CEACAMs found on epithelial cells, such as CEACAM1, CEA or CEACAM6, differs mechanistically from phagocytosis mediated by CEACAM3, a CEACAM family member expressed selectively by human granulocytes. PRINCIPAL FINDINGS: We find that CEACAM1- as well as CEACAM3-mediated bacterial internalization are accompanied by a rapid increase in phosphatidylinositol-3,4,5 phosphate (PI(3,4,5)P) at the site of bacterial entry. However, pharmacological inhibition of phosphatidylinositol-3' kinase (PI3K) selectively affects CEACAM1-mediated uptake of Neisseria gonorrhoeae. Accordingly, overexpression of the PI(3,4,5)P phosphatase SHIP diminishes and expression of a constitutive active PI3K increases CEACAM1-mediated internalization of gonococci, without influencing uptake by CEACAM3. Furthermore, bacterial uptake by GPI-linked members of the CEACAM family (CEA and CEACAM6) and CEACAM1-mediated internalization of N. meningitidis by endothelial cells require PI3K activity. Sensitivity of CEACAM1-mediated uptake toward PI3K inhibition is independent of receptor localization in cholesterol-rich membrane microdomains and does not require the cytoplasmic or the transmembrane domain of CEACAM1. However, PI3K inhibitor sensitivity requires the Ig(C2)-like domains of CEACAM1, which are also present in CEA and CEACAM6, but which are absent from CEACAM3. Accordingly, overexpression of CEACAM1 Ig(C2) domains blocks CEACAM1-mediated internalization. CONCLUSIONS: Our results provide novel mechanistic insight into CEACAM1-mediated endocytosis and suggest that epithelial CEACAMs associate in cis with other membrane receptor(s) via their extracellular domains to trigger bacterial uptake in a PI3K-dependent manner.

The human G1m1 allotype associates with CD4+ T-cell responsiveness to a highly conserved IgG1 constant region peptide and confers an asparaginyl endopeptidase cleavage site.

The human G1m1 allotype comprises two amino acids, D12 and L14, in the CH3 domain of IGHG1. Although the G1m1 allotype is prevalent in human populations, ~40% of Caucasiods are homozygous for the nG1m1 allotype corresponding to E12 and M14. Peptides derived from the G1m1 region were tested for their ability to induce CD4+ T-cell proliferative responses in vitro. A peptide immediately downstream from the G1m1 sequence was recognized by CD4+ T cells in a large percentage of donors (peptide CH315-29). CD4+ T-cell proliferative responses to CH315-29 were found at an increased frequency in nG1m1 homozygous donors. Homozygous nG1m1 donors possessing the HLA-DRB1*07 allele displayed the highest magnitudes of proliferation. CD4+ T cells from donors homozygous for nG1m1 proliferated to G1m1-carrying Fc-fragment proteins, whereas CD4+ T cells from G1m1 homozygous donors did not. The G1m1 sequence creates an enzymatic cleavage site for asparaginyl endopeptidase in vitro. Proteolytic activity at D12 may allow the presentation of the CH315-29 peptide, which in turn may result in the establishment of tolerance to this peptide in G1m1-positive donors. Homozygous nG1m1 patients may be more likely to develop CD4+ T-cell-mediated immune responses to therapeutic antibodies carrying the G1m1 allotype.

The critical role of the constant region in thermal stability and aggregation of amyloidogenic immunoglobulin light chain.

Light chain (LC) amyloidosis (AL) is a fatal disease in which immunoglobulin LC deposit as fibrils. Although the LC amyloid-forming propensity is attributed primarily to the variable region, fibrils also contain full-length LC comprised of variable-joining (V(L)) and constant (C(L)) regions. To assess the role of C(L) in fibrillogenesis, we compared the thermal stability of full-length LC and corresponding V(L) and C(L) fragments. Protein unfolding and aggregation were monitored by circular dichroism and light scattering. A full-length λ6 LC purified from urine of a patient with AL amyloidosis showed irreversible unfolding coupled to aggregation. The transition temperature decreased at slower heating rates, indicating kinetic effects. Next, we studied five recombinant λ6 proteins: full-length amyloidogenic LC, its V(L), germline LC, germline V(L), and C(L). Amyloidogenic and germline proteins showed similar rank order of stability, V(L) < LC < C(L); hence, in the full-length LC, V(L) destabilizes C(L). Amyloidogenic proteins were less stable than their germline counterparts, suggesting that reduction in V(L) stability destabilizes the full-length LC. Thermal unfolding of the full-length amyloidogenic and germline LC required high activation energy and involved irreversible aggregation, yet the unfolding of the isolated V(L) and C(L) fragments was partially reversible. Therefore, compared to their fragments, full-length LCs are more likely to initiate aggregation during unfolding and provide a template for the V(L) deposition. The kinetic barrier for this aggregation is regulated by the stability of the V(L) region. This represents a paradigm shift in AL fibrillogenesis and suggests C(L) region as a potential therapeutic target.

Channel catfish soluble FcmuR binds conserved linear epitopes present on Cmu3 and Cmu4.

A linear epitope on catfish IgM has been identified as the docking site for the catfish soluble FcmuR (IpFcRI). Western blot analyses and latex bead binding assays identified the consensus octapeptide motif FxCxVxHE located at the second cysteine that forms the intrachain disulfide bond of the catfish Cmu3 and Cmu4 immunolglobulin (Ig) domains as the IpFcRI binding sites. Furthermore, molecular modeling of catfish Cmu3 and Cmu4 confirmed that the octapeptide in both of these domains is accessible for IpFcRI interactions. In addition, since this octapeptide motif is also found in other vertebrate Ig domains, IpFcRI binding to Ig heavy (H) and light (L) chains from rainbow trout, chicken, mouse, rabbit, and goat were examined by Western blot analyses and latex bead binding assays. IpFcRI readily bound reduced rainbow trout (Igmu), chicken (Ignu), mouse (Igmu, Iggamma1, Iggamma2a, Iggamma2b, and Igalpha), rabbit (Igmu and Iggamma) and goat (Iggamma) IgH chains, and mouse Igkappa and Iglambda, and chicken Iglambda IgL chains. IpFcRI also bound mouse IgM, IgA and IgG subclasses when examined under native conditions.

Engineered human antibody constant domains with increased stability.

The immunoglobulin (Ig) constant CH2 domain is critical for antibody effector functions. Isolated CH2 domains are promising as scaffolds for construction of libraries containing diverse binders that could also confer some effector functions. However, previous work has shown that an isolated murine CH2 domain is relatively unstable to thermally induced unfolding. To explore unfolding mechanisms of isolated human CH2 and increase its stability gamma1 CH2 was cloned and a panel of cysteine mutants was constructed. Human gamma1 CH2 unfolded at a higher temperature (T(m) = 54.1 degrees C, as measured by circular dichroism) than that previously reported for a mouse CH2 (41 degrees C). One mutant (m01) was remarkably stable (T(m) = 73.8 degrees C). Similar results were obtained by differential scanning calorimetry. This mutant was also significantly more stable than the wild-type CH2 against urea induced unfolding (50% unfolding at urea concentration of 6.8 m versus 4.2 m). The m01 was highly soluble and monomeric. The existence of the second disulfide bond in m01 and its correct position were demonstrated by mass spectrometry and nuclear magnetic resonance spectroscopy, respectively. The loops were on average more flexible than the framework in both CH2 and m01, and the overall secondary structure was not affected by the additional disulfide bond. These data suggest that a human CH2 domain is relatively stable to unfolding at physiological temperature, and that both CH2 and the highly stable mutant m01 are promising new scaffolds for the development of therapeutics against human diseases.

Engineered CH2 domains (nanoantibodies).

Currently, almost all FDA approved therapeutic antibodies (except ReoPro, Lucentis and Cimzia which are Fabs), and the vast majority of those in clinical trials are full-size antibodies mostly in IgG1 format of about 150 kDa size. A fundamental problem for such large molecules is their poor penetration into tissues (e.g., solid tumors) and poor or absent binding to regions on the surface of some molecules (e.g., on the HIV envelope glycoprotein) which are fully accessible only by molecules of smaller size. Therefore, much work especially during the last decade has been aimed at developing novel scaffolds of much smaller size and high stability. Here I briefly describe a proposition to use the immunoglobulin (Ig) constant CH2 domain (CH3 for IgE and IgM) as a scaffold. CH2 is critical for the Ig effector functions. Isolated CH2 is stable monomer in contrast to all other constant domains and most of the variable domains. CH2 and engineered CH2 domains with improved stability can be used as scaffolds for construction of libraries containing diverse binders to various antigens. Such binders based on a CH2 scaffold could also confer some effector functions. Because the CH2 domains are the smallest independently folded antibody domains that can be engineered to contain simultaneously antigen-binding sites and binding sites mediating effector and stability functions, and to distinguish them from domain antibodies which are used to denote engineered VH or VL domains or nanobodies which are used to denote camelid VHH, I termed them nanoantibodies (nAbs).

Production of therapeutic antibodies with controlled fucosylation.

The clinical success of therapeutic antibodies is demonstrated by the number of antibody therapeutics that have been brought to market and the increasing number of therapeutic antibodies in development. Recombinant antibodies are molecular-targeted therapeutic agents and represent a major new class of drugs. However, it is still very important to optimize and maximize the clinical efficacy of therapeutic antibodies, in part to help lower the cost of therapeutic antibodies by potentially reducing the dose or the duration of treatment. Clinical trials using therapeutic antibodies fully lacking core fucose residue in the Fc oligosaccharides are currently underway, and their remarkable physiological activities in humans in vivo have attracted attention as next-generation therapeutic antibody approaches with improved efficacy. Thus, an industrially applicable antibody production process that provides consistent yields of fully non-fucosylated antibody therapeutics with fixed quality has become a key goal in the successful development of next-generation therapeutic agents. In this article, we review the current technologies for production of therapeutic antibodies with control of fucosylation of the Fc N-glycans.

Design of the artificial acellular feeder layer for the efficient propagation of mouse embryonic stem cells.

Embryonic stem (ES) cells are pluripotent-undifferentiated cells that have a great interest for the investigation of developmental biology. Murine ES cells maintain their pluripotency by the supplementation of the leukemia inhibitory factor (LIF). LIF is reported to act as a matrix-anchored form, and immobilized cytokines are useful to sustain their signaling on target cells. In this study, we used the immobilizable fusion protein composed of LIF and IgG-Fc region, which was used as a model of the matrix-anchored form of LIF to establish a novel system for ES cell culture and to investigate the effect of immobilized LIF on maintenance of ES cell pluripotency. Mouse ES cells maintained their undifferentiated state on the surface coated with LIF-Fc. Furthermore, when cultured on the co-immobilized surface with LIF-Fc and E-cadherin-Fc, mouse ES cells showed characteristic scattering morphologies without colony formation, and they could maintain their undifferentiated state and pluripotency without additional LIF supplementation. The activation of LIF signaling was sustained on the co-immobilized surface. These results indicate that immobilized LIF and E-cadherin can maintain mouse ES cells efficiently and that the immobilizable LIF-Fc fusion protein is useful for the investigation of signaling pathways of an immobilized form of LIF in the maintenance of ES cell pluripotency.

An investigation of the ligand binding properties and negative cooperativity of soluble insulin-like growth factor receptors.

To investigate the interaction of the insulin-like growth factor (IGF) ligands with the insulin-like growth factor type 1 receptor (IGF-1R), we have generated two soluble variants of the IGF-1R. We have recombinantly expressed the ectodomain of IGF-1R or fused this domain to the constant domain from the Fc fragment of mouse immunoglobulin. The ligand binding properties of these soluble IGF-1Rs for IGF-I and IGF-II were investigated using conventional ligand competition assays and BIAcore biosensor technology. In ligand competition assays, the soluble IGF-1Rs both bound IGF-I with similar affinities and a 5-fold lower affinity than that seen for the wild type receptor. In addition, both soluble receptors bound IGF-II with similar affinities to the wild type receptor. BIAcore analyses showed that both soluble IGF-1Rs exhibited similar ligand-specific association and dissociation rates for IGF-I and for IGF-II. The soluble IGF-1R proteins both exhibited negative cooperativity for IGF-I, IGF-II, and the 24-60 antibody, which binds to the IGF-1R cysteine-rich domain. We conclude that the addition of the self-associating Fc domain to the IGF-1R ectodomain does not affect ligand binding affinity, which is in contrast to the soluble ectodomain of the IR. This study highlights some significant differences in ligand binding modes between the IGF-1R and the insulin receptor, which may ultimately contribute to the different biological activities conferred by the two receptors.