Robust production of monovalent bispecific IgG antibodies through novel electrostatic steering mutations at the CH1-Cλ interface.

Bispecific antibodies represent an increasingly large fraction of biologics in therapeutic development due to their expanded scope in functional capabilities. Asymmetric monovalent bispecific IgGs (bsIgGs) have the additional advantage of maintaining a native antibody-like structure, which can provide favorable pharmacology and pharmacokinetic profiles. The production of correctly assembled asymmetric monovalent bsIgGs, however, is a complex engineering endeavor due to the propensity for non-cognate heavy and light chains to mis-pair. Previously, we introduced the DuetMab platform as a general solution for the production of bsIgGs, which utilizes an engineered interchain disulfide bond in one of the CH1-CL domains to promote orthogonal chain pairing between heavy and light chains. While highly effective in promoting cognate heavy and light chain pairing, residual chain mispairing could be detected for specific combinations of Fv pairs. Here, we present enhancements to the DuetMab design that improve chain pairing and production through the introduction of novel electrostatic steering mutations at the CH1-CL interface with lambda light chains (CH1-Cλ). These mutations work together with previously established charge-pair mutations at the CH1-CL interface with kappa light chains (CH1-Cκ) and Fab disulfide engineering to promote cognate heavy and light chain pairing and enable the reliable production of bsIgGs. Importantly, these enhanced DuetMabs do not require engineering of the variable domains and are robust when applied to a panel of bsIgGs with diverse Fv sequences. We present a comprehensive biochemical, biophysical, and functional characterization of the resulting DuetMabs to demonstrate compatibility with industrial production benchmarks. Overall, this enhanced DuetMab platform substantially streamlines process development of these disruptive biotherapeutics.

Conformation-selective rather than avidity-based binding to tumor associated antigen derived peptide-MHC enables targeting of WT1-pMHC low expressing cancer cells by anti-WT1-pMHC/CD3 T cell engagers.

T cell engagers, a category of T cell-retargeting immunotherapy, are rapidly transforming clinical cancer care. However, the lack of tumor-specific targets poses a significant roadblock for broad adaptation of this therapeutic modality in many indications, often resulting in systemic on-target off-tumor toxicity. Though various tumor-derived intracellular mutations provide a massive pool of potential tumor-specific antigens, targeting them is extremely challenging, partly due to the low copy number of tumor associated antigen (TAA)-derived pMHC on tumor cell surface. Further, the interplay of binding geometry and format valency in relation to the capacity of a T cell engager to efficiently target low density cell-surface pMHC is not well understood. Using the Wilms' tumor 1 (WT1) oncoprotein as a proof-of-principle TAA, combined with an array of IgG-like T cell engager modalities that differ in their anti-TAA valency and binding geometry, we show that the ability to induce an immunological synapse formation, resulting in potent killing of WT1 positive cancer cell lines is primarily dependent on the distinct geometrical conformations between the Fab arms of anti-WT1-HLA-A*02:01 and anti-CD3. The augmented avidity conferred by the binding of two anti-WT1-HLA-A*02:01 Fab arms has only minimal influence on cell killing potency. These findings demonstrate the need for careful examination of key design parameters for the development of next-generation T cell engagers targeting low density TAA-pMHCs on tumor cells.

Design and Efficacy of a Monovalent Bispecific PD-1/CTLA4 Antibody That Enhances CTLA4 Blockade on PD-1+ Activated T Cells.

The clinical benefit of PD-1 blockade can be improved by combination with CTLA4 inhibition but is commensurate with significant immune-related adverse events suboptimally limiting the doses of anti-CTLA4 mAb that can be used. MEDI5752 is a monovalent bispecific antibody designed to suppress the PD-1 pathway and provide modulated CTLA4 inhibition favoring enhanced blockade on PD-1+ activated T cells. We show that MEDI5752 preferentially saturates CTLA4 on PD-1+ T cells versus PD-1- T cells, reducing the dose required to elicit IL2 secretion. Unlike conventional PD-1/CTLA4 mAbs, MEDI5752 leads to the rapid internalization and degradation of PD-1. Moreover, we show that MEDI5752 preferentially localizes and accumulates in tumors providing enhanced activity when compared with a combination of mAbs targeting PD-1 and CTLA4 in vivo. Following treatment with MEDI5752, robust partial responses were observed in two patients with advanced solid tumors. MEDI5752 represents a novel immunotherapy engineered to preferentially inhibit CTLA4 on PD-1+ T cells. SIGNIFICANCE: The unique characteristics of MEDI5752 represent a novel immunotherapy engineered to direct CTLA4 inhibition to PD-1+ T cells with the potential for differentiated activity when compared with current conventional mAb combination strategies targeting PD-1 and CTLA4. This molecule therefore represents a step forward in the rational design of cancer immunotherapy.See related commentary by Burton and Tawbi, p. 1008.This article is highlighted in the In This Issue feature, p. 995.

Regulation of antibody-mediated complement-dependent cytotoxicity by modulating the intrinsic affinity and binding valency of IgG for target antigen.

Complement-dependent cytotoxicity (CDC) is a potent effector mechanism, engaging both innate and adaptive immunity. Although strategies to improve the CDC activity of antibody therapeutics have primarily focused on enhancing the interaction between the antibody crystallizable fragment (Fc) and the first subcomponent of the C1 complement complex (C1q), the relative importance of intrinsic affinity and binding valency of an antibody to the target antigen is poorly understood. Here we show that antibody binding affinity to a cell surface target antigen evidently affects the extent and efficacy of antibody-mediated complement activation. We further report the fundamental role of antibody binding valency in the capacity to recruit C1q and regulate CDC. More specifically, an array of affinity-modulated variants and functionally monovalent bispecific derivatives of high-affinity anti-epidermal growth factor receptor (EGFR) and anti-human epidermal growth factor receptor 2 (HER2) therapeutic immunoglobulin Gs (IgGs), previously reported to be deficient in mediating complement activation, were tested for their ability to bind C1q by biolayer interferometry using antigen-loaded biosensors and to exert CDC against a panel of EGFR and HER2 tumor cells of various histological origins. Significantly, affinity-reduced variants or monovalent derivatives, but not their high-affinity bivalent IgG counterparts, induced near-complete cell cytotoxicity in tumor cell lines that had formerly been shown to be resistant to complement-mediated attack. Our findings suggest that monovalent target engagement may contribute to an optimal geometrical positioning of the antibody Fc to engage C1q and deploy the complement pathway.

Correlation between root respiration and the levels of biomass and glycyrrhizic acid in Glycyrrhiza uralensis.

The aim of the present study was to investigate the correlation between root respiration and the levels of biomass and glycyrrhizic acid in Glycyrrhiza uralensis. Root respiration was determined using a biological oxygen analyzer. Respiration-related enzymes including glucose-6-phosphate dehydrogenase plus 6-phosphogluconate dehydrogenase, phosphohexose isomerase and succinate dehydrogenase, and respiratory pathways were evaluated. Biomass was determined by a drying-weighing method. In addition, the percentage of glycyrrhizic acid was detected using high-performance liquid chromatography. The association between root respiration and the levels of biomass and glycyrrhizic acid was investigated. The glycolysis pathway (EMP), tricarboxylic acid cycle (TCA) and pentose phosphate (PPP) pathway acted concurrently in the roots of G. uralensis. Grey correlation analysis showed that TCA had the strongest correlation (correlation coefficient, 0.8003) with biomass. Starch and acetyl coenzyme A had the closest association with above-ground biomass, while soluble sugar correlated less strongly with above-ground biomass. Grey correlation analysis between biochemical pathways and the intermediates showed that pyruvic acid had the strongest correlation with EMP, while acetyl coenzyme A correlated most strongly with TCA. Among the intermediates and pathways, pyruvic acid and EMP exhibited the greatest correlation with glycyrrhizic acid, while acetyl coenzyme A and TCA correlated with glycyrrhizic acid less closely. The results of this study may aid the cultivation of G. uralensis. However, these results require verification in further studies.

Enhanced tumor-targeting selectivity by modulating bispecific antibody binding affinity and format valence.

Bispecific antibodies are considered attractive bio-therapeutic agents owing to their ability to target two distinct disease mediators. Cross-arm avidity targeting of antigen double-positive cancer cells over single-positive normal tissue is believed to enhance the therapeutic efficacy, restrict major escape mechanisms and increase tumor-targeting selectivity, leading to reduced systemic toxicity and improved therapeutic index. However, the interplay of factors regulating target selectivity is not well understood and often overlooked when developing clinically relevant bispecific therapeutics. We show in vivo that dual targeting alone is not sufficient to endow selective tumor-targeting, and report the pivotal roles played by the affinity of the individual arms, overall avidity and format valence. Specifically, a series of monovalent and bivalent bispecific IgGs composed of the anti-HER2 trastuzumab moiety paired with affinity-modulated VH and VL regions of the anti-EGFR GA201 mAb were tested for selective targeting and eradication of double-positive human NCI-H358 non-small cell lung cancer target tumors over single-positive, non-target NCI-H358-HER2 CRISPR knock out tumors in nude mice bearing dual-flank tumor xenografts. Affinity-reduced monovalent bispecific variants, but not their bivalent bispecific counterparts, mediated a greater degree of tumor targeting selectivity, while the overall efficacy against the targeted tumor was not substantially affected.

Element-tracing of mineral matters in Dendrobium officinale using ICP-MS and multivariate analysis.

Rare studies have been performed to trace the mineral elements in Dendrobium officinale. In this study, we aim to trace the mineral elements in D. officinale collected from ten geographical locations in China. ICP-MS system was used for simultaneous determination of mineral elements. Principal component analysis was performed using the obtained data in the quantification of mineral contents. Cluster analysis was performed using the Ward's method. Several of essential microelments were detected in D. officinale, including ferrum (Fe), manganese (Mn), zinc (Zn), chromium (Cr), nickel (Ni) and vanadium (V). Among these elements, three elements (i.e. Fe, Mn and Zn) were highly and simultaneously detected in the D. officinale collected from the ten locations. The level of Ni was positively associated with that of Zn (r = 0.986, P < 0.01). The level of titanium (Ti) was positively associated with that of V (r = 0.669, P < 0.05), and negatively associated with Cr (r = -0.710, P < 0.05). In addition, the level of Mn was positively associated with that of barium (r = 0.749, P < 0.05). Further, the level of Fe was positively associated with that of Ni (r = 0.664, P < 0.05), Zn (r = 0.742, P < 0.05), and rare earth elements (r = 0.847, P < 0.01), respectively. Three eigenvalues explained about 86.60 % of the total variance, which contributed significantly to the explanation of cumulative variance. Cluster analysis indicated the cultivars were categorized into 3 clusters. Ni, Zn, Fe, Cr, Ti and rare earth elements were designated as the characteristic elements. Cultivars collected from Yulin, Menghai, and Shaoguan ranked the top 3 in the comprehensive scores, indicating the content of the mineral elements was comparatively higher in these locations.

Enhancement of Immune Effector Functions by Modulating IgG's Intrinsic Affinity for Target Antigen.

Antibody-mediated immune effector functions play an essential role in the anti-tumor efficacy of many therapeutic mAbs. While much of the effort to improve effector potency has focused on augmenting the interaction between the antibody-Fc and activating Fc-receptors expressed on immune cells, the role of antibody binding interactions with the target antigen remains poorly understood. We show that antibody intrinsic affinity to the target antigen clearly influences the extent and efficiency of Fc-mediated effector mechanisms, and report the pivotal role of antibody binding valence on the ability to regulate effector functions. More particularly, we used an array of affinity modulated variants of three different mAbs, anti-CD4, anti-EGFR and anti-HER2 against a panel of target cell lines expressing disparate levels of the target antigen. We found that at saturating antibody concentrations, IgG variants with moderate intrinsic affinities, similar to those generated by the natural humoral immune response, promoted superior effector functions compared to higher affinity antibodies. We hypothesize that at saturating concentrations, effector function correlates most directly with the amount of Fc bound to the cell surface. Thus, high affinity antibodies exhibiting slow off-rates are more likely to interact bivalently with the target cell, occupying two antigen sites with a single Fc. In contrast, antibodies with faster off-rates are likely to dissociate each binding arm more rapidly, resulting in a higher likelihood of monovalent binding. Monovalent binding may in turn increase target cell opsonization and lead to improved recruitment of effector cells. This unpredicted relationship between target affinity and effector function potency suggests a careful examination of antibody design and engineering for the development of next-generation immunotherapeutics.

A Potent HER3 Monoclonal Antibody That Blocks Both Ligand-Dependent and -Independent Activities: Differential Impacts of PTEN Status on Tumor Response.

HER3/ERBB3 is a kinase-deficient member of the EGFR family receptor tyrosine kinases (RTK) that is broadly expressed and activated in human cancers. HER3 is a compelling cancer target due to its important role in activation of the oncogenic PI3K/AKT pathway. It has also been demonstrated to confer tumor resistance to a variety of cancer therapies, especially targeted drugs against EGFR and HER2. HER3 can be activated by its ligand (heregulin/HRG), which induces HER3 heterodimerization with EGFR, HER2, or other RTKs. Alternatively, HER3 can be activated in a ligand-independent manner through heterodimerization with HER2 in HER2-amplified cells. We developed a fully human mAb against HER3 (KTN3379) that efficiently suppressed HER3 activity in both ligand-dependent and independent settings. Correspondingly, KTN3379 inhibited tumor growth in divergent tumor models driven by either ligand-dependent or independent mechanisms in vitro and in vivo Most intriguingly, while investigating the mechanistic underpinnings of tumor response to KTN3379, we discovered an interesting dichotomy in that PTEN loss, a frequently occurring oncogenic lesion in a broad range of cancer types, substantially blunted the tumor response in HER2-amplified cancer, but not in the ligand-driven cancer. To our knowledge, this represents the first study ascertaining the impact of PTEN loss on the antitumor efficacy of a HER3 mAb. KTN3379 is currently undergoing a phase Ib clinical trial in patients with advanced solid tumors. Our current study may help us optimize patient selection schemes for KTN3379 to maximize its clinical benefits. Mol Cancer Ther; 15(4); 689-701. ©2016 AACR.

Comparison of photosynthesis and fluorescent parameters between Dendrobium officinale and Dendrobium loddigesii.

OBJECTIVE: To investigate the photosynthesis and fluorescent parameters between Dendrobium officinale and Dendrobium loddigesii, based on which to provide helpful information for the artificial cultivation of these cultivars. METHODS: Seeds were placed on the MS medium supplemented with 0.2 mg/L NAA, 2% (w/v) sucrose, 15% (v/v) potato extracts and powered agar (pH 5.8). Two months after germination, seedlings (n = 10) were transferred onto rooting medium containing MS medium supplemented with 0.5 mg/L NAA, 3% (w/v) sucrose, 20% (v/v) potato extracts and 1‰ (w/v) activated carbon (pH 5.8) in a glass bottle (6.5 cm in diameter and 9.5 cm in height) with a white transparent plastic cap. Chlorophyll content was determined using the UV-Vis spectrophotometric method. In addition, rates of oxygen evolution and uptake were measured. The chlorophyll fluorescence was determined at room temperature using PAM 2000 chlorophyll fluorometer (Heinz Walz GmbH, Germany). RESULTS: From month 5 to month 10, the overall contents of both chlorophyll a and chlorophyll b were higher in D. loddigesii compared with those in D. officinale. No statistical differences were observed in the apparent photosynthetic rate (APR) between D. loddigesii and D. officinale. No statistical difference was noticed in the Fo, Fm and Fv between D. loddigesii and D. officinale (P > 0.05). Significant increase was noticed in the oxygen consuming in PSI in month-8 and month-10 compared with that of month-6 in D. loddigesii. Nevertheless, in the D. officinale, the oxygen consuming in PSI in month-6 was remarkably increased with those of month-8 and month-10, respectively. CONCLUSIONS: The photosynthesis and fluorescence parameters varied in the seedling of D. loddigesii and D. officinale. Such information could contribute to the artificial cultivation of these cultivars.

Structural insights into the interaction of human IgG1 with FcγRI: no direct role of glycans in binding.

The three-dimensional structure of a human IgG1 Fc fragment bound to wild-type human FcγRI is reported. The structure of the corresponding complex was solved at a resolution of 2.4 Šusing molecular replacement; this is the highest resolution achieved for an unmutated FcγRI molecule. This study highlights the critical structural and functional role played by the second extracellular subdomain of FcγRI. It also explains the long-known major energetic contribution of the Fc `LLGG' motif at positions 234-237, and particularly of Leu235, via a `lock-and-key' mechanism. Finally, a previously held belief is corrected and a differing view is offered on the recently proposed direct role of Fc carbohydrates in the corresponding interaction. Structural evidence is provided that such glycan-related effects are strictly indirect.

Insights into the molecular basis of a bispecific antibody's target selectivity.

Bispecific antibodies constitute a valuable class of therapeutics owing to their ability to bind 2 distinct targets. Dual targeting is thought to enhance biological efficacy, limit escape mechanisms, and increase target selectivity via a strong avidity effect mediated by concurrent binding to both antigens on the surface of the same cell. However, factors that regulate the extent of target selectivity are not well understood. We show that dual targeting alone is not sufficient to promote efficient target selectivity, and report the substantial roles played by the affinity of the individual arms, overall avidity and valence. More particularly, various monovalent bispecific IgGs composed of an anti-CD70 moiety paired with variants of the anti-CD4 mAb ibalizumab were tested for preferential binding and selective depletion of CD4(+)/CD70(+) T cells over cells expressing only one of the target antigens that resulted from antibody dependent cell-mediated cytotoxicity. Variants exhibiting reduced CD4 affinity showed a greater degree of target selectivity, while the overall efficacy of the bispecific molecule was not affected.

Improving target cell specificity using a novel monovalent bispecific IgG design.

Monovalent bispecific IgGs cater to a distinct set of mechanisms of action but are difficult to engineer and manufacture because of complexities associated with correct heavy and light chain pairing. We have created a novel design, "DuetMab," for efficient production of these molecules. The platform uses knobs-into-holes (KIH) technology for heterodimerization of 2 distinct heavy chains and increases the efficiency of cognate heavy and light chain pairing by replacing the native disulfide bond in one of the CH1-CL interfaces with an engineered disulfide bond. Using two pairs of antibodies, cetuximab (anti-EGFR) and trastuzumab (anti-HER2), and anti-CD40 and anti-CD70 antibodies, we demonstrate that DuetMab antibodies can be produced in a highly purified and active form, and show for the first time that monovalent bispecific IgGs can concurrently bind both antigens on the same cell. This last property compensates for the loss of avidity brought about by monovalency and improves selectivity toward the target cell.

Targeting the junction of CɛmX and ɛ-migis for the specific depletion of mIgE-expressing B cells.

Monoclonal antibodies targeting the extracellular region of the human IgE heavy chain membrane-tethering domain have been proposed for treating allergies caused by hyperproliferative monoclonal expansion of IgE-producing B cells. Antibodies against this target are expected to deplete membrane IgE (mIgE) displaying B cells and leave B cells of other immunoglobulin isotypes intact. Because of alternative splicing, the mIgE heavy chain has two isoforms that differ in their membrane-proximal segment. In the long isoform, the CH4 domain is followed by a 67-amino acid-long extracellular portion. Out of these 67 amino acids, the first 52 amino acids following the CH4 domain constitute the CɛmX segment while the rest of the 15 amino acids immediately adjacent to the membrane constitute the ɛ-migis. In the short isoform the CɛmX segment is absent and the CH4 domain is followed only by the 15-amino acid-long ɛ-migis segment. Using antibodies derived from a phage display library, we investigated: (1) ɛ-migis and (2) the junction of CɛmX and ɛ-migis (CɛmX.migis), as potential therapeutic antibody targets. Our results indicate that antibodies obtained from our phage library that target ɛ-migis bind to a variety of human cells irrespective of mIgE expression, possibly due to homology between ɛ-migis and a region of phosphoinositide-binding protein (ARAP3). In contrast, antibodies specific for the CɛmX.migis junctional region, bound specifically to transfected and primary B cells expressing human mIgE and elicited antibody-dependent cellular cytotoxicity and reduction in IgE production. These antibodies did not bind secreted IgE or the mIgE isoform in which CɛmX is absent. These results suggest that CɛmX.migis junctional region is a promising antibody target and the human antibodies we describe warrant further evaluation.

Deletions in the transmembrane domain of a sindbis virus glycoprotein alter virus infectivity, stability, and host range.

The alphaviruses are composed of two icosahedral protein shells, one nested within the other. A membrane bilayer derived from the host cell is sandwiched between the protein shells. The protein shells are attached to one another by protein domains which extend one of the proteins of the outer shell through the membrane bilayer to attach to the inner shell. We have examined the interaction of the membrane-spanning domain of one of the membrane glycoproteins with the membrane bilayer and with other virus proteins in an attempt to understand the role this domain plays in virus assembly and function. Through incremental deletions, we have reduced the length of a virus membrane protein transmembrane domain from its normal 26 amino acids to 8 amino acids. We examined the effect of these deletions on the assembly and function of virus particles. We found that progressive truncations in the transmembrane domain profoundly affected production of infectious virus in a cyclic fashion. We also found that membrane composition effects protein-protein and protein-membrane interactions during virus assembly.